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1. Cells of the ceco-colonic epithelium that use butyrate as a major substrate for the
maintenance of energy producing path-ways; 2.
Liver cells that metabolize residual butyrate with propionate used for gluconeogenesis; 50 to 70 of acetate is also taken up by the liver;
3. Muscle cells that generate energy from the oxidation of residual acetate.
In ruminants and other herbivores, SCFA are absorbed and transported via the portal vein to the liver, and the fraction not absorbed is distributed to the other body organs and tissues for
metabolism. In herbivores, peripheral venous SCFA concentrations are high due to comparatively low visceral extraction and high rates of absorption into the circulation. However, human
peripheral venous blood concentrations are normally low, and only acetate is present in measurable amounts. This profile reflects the lower SCFA production rates and greater visceral
extraction in omnivores, meaning that human peripheral venous SCFA are not representative of those in the portal circulation. Human experimentation has been confined largely to fecal
measurements, which are also limited as 95 of SCFA are produced and absorbed within the colon Topping and Clifton 2001.
Purwani, et. al 2012 study revealed that type 3 resistant starch RS3 derived from sago and rice starch that treated with pullulanase could be well utilized as substrate by Clostridium
butyricum and showed good proportion of acetate: propionate: butyrate in the bacterial culture filtrate. The fermentation of resistant starch type 3 RS3 derived from sago starch treated with
pullulanase RSSP by C. butyricum produced 17.65 mM of butyrate, and fermentation of RS3 derived from rice starch treated with pullulanase RSRP by C. butyricum produced 21.76 mM,
higher concentration than fermentation of RSSP. This study implied that difference of starch source resulted difference of SCFA concentration.
C. APOPTOSIS
Apoptosis “normal” or “programmed” cell death is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological
processes. Apoptosis is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise “cellular suicide” Wyllie, et. al 1998.
Apoptosis is a form of programmed cell death characterized by cytoplasmic condensation, plasma membrane blebbing and nuclear pycnosis, leading to nuclear DNA breakdown into multiples of
~200 bp oligonucleosomal size fragments Chen and Ioannou 1996.
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Figure 1 Illustration of the morphological features of apoptosis Wyllie, et. al 1998 Apoptosis is characterised by an initial shrinkage of the cell, which in a tissue means
breaking cell-to-cell contacts with neighbours and rounding up. The consequence of this stage tends to be that the volume of the cell becomes smaller, and the cytoplasmic internal membranes,
ribosomes, mitochondria, and other cytoplasmic organelles are more concentrated in the cytoplasm, which then consequently looks darker. The organelles remain intact and healthy
looking very late into the process of death, suggesting that the cell continues metabolic activity for some considerable time. This indeed can be demonstrated in many cell systems by using various
inhibitors of metabolic processes RNA and protein synthesis inhibitors, which delay the progression of a cell through the apoptotic sequence. Apoptosis characteristically involves single
isolated cells and not clusters. The DNA or chromatin material in the nucleus condenses very extreme Potten and Wilson 2004. Pyknosis is the result of chromatin condensation and this is the
most characteristic feature of apoptosis. On histologic examination with hematoxylin and eosin stain, apoptosis involves single cells or small clusters of cells. The apoptotic cell appears as a
round or oval mass with dark eosinophilic cytoplasm and dense purple nuclear chromatin fragments. Electron microscopy can better define the subcellular changes. Early during the
chromatin condensation phase, the electron-dense nuclear material characteristically aggregates peripherally under the nuclear membrane although there can also be uniformly dense nuclei.
Extensive plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies during a process
called “budding.” Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment. The organelle
integrity is still maintained and all of this is enclosed within an intact plasma membrane. These bodies are subsequently phagocytosed by macrophages, parenchymal cells, or neoplastic cells and
degraded within phagolysosomes. Macrophages that engulf and digest apoptotic cells are called “tingible body macrophages” and are frequently found within the reactive germinal centers of
lymphoid follicles or occasionally within the thymic cortex. The tingible bodies are the bits of nuclear debris from the apoptotic cells. There is essentially no inflammatory reaction associated
with the process of apoptosis nor with the removal of apoptotic cells because: 1 apoptotic cells
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do not release their cellular constituents into the surrounding interstitial tissue; 2 they are quickly phagocytosed by surrounding cells thus likely preventing secondary necrosis; and 3 the engulfing
cells do not produce anti-inflammatory cytokines Savill and Fadok 2000; Kurosaka, et. al 2003. The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an
irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability prelytic DNA fragmentation. The fragmentation of chromosomal DNA is a
hallmark of apoptosis and may facilitate apoptosis by terminating DNA replication and gene transcription. In many systems, this DNA fragmentation has been shown to result from activation
of an endogenous Ca
2+
and Mg
2+
dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between nucleosomal units linker DNA generating mono- and
oligonucleosomal DNA fragments Arends et. al, 1990; Wyllie, et. al 1998. In priciple, there are two main apoptotic pathways: the extrinsic or death receptor pathway
and the intrinsic or mitochondrial pathway. In both pathways, cysteine aspartyl-specific proteases caspases are activated that cleave cellular substrates, and leads to the biochemical and
morphological changes that are characteristic of apoptosis. However, there is now evidence that the two pathways are linked and that molecules in one pathway can influence the other Igney and
Krammer 2002. Figure 2 shows the two main apoptotic signalling pathways.
Figure 2. The two main apoptotic signalling pathwaysIgney and Krammer 2002
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Apoptotic cells exhibit several biochemical modifications such as protein cleavage, protein cross-linking, DNA breakdown, and phagocytic recognition that together result in the distinctive
structural pathology described previously Hengartner 2000. Caspases are widely expressed in an inactive proenzyme form in most cells and once activated can often activate other procaspases,
allowing initiation of a protease cascade. Some procaspases can also aggregate and autoactivate. This proteolytic cascade, in which one caspase can activate other caspases, amplifies the apoptotic
signaling pathway and thus leads to rapid cell death Elmore 2007. Although DNA fragmentation into oligonucleosomal ladders is characteristic of apoptosis,
recent evidence indicates that not all cells undergo such extensive DNA fragmentation Cohen, et. al1. 1992 in Chen and Ioannou 1996. In fact, fragmentation of DNA into kilobase-size fragments
appears to be an early event in apoptosis, preceding the complete digestion of DNA into multiples of nucleosomal size fragments Cohen and Sun 1994 in Chen and Ioannou 1996.
D. DNA LADDER ASSAY
