Journal of Life Sciences Volume 8 Number (1)
J LS
Journal of Life Sciences
Volume 8, Number 5, May 2014 (Serial Number 73)
Contents
Physiology and Biochemistry
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the Seldinger Technique Ultrasound-Guided
Gustavo Henrique Pereira Dutra, Fábio Futema, Flávio Augusto Marques dos Santos and Cristiane Lassalvia Nascimento
Arg-X Activity in Trypsin-like Complexes of the Nuclear Rroteins in the Suprastructures of Interphase Chromatin During Induction of Growth Morphogenesis Mature Germs of Wheat
Ivanova Evilina, Vafina Gulnara, Ivanov Ruslan and Tereshchenko Lidia 399
The Effect of Mixture of Alpinia Galanga, Eurycoma Longifolia Jack and Syzygium Aromaticum Crude Extract on the Growth of Saccharomyces Cerevisiae and Escherichia Coli
Nur Jasrina Jaafar, Kaswandi Md. Ambia, Hing Hiang Lian and Rahim Md. Noah 404
Association Between Interleukin-6 (IL-6) and Iron Status in Rheumatoid Arthritis Patients
Abbas Sabbar Dkhil and Musa Nima Mezher
Botany and Zoology
Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F1 Hybrids Using Random Amplified Polymorphic DNA in Indonesia
Sukaya, Nandariyah and Bambang Pujiasmanto 418
Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture, Example of Two Tunisian Cultivars
Sana Gammoudi, René Lecoustre and Mohamed Ben Salah 425
Architecter Study of the Young Date Palm (Phoenix dactylifera L.) Root System
JRAD Amira and BEN SALAH Mohamed
Study on Development of Formulated Feed for Improving Growth and Pigmentation of Koi Carp (Cyprinus carpio L., 1758) Juveniles
Nguyen Van Nguyen, Tran Van Khanh and Pham Duy Hai 442
Study of Caspian Goby Neogobius sp. Karyotype Flexibility from Several Biotops
Aithazha Bigaliev, Saidina Kobegenova, Viktor Vasil’ev, Elena Vasil’eva, Aiman Imentai and Ashan Shametov
Interdisciplinary Researches
Diel Vertical Distribution of Zooplankton in Lake Baringo, Kenya
Reuben Omondi, Andrew W. Yasindi and Adiel M. Magana 461
Heavy Metals in Dust Deposition in the Vicinity of Coal Ash Disposal Site Divkovici II
Abdel Dozic, Vahida Selimbasic, Amira Cipurkovic, Aida Crnkic, Zorica Hodzic and Ilvana Trumic
A Potential Weed Control—Using Robotic Implement
Kwang Ho Park, Soo Hyun Kim, Young Kuk Kim, Han Jong Joo, Yoon Shik Hong, Jee Hyong Kim and Keun Mo Koo
May 2014, Vol. 8, No. 5, pp. 385-393
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the Seldinger Technique Ultrasound-Guided
1 2 Gustavo Henrique Pereira Dutra 3 , Fábio Futema , Flávio Augusto Marques dos Santos and Cristiane Lassalvia Nascimento 1
1. Aquário Municipal de Santos, Av. Bartolomeu de Gusmão s n. Ponta da Praia. Santos-SP 11030-500, Brazil 2. Universidade de Guarulhos, Av. Anton Philips, 01, Vila Hermínia, Guarulhos 07030-010, Brazil 3. Universidade Anhembi Morumbi, Rua Casa do Ator, 294. Vila Olímpia 04546-000-Sao Paulo, SP, Brasil
Received: March 12, 2014/ Accepted: May 12, 2014 / Published: May 30, 2014.
Abstract: Strand-ed turtles need fluid parenterally. The jugular access is best for the maintenance and patency of the catheter. The Seldinger technique guided by ultrasound seems to be the safest for catheter insertion. Five juveniles Chelonia mydas with fibropapillomatosis and an adult male leatherback turtle (Dermochelys coriacea) with altered buoyancy were sent to Santos Municipal Aquarium for rehabilitation. Turtles underwent catheterization of the jugular vein using the Seldinger technique with central venous polyurethane catheter monolumen 14 G to Dermochelys and 18 G for Chelonia, guided by ultrasound with 10 MHz transducer catheters were sutured to the skin and animals were subjected to fluid. In all turtles, the catheters were patency and were well established. There were no granulomatous reactions or related infections. The Dermochelys improved clinically after fluid resuscitation, and the catheter was removed one week after. In one Chelonia excision was 21% of its weight in tumors, and the animals received colloid catheter by enabling the mitigation of bleeding due to removal. The use of ultrasonography has enabled monitoring of all access as the patency of the catheter and fluid diffusivity.
Key words: Sea turtles, Chelonia, Dermochelys, ultrassonography, Seldinger technique.
1. Introduction emergency therapy. However, the placement and maintenance of catheters in these locations can be
Stranded sea turtles usually require hydration care; technically challenging, especially in aquatic species, they have varying degrees of gastrointestinal and should be reserved for patients who are obstruction, cold stunning paralysis, trauma, unconscious or minimally sensitive [3]. A constant infectious diseases, parasitism and fibropapillomatosis. infusion of fluids is preferable over intermittent bolus, Hypoglycemic turtles need to receive fluid but any effort should be made to place an intravenous resuscitation with glucose. Most stranded animals catheter, or alternatively intraosseous. Intraosseous have hydroelectrolytic imbalances such as catheters can be placed in the distal humerus, femur or hyperkalemia and hyponatremia should receive fluids bone bridges. The main disadvantages of intraosseous parenterally [1, 2]. In severely compromised turtles, catheters are limited flow rate of fluid due to the short routes of administration of IV (intravenous fluids) or bone marrow, administration of drugs possibly painful IO (intraosseous) allow rapid rehydration and and the needle may undergo metal fatigue and rupture
[4, 5]. The catheter should not remain in place for Corresponding author: Gustavo Henrique Pereira Dutra,
more than 72 h. The catheter site should be prepared research fields: experimental and comparative pathology,
pathology of testudines. E-mail: dutravet@ig.com.br. aseptically [6]. Intraosseous fluids are best provided to
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
patients with poorly calcified shells. It is indicated for whenever a patient is anesthetized. Severely adult species of tortoises, drilling the bone bridge
dehydrated and hypotensive patients may present as a before than needle is inserted. The usual site is the
challenge. Whenever possible, the patient should be bone bridge and limb bones [7]. Young et al. [8]
adequately heated before cutting to be done. The best evaluated the effectiveness of intraosseous place to install an intravenous catheter in turtles is the catheterization in Gopherus agassizii, comparing four
jugular vein. In animals with poor peripheral possible locations of maintenance intraosseous perfusion, skin thickening or swelling around the neck, catheters (humerus, femur, bridge and plastron gular
a procedure for dissection may be necessary [11]. region) to the jugular vein. Compared to the jugular
Fluid can be administered via IV infusion set device vein, the intraosseous sites of the humerus and femur
or infusion pumps. The catheter should be inspected were the best access, near 84.4 and 61.8% respectively
daily and removed in 96 hours of use. Once the of the distribution by jugular vascular access. catheter inserted in its place, it must be sutured and Insertion sites in the bridge and gular shell were less
washed with heparinized solution whenever required. effective with only 41.9 and 40.8% of the systemic
Fluid resuscitation with crystalloids or colloids to activity of flow, respectively. The subcarapacial vein
increase osmotic pressure as dextran and hydroxyethyl and the cervical sinus are most commonly used in
starch, blood transfusions, medications and parenteral marine turtles for venipuncture and bolus of fluids
nutrition may be administered via catheter [3]. Fluids drugs. Benefits for the IV bolus method include easy
before and after surgery help to maintain liver accessibility of the vessel and minimal stress for the
function and glomerular filtration rate. Fluids patient [5]. In clinical practice with sea turtles, it is
maximize metabolism and excretion rate of anesthetic often necessary to maintain a permanent venous
agents by the kidney [7]. In a retrospective study, access for rapid rehydration and emergency therapy in
Pittiruti et al. [12] suggested a lateral approach to the critically ill turtles. In addition, venous catheterization
internal jugular vein, using the Seldinger technique, can be used to administer drugs and fluids during and
seems to be the easiest and safest technique for after surgery [9]. The supravertebral sinus is percutaneous insertion of central venous access, commonly used to collect blood in sea turtles. Blood
characterized by a lower incidence of accidental is drawn from adults and juveniles in such access. If
arterial puncture, malposition, pneumothorax, and an necessary, an ultrasound can be used to identify the
extremely low rate of repeated attempts. Although the location of the sinus, especially useful in large patients.
PVAD (long-term venous access device) are used in Long spinal needles may be necessary to achieve
long term intravenous therapy, use of these devices is supravertebral sinus and sedation may be necessary to
associated with complications. A retrospective review prevent injury to the medulla of the animal. In
of 225 PVAD implanted in 217 patients using the addition, incidental aspiration of lymph within the
method percutaneous access of Seldinger or by cutting sample is not uncommon and essentially invalidates
access to the subclavian vein was done by Yildizeli et the sample parameters. This site is important for
al. [13]. Complications observed were poor obtaining samples, but for the maintenance and
positioning of the catheter, pneumothorax, bleeding, patency of the vessel to fluid therapy, the best access
thromboembolism, catheter fracture and infections. is from the jugular vein [3, 5, 7, 10]. The skin of the
Patients undergoing insertion of central venous access site should be cleaned aseptically. In some
catheters for Seldinger technique guided by ultrasound patients, anesthesia must be made, even if local
had fewer complications [14, 15]. Di Bello et al. [9] anesthesia. The risk versus benefit should be assessed
described a simple procedure nontraumatic guided by
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
ultrasound to catheterization of the jugular vein and cephalic in Caretta caretta. The authors worked with
22 loggerhead turtles referred for surgery of the gastrointestinal tract. The ultrasound examinations were performed with a device connected to a transducer of ultrasound of linear multifrequency with frequency set at 11 MHz. Once locating the vein during the longitudinal ultrasound examination, a catheter was inserted into the skin below the transducer, 15-25 degrees relative to the skin surface. For the cephalic vein, a catheter 51 mm 20 gauge or
22 is inserted and proximally of this dorso-ventrally, to the jugular vein caliber of 51 mm 16 or 18 g
inserted into the craniocaudal direction. Fig. 1 Transducer on skin of dorso-lateral region of neck.
inside the vessel was slipped the metallic flexible
2. Materials and Methods
guide wire with tip in J inside the lumen of the needle, Five juvenile turtles of undetermined sex of the
until the end of the guide wire, with no resistance to species Green turtle (Chelonia mydas) and an adult
the passage of the guide wire (Fig. 4). This
male individual of the species leatherback turtle procedure allowed the maintenance of the position (Dermochelys coriacea) with 360 kg and 2.2 m long
of the jugular vein. The needle was withdrawn, were used. The animals arrived at the shore of
leaving the guide wire (Fig. 5) and an vascular
stranded beach, and were sent to Santos Municipal expander (dilator) immediately inserted into the
Aquarium for rehabilitation. Green turtles were jugular vein, slipped around the guide wire by admitted to Santos Municipal Aquarium in different
expander lumen to dilate the path of the catheter (Fig. degrees and clinical stages of fibropapillomatosis for
6). After using the expander, it was withdrawn. Later surgical excision of tumors in different dates.
a central venous catheter polyurethane monolumen
14 L of 20 cm it was fully inserted around the guide the Port of Santos, with changes in buoyancy on
The leatherback turtle was found in the channel of
wire toward the jugular vein (Fig. 7). Withdrawing the March 17, 2011. Blood was collected from the hind
guide wire and the catheter was fixed to the skin of the flippers and the animal was subjected to subcutaneous
animal. After catheterization, the catheter was tapped fluid therapy. The animal was transferred via crane to
with luer device (PRN Luer Lock ® BD device),
a room with a pool of 5.000 L, and underwent digital sutured to the skin with nylon monofilament and the radiography for the evaluation of its postural change.
animal was subjected to intravenous fluid with 1
On March 19, the animal underwent jugular vein L of saline solution + 500 mL of glucose solution, 250 catheterization using the Seldinger technique, guided mL ringer’s lactate solution, 200 mL metronidazole,
by an ultrasound machine SONOSITE NanoMaxx 50 mL albumin, 30 mg metoclopramide and 10 mL of ® L38n connected to transducer 10-5 MHz with depth
nutraceutical (Mercepton®) IV and 4 g of ceftazidime scan of 9.0 cm (Fig. 1). After viewing the jugular vein
IV. The treatment was continued intravenously until in a sagittal section by transducer, a 14 G catheter it
the release of the animal. Venipunctures in two was inserted under visual ultrasound monitoring of the
days were made for hematology and biochemistry. catheter within the vessel (Figs. 2 and 3). The needle
Hematology was done manually, with the red blood
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
Fig. 2 Inserting a catheter 14 G in jugular vein. Fig. 5 Withdraw of needle, leaving patent the wire guide.
Fig. 3 US image of needle within the vessel. Fig. 6 Insertion of vascular expander (dilator).
Fig. 7 Catether inserted and intravenous fluid therapy in Fig. 4 Wire guide slipped inside the lumen needle.
Dermochelys coriacea.
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
cells and total leukocyte counts made in a Neubauer tips. The animals were kept with their jugular vein chamber with diluted aliquots in Natt and Herrick
accessed after the procedure.
solution at 1:100 dilutions. Hematocrit was obtained
3. Results
by rotating closed microcapillary filled with heparinized whole blood at 5000 rpm for 5 min, and
The results of digital radiography of leatherback the hemoglobin concentration was obtained from an
turtle showed only radiopaque and distended bowel photocolorimetric method (CELM HB ®) at 530 nm.
loops, for this exam was possible to observe the lungs For the green sea turtles, the animals were found at
with dense parenchyma, typically with a fibrous different dates and shores and were taken to rehab at
appearance. During the rehabilitation period, the Santos Municipal Aquarium, all turtles suffering from
animal may be hydrated and receive medication different grades and clinical stages of quickly and efficiently. The animal defecated five fibropapillomatosis. The animals were taken to the
times in the enclosure, and improved survivability. University of Guarulhos to remove the tumors and the
The animal was kept in rehab in the enclosure until it use of cryosurgery on June 30, 2011. The animals
started to hurt itself in the edges of the pool, as the were initially induced with intravenous bolus of
animal already had better buoyancy, to prevent propofol at a dose of 5.0 mg/kg to allow intubation
bacterial contamination lacerations to the patent. The with an endotracheal tube. All animals had the jugular
animal received the metal tags on the hind limbs (BR vein catheterized using the Seldinger technique with a
75672 and BR 75673), and the catheter was removed central venous catheter polyurethane monolumen 18 L
and the chelonian was released in the vicinity of
20 cm peripherally inserted, with wire guide, Parque Estadual Marinho da Lage de Santos ultrasound guided (MyLab30VetGold ®) frequency
(24°20'503" S 46°07'317" W) at 03/25/2011. often set at 10 Mhz, in the same way (Figs. 8 to 13) as
For green turtles, the catheters maintained adequate described above for Dermochelys coriacea. The tube
patency and it was well established. There was no
was tapped with luer device (PRN Luer Lock ® BD granulomatous or infectious changes related to
device) and sutured to the skin with nylon catheters reaction. Just one green turtle died after the monofilament. All animals were anesthetized with
procedure. At necropsy of this turtle showed grayish isoflurane in veterinary vaporizer (HB Conquest
tumor of firm consistency of approximately two
Shape ®) with isoflurane minimum alveolar centimeters in the caudal pole of the left lung. Tumor concentration at 2.5%. A single 25 kg turtle was
of similar consistency and color, with 0.5cm was anesthetized with propofol 1% (10 mg/mL) with a
located in the cranial pole of the right lung, which had syringe infusion pump (Baxter ® Model AS40A), in
a neoplasia of 0.7 cm in diameter in the caudal pole. the ratio of 30 mL/h, at a dose of 200 mg/kg/min, a
The corpse showed clear cardiomegaly, whose organ three-way stopcock device connected to a central
was ten centimeters in diameter (for a 7.0 kg green sea venous catheter and peripherally inserted. Associated
turtle). After cutting the heart, there was a large mass with the three-way stopcock device was an infusion
of firm consistency, whitish in color, about 2.0 cm in set providing 25 mL/kg of sodium chloride solution at
the region of ventricular cavum pulmonale (Fig. 14) 0.9%. In this way, the animal received hetastarch at
and loads of blood clot in the atrial chambers. Spleen dosage of 4.0 mL/kg. All animals had their tumors
was with rounded edges, filled with blood. The liver, excised with an electronic multiprocessor bipolar
with rounded edges, increased volume, and red color, scalpel and underwent cryosurgery with Cryogun ®
when cutted; there was drainage of large amounts of device inserted into 500 mL liquid nitrogen with metal
blood through the sinusoidal trabeculae. To that
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
Fig. 8 Insertion of wire guide within catheter lumen. Fig. 11 Insertion of polyurethane monolumen catheter.
Fig. 9 After withdraw of the needle, maintaining the wire Fig. 12 Proving of patency of catheter, with blood guide patent.
drainage.
Fig. 10 Insertion of vascular dilator in C. mydas. Fig. 13 US sagital view of catheter within jugular vein.
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
in the population; this reduces the potential adaptive, and increases the likelihood of extinction [17]. Therefore, any conservation effort, including clinical care, to keep individuals of hazardous situations, whether or not anthropogenic nature, it is important for the turtle population as a whole [18].
Similar to the findings of Di Bello et al. [9], progress on the catheter until insertion were successful in veins and were monitored by ultrasound. A bolus of sterile saline was injected to test the correct
Fig. 14 Neoplasia in ventricular cavum pulmonale.
positioning of the catheter. In all animals, the catheters were maintained successfully. For the leatherback
turtle, the authors believed to be the most effective and most appropriate (perhaps the only) for volume replacement in this species of large chelonian, unable to stay in the dorsal recumbency to receive intracelomic fluid. As this species critically endangered, all efforts should be made in the conservation of this adult male. The results corroborate what has been postulated by Pittiruti et al. (2000) [12], for being the Seldinger technique the easiest and safest technique of percutaneous
Fig. 15 Propofol anesthesia and fluid therapy under
percussion. Despite reporting Yildizeli et al. (2004)
infusion pump in Chelonia mydas.
[13], there was not contamination of the site of green turtle of 25 kg, the excision was 5.4 kg of skin
insertion into the jugular vein in this specie, with over fibropapillomas and after the procedure, the animal
a week of catheterization, confirming the findings of received hydroxyethyl starch at dosage of 4.0 mL/kg
Pittiruti et al (2000) [12], in a marine animal of
IV (Fig. 15). Despite extensive areas of neoplastic absolutely pelagic habits. The use of ultrasonography tissue excised, preventing a wound healing by first
has enabled the monitoring of safe and adequate intention, there was excellent healing and repair of
access from the jugular vein, confirming anterior surgical wounds and the use of cryosurgery was
studies [14, 15]. The animal improved buoyancy responsible for fibropapillomas.
during treatment and in this way was released in pelagic environment.
4. Discussion
For green sea turtles , the results were coincident According to the Red List of Threatened Animals
and corroborated the works of DiBello et al. (2010) [16], there are 129 species of turtles (62.3% of 207
with respect to monitoring access by ultrasound, listed species, 39.3% of all species) officially catheter patency [14, 15] and diffusivity of drugs and considered globally threatened, especially marine
fluid administration. The death occurred in one of the species [16]. The low-density population decline
turtles was due to a right congestive heart failure associated with loss of alleles leads to a loss of genetic
caused by neoplastic growth of a probable cardiac variability and consequent fixing of deleterious alleles
fibroma in ventricular cavum pulmonale, an area
Intravascular Access in Chelonia mydas and Dermochelys coriacea Using the
Seldinger Technique Ultrasound-Guided
which receives blood from the right atrium (this in Reptilian and Amphibian Veterinarians Conference, Houston, ARAV, 1997, p. 125.
turn receives blood from the vena pre cava and jugular [5] T.M. Norton, Chelonian emergency and critical care, catheter was inserted where). At the time of fluid
Seminars in Avian and Exotic Pet Medicine 14 (2) (2005) administration during the surgical procedure, there
106-130.
was overhead in large-circulation return, the systolic [6] D. Martinez-Jimenez, S.J. Hernandez-Divers, Emergency mechanical impossibility, due to the presence of heart care of reptiles, Veterinary Clinics of North America- Exotic Animal Practice 10 (2007) 557-585. disease at cavum pulmonale, generating an increase in
[7] S. McArthur, Problem-solving Approach to Conditions of preload, an insufficiency in cardiac pump, an increase
Marine Turtles, in: S. McArthur, R. Wilkinson, J. Meyer the hydrostatic pressure in wide circulation,
(Eds.), Medicine and Surgery of Turtles and Tortoises, generating the events of hepatic and splenic Blackwell Publishing, Oxford, 2004, pp. 301-307. [8] B.D. Young, N. Stegeman, B. Norby, J.J. Heatley,
congestion visualized at autopsy. Visceral fibromas Comparison of intraosseous and peripheral venous fluid are reported in some turtles that have fibropapillomas
dynamics in the desert tortoise (Gopherus agassizii), skin and major organs are lung (which was verified in
Journal of Zoo and Wildlife Medicine 43 (1) (2012) this case), kidneys, liver and heart. For the green turtle 59-66. [9] A. Di Bello, C. Valastro, D. Freggi, V. Saponaro, D.
that underwent excision of 5.4 kg of tumors, we Grimaldi, Ultrasound-guided vascular catheterization in believe that the loss of blood suffered due to excision
loggerhead sea turtles (Caretta caretta), Journal of Zoo was quite significant and this animal was only kept
and Wildlife Medicine 41 (3) (2010) 516-518. alive today because of the patency of central venous
[10] S.J. Divers, Emergency care of the critically ill reptiles, in: Proceedings of Association of Reptilian and Amphibian catheter peripherally inserted, which allowed
Veterinarians, 1997, pp. 153-161. administering to restore colloidal plasma volume
[11] J.A. Briscoe, R. Syring, Techniques for emergency suitable for infusion.
airway and vascular access in special species, Seminars in Avian and Exotic Pet Medicine 13 (3) (2004) 118-131.
[12] M. Pittiruti, M. Buononato, M. Malerba, C. Carriero, L. Tazza, D. Gui, Which is the easiest and safest technique The authors concludes that the Seldinger technique,
5. Conclusions
for central venous access? A retrospective survey of more guided by ultrasound is the most appropriate, safe and
than 5,400 cases, Journal of Vascular Access 1 (3) (2000) effective vascular access for fluid replacement in
100-107.
marine turtles needing veterinary care and intensive [13] B. Yildizeli, T. Lacin, H.F. Batirel, M. Yüksel, Complications and management of long-term central care.
venous access catheters and ports, Journal of Vascular Access 5 (4) (2004) 174-178.
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[17] A.T. Hahn, J.C. Castilho, L. Soares, S.L. Bonatto, There [18] Projeto Tamar, Espécies-Bandeira, in: Projeto Tamar, was a significant loss of genetic variability of olive turtle
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May 2014, Vol. 8, No. 5, pp. 394-398
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Arg-X Activity in Trypsin-like Complexes of the Nuclear Rroteins in the Suprastructures of Interphase Chromatin During Induction of Growth Morphogenesis Mature Germs of Wheat
Ivanova Evilina, Vafina Gulnara, Ivanov Ruslan and Tereshchenko Lidia Institute of Biology Ufa Science Centre, Russian Academy of Science, Ufa, Bashkortostan 450054, Russia
Received: March 05, 2014/ Accepted: April 11, 2014 / Published: May 30, 2014.
Abstract: The purpose of this study was to analyze spatio-temporal dynamics of localization of protease-sensitive sites Arg-X in non-histone and histone blocks of heteropolymer suprastructures (nucleoplasm, chromatin, nuclear matrix) as possible zones affecting the conformational rearrangements of the total interphase chromatin at the induction of increasing morphogenesis of mature embryos-germs of spring and transformed from its winter wheat. Germinated embryos-germs were detached from endosperm after 24 hours from the start of soaking. Cell nuclei have been allocated from embryos-germs and cleared, and then from their heteropolymer suprastructures (nucleoplasm, chromatin loosely bound with nuclear matrix and chromatin tightly bound with nuclear matrix, and nuclear matrix) were extracted by increasing ionic strength of solution. From isolated nuclear suprastructures, non-histone proteins were separated from histones using ion exchange chromatography. Trypsin-like complexes from non-histone proteins and histone blocks were isolated using the affinity chromatography. The Arg-X (tryptase) activity was assessed by cleavage of Arg-X bonds in the arginine-enriched protein protamine. Hypersensitivity to the Arg-X proteolysis in trypsin-like complexes detected at the level suprastructures of chromatin tightly bound with the nuclear matrix was shown. The most active changes of the nuclear proteome have occurred at the level of the non-histone proteins and the core histones (H2A + H2B) (H3 + H4) of induced to growth embryos-seedlings of winter wheat (compared to the initial spring form of wheat). Perhaps hypersensitivity to the Arg-X activity of the trypsin-like complexes in the non-histone proteins and the core blocks of chromatin tightly bound with nuclear matrix have been entrenched during the transforming of the winter wheat from the initial spring wheat.
Key words: Arg-X protease-sensitive, trypsin-like complexes, non-histones, histones, supramolecular structures, cell nuclei, spring and winter wheat.
1. Introduction includes numerous processes. In the literature, there is no report on the classification of morphological
In recent years, interests have increased to the processes. The modern biology has established morphogenesis significantly. Evidence of this re-edit membership in the number of morphological processes: the book Rene Thom "Structural Stability and molecular and supramolecular morphogenesis. Morphogenesis" in 2002 [1]. Retrospective, active Processing programmed molecular (or covalent) interests to the problem of morphogenesis arise in information assume the transition to self-organization connection with well-known successes in the fields of of supramolecular architecture from individual molecular biology and genetics, morphogenesis compound components based on non-covalent
Corresponding author: Evilina Ivanova, Ph.D., chief intermolecular interactions [2]. Currently, some
research officer, research field: biochemistry. E-mail: progress has been made in the study of chromatin evilina@anrb.ru.
Arg-X Activity in Trypsin-like Complexes of the Nuclear Proteins in the Suprastructures of Interphase 395
Chromatin During Induction of Growth Morphogenesis Mature Germs of Wheat
structure on the level of the whole nucleus [3]. In suprastructures (nucleoplasm, chromatin, nuclear plants, there is a highly organized epigenetic system.
matrix) as possible zones affecting the conformational They are capable to modify the histone proteins and
rearrangements of the total interphase chromatin at the DNA almost the same way as well as do animals.
induction of growth morphogenesis of mature Plants use the same epigenetic enzymes like that in
embryos-germs of spring and transformed from its animals and even humans. Many of plants winter wheat.
responsing to the environmental conditions are
2. Materials and Methods
directly related with the change program of cells. However, still now the molecular mechanisms of
Highest-quality seeds of wheat (Triticum aestivum adaptive evolution of features have been studied less
L.) cv.: Artemovka (spring), and transformed from it considerably [4]. At present, a certain interest appears
Mironovsky 808 (winter) were selected for this to elucidate the role of proteolysis at the level of
investigation. The seeds were kindly provided from histones [5], and even modified proteolytic of histones
the collection of “All-Russian Research Institute of by tryptase [6], as well as epigenetic regulation of the
Plant Industry”. Detailed description the method of expression of genes vernalization [7]. Morphological
transformation the Mironovsky 808 (winter) from limits and epigenetic influences on the nuclear
Artemovka (spring) was submitted in the work [9]. architecture, on the evolutionary stability of Germinated embryos-germs were detached from chromosome territory mechanisms, and on the endosperms after 24 h from the start of soaking. From changes in their architecture during the transcription
these embryos-germs were isolated with cell nuclei and reparation were discussed [8]. Thus, the dynamics
[10]. Supramolecular structures: Np (nucleoplasm), of chromosomes in the interphase nuclei is still not
Chr I (chromatin loosely bound with nuclear matrix) well studied to build more than adequate mathematical
and Chr II (chromatin tightly bound with nuclear logic circuits of the theory of biological specificity of
matrix), and NM (nuclear matrix) were isolated from development. The cell nuclei of mature embryos
cell nuclei by increasing ionic strength of solution by initial spring and transformed from its winter wheat
the method, described in detail [11]. From isolated used as a model system, in which there was an
suprastructures of the cell nuclei, NHP (non-histone adaptive formation of the functional group of proteins) were separated from histones by method [12]. coordinately expressed genes. Problems of The resin for the ion exchange chromatography was self-organization of biological systems are in the focus
prepared by method [13]. Heteropolymeric blocks of of specialists in macromolecular compounds, in
suprastructures (Np, Chr I, Chr II, NM) were passed particular biopolymers [2]. Chromatin structure is a
through polymethacrylic synthetic resin IRC-50 supra-hetero-polymer "coherent and extremely viable
(Amberlite, IRC-50, Serva, Heidelberg) column. Thus, organism" where effected intermolecular interactions
NHP and histone (H1, H2A + H2B, H3 + H4) blocks of different supramolecular structures: DNA, RNA,
were eluted using step gradient (6.0, 8.9, 10.6, 13, proteins and polysaccharides. They have spatial
40%) of guanidine hydrochloride (Dia-M). In the (geometrical, topological) characteristics, which present study, the data on 13% and 40% fractions are depend on the surrounding environmental conditions
pooled because they are enriched blocks of histone H3 exhibiting their ability to adapt. The purpose of this
+ H4. Trypsin-like complexes from NHP and histone work was the analysis of spatial-temporal dynamics of
blocks were isolated using the affinity localization of protease-sensitive sites Arg-X in
chromatography by the method [11]. Protease non-histone and histone blocks of heteropolymer
sensitivity to Arg-X in NHP and histone blocks was
Arg-X Activity in Trypsin-like Complexes of the Nuclear Proteins in the Suprastructures of Interphase
Chromatin During Induction of Growth Morphogenesis Mature Germs of Wheat
evaluated by cleavage Arg-X bonds in the arginine—enriched protein-protamine Salmine-A-1 («Merk»)—the molecule of which consists of 33 amino acids (22 molecules of Arg, 4-Ser, 3-Pro, 2-Glu and Val) in all fractions of the nuclei [11]. Activity of Arg-X proteolysis was calculated in nanomoles of arginine per second per microgram of protein (n mol/s·mkg protein).
3. Results and Discussion
3.1. Physiology-morphological state of embryos-germs The growth of morphogenesis begins with the
initiation of physiological processes of growth and development of the germs. This period of differentiated growth at the expense of stretching cells is characterized the root emergence. In the 24 h embryos-germs, the distal part of the root is pushed on account of stretching the cells of the embryonic axis of coleoptile and the root. The apex of coleoptile and the root tip are the sensory organs, which are specifically responding to one or another inducible influence. The metabolic system of regulation at the level of signaling "language" of the apical meristem of shoot and root are formed in the period preceding the root emergence. It provides the plasticity, coordinating and simultaneous growth of different tissues of the germ in the close interrelation between them. During the formation of the root meristem, the final "destination of the cells" was fixed only after germination. In growing continuous cellular-tissue masses there will always be mechanical tension due to passive-smoothly deformable cell layers [14]. Moreover, cell-tissue layers may lead to the formation of geometrically striking complex forms. In plants this is observed during the morphogenesis of growth shoot cones. Interrelation of growth with mechanical tensions, naturally distributed in the wide areas of growing tissues, suggests that growth is undoubtedly obeying certain forms of holistic control of the
organism [15].
3.2. Arg-X activity of trypsin-like complexes of the nuclear proteins
Earlier, at the level of suprastructures of the cell nuclei of embryos induced to growth (21 h) [16], and on identifying of sites of hypersensitive to proteolysis at the level of the linker and core histones were shown the dynamics of functioning of Arg-X proteolysis [17]. In the next step of this experiment, Arg-X proteolysis activity was decided to isolate the TLC (trypsin-like complexes) from the proteome of all these suprastructures cell nuclei, and identify in these complexes the sites of sensitivity to the Arg-X protease. Sites of hypersensitivity to the Arg-X proteolysis in TLC are at the level suprastructures of chromatin tightly bound with the nuclear matrix (Chr
II) which was shown in Fig. 1. This fraction-suprastructure of chromatin differs from other identified fractions-suprastructures not only by localization in the nucleus, but also by the ratio of active/inactive genes, by sensitivity to effects of dissociating substances, nucleases and by the ability enduring conformational transitions [18]. As it can be seen from our data, the feature TLC of this suprastructure is the high sensitivity to Arg-X proteolysis. "Background" of the possibility of manifestation proteolytic activity was separated specially (subjectively) in Fig. 1. When induced to the growth of embryos-germs of winter wheat (compared with the initial spring form of wheat), the most active change of the nuclear proteome has occurred at the level of the NHP and the core histones (H2A + H2B) (H3 + H4) as it has shown in Fig. 1. Linker histones (HI) of winter wheat remained at the level of spring forms. NHP is an extensive and very diverse group of proteome of the cell nucleus. This group includes enzymes of remodeling the chromatin and proteins directly involved in the formation of chromatin fibrils [19].
Arg-X Activity in Trypsin-like Complexes of the Nuclear Proteins in the Suprastructures of Interphase 397
Chromatin During Induction of Growth Morphogenesis Mature Germs of Wheat
Fig. 1 Arg-X protease sensitivity in TLC of proteins from the suprastructures cell nuclei of embryos-germs wheat (Triticum aestivum L.) cv.: Artemovka (spring) (1) and Mironovsky 808 (winter) (2) after 24 h from the start of soaking. TLC: Trypsin-Like Complexes isolated using the affinity chromatography. Blocks of proteins of the cell nucleus: NHP: non-histone proteins; HI: lysine-rich histone, H2A + H2B: moderately lysine-rich histones, H3 + H4: arginine-rich histones; Suprastructures of cell nucleus: Np: nucleoplasm, Chr I: chromatin loosely bound with the NM, Chr II: chromatin tightly bound with NM, NM: nuclear matrix.
4. Conclusions
Acknowledgments
In this paper, features of Arg-X proteolysis were The reported study was supported by RFBR, considered as a possible mechanism for the research project No. 14-04-3124314. architecture reorganization of chromatin matrix in
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Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
The Effect of Mixture of Alpinia Galanga, Eurycoma Longifolia Jack and Syzygium Aromaticum Crude Extract on the Growth of Saccharomyces Cerevisiae and Escherichia Coli
Nur Jasrina Jaafar 1 , Kaswandi Md. Ambia 1 , Hing Hiang Lian 2 and Rahim Md. Noah 1 1. University Kuala Lumpur, Institute of Medical Science, Blok A1-1, Jalan TKS 1, Taman Kajang Sentral, 43000 Kajang, Selangor
Darul Ehsan, MALAYSIA
2. School of Diagnostic & Applied Health Sciences, Faculty of Health Science, Universiti Kebangsaan Malaysia, 50300 Jalan Raja Muda, Kuala Lumpur, MALAYSIA
Received: March 05, 2014/ Accepted: April 25, 2014 / Published: May 30, 2014.
Abstract: Alpinia galanga, Eurycoma longifolia Jack and Syzygium aromaticum have been widely used in traditional medicine for decades. Antimicrobial activities for individual crude extract were well established. Crude methanolic extracts of Alpinia galanga, Eurycoma longifolia Jack and Syzygium aromaticum and the combination for all extracts were tested using well diffusion techniques against Saccharomyces cerevisiae (ATCC 9763) and Escherichia coli (ATCC 25922). The mixed extracts were prepared based on the concentration ratio of 50 µg/µL which are 1:1, 1:1:1 and 1:2:2. Single Syzygium aromaticum extract showed higher inhibition zone on Saccharomyces cerevisiae compared to Escherichia coli. There is reduction in diameter of inhibition zone for single extract and mixture extracts either in combination of two or three extracts tested on Saccharomyces cerevisiae and Escherichia coli growth but they are not significant. In conclusion, Syzygium aromaticum showed highest activity against Saccharomyces cerevisiae and Escherichia coli. Reduction in diameter of inhibition zone indicated that Alpinia galanga and Eurycoma longifolia Jack extracts had antagonistic effect with Syzygium aromaticum.
Keywords: Antifungal, antibacterial, extract, Alpinia galanga, Eurycoma longifolia, Syzygium aromaticum, Saccharomyces cerevisiae and Esherichia coli.
1. Introduction [4] and sea sickness [5]. Different parts of A. galanga are traditionally claimed to be used for the treatment
The medicinal plants that are frequently used including anti-fungal, anti-tumor, anti-used as a locally such as langkuas galanga (Alpinia galanga) [1], carminative, to increase hydrochloric acid in the Tongkat Ali (Eurycoma longifolia Jack) [2] and clove stomach and to improve peristalsis [6]. It was also (Syzygium aromaticum) [3] exhibited many used in dentistry where the essential oil of clove was antimicrobial effects. used as anodyne for dental emergencies [7]. Alpinia galanga (L.) Willd or Lengkuas a plant in Eurycoma longifolia Jack also known as Tongkat the ginger (Zingiberaceae) family was commonly used Ali, a flowering plant in the Simaroubaceae family in the Arabian and in Unani systems of medicine for claimed to have aphrodisiac properties was mainly the treatment of dyspepsia, gastralgia, chronic enteritis used as aphrodisiac, anti-cancer, anti-pyretic and
Corresponding author: Kaswandi Md. Ambia, Ph.D.,
anti-malarial remedy [8].
associate professor, research field: natural Product. E-mail: kaswandi@mestech.unikl.edu.my.
The effect of mixture of Alpinia galanga, Eurycoma longifolia Jack and Syzygium aromaticum crude
extract on the growth of Saccharomyces cerevisiae and Escherichia coli
A wide range of medical plants parts was used for cerevisiae was prepared using the same procedure but extract as raw drugs and they possess varied medical
at different temperature incubation i.e. 30 C for 48 h. properties [9]. Many of bacteria species are resistant
2.3 The survival test
to multiple agents, making choosing an antibiotic to treat such infections difficult [10]. The increase effects
The survival test was conducted using well were associated with conventional treatments [11].
diffusion method to detect the antibacterial and This paper described the mixture of Alpinia galanga,
antifungal activity towards the extract solution [13]. A Eurycoma longifolia Jack and Syzygium aromaticum
sterile cotton swap was dipped in a bacterial extract and their effect on Escherichia coli and
suspension of E. coli and streaked on nutrient agar. Saccharomyces cerevisiae.
The inoculums were allowed to dry for 3 to 5 min to
2. Materials and Methods allow diffusion of bacteria into the agar. A sterilized
Durham tube was used to punch wells on the agar
2.1 Preparation of extracts plates with maximum 4 wells per plate. A total of 50 Methanolic plant extraction for this experiment was
L of the single extracts, mixture of extracts, positive prepared by Faculty of Health Science, Universiti
controls and negative control respectively were filled Kebangsaan Malaysia using modified technique of
into the labeled wells. The test was repeated five times Shimada [12]. A. galanga, E. longifolia and S.
and the mean value was recorded. The same procedure aromaticum extracts weighted respectively (0.5 g) and
was applied for S. cerevisiae. All the plates used, dissolved into 0.5 m/L of 5% DMSO and 9.5 m/L of
containing E. coli are incubated at 37 C for 24 h, distilled water to obtain the stock concentration of 500