Enzyme aspects of acute intermittent porphyria

Summary Patients with acute intermittent porphyria are now known to have a decrease of the third enzyme and, in liver, an increase of the first enzyme of the heme biosynthetic pathway. It is possible that the induction of the first enzyme (ALA synthetase) in the liver of these patients results from the partial block in heme synthesis, since heme is known to be involved in the repression of hepatic ALA synthetase via a closed negative feedback loop. Presumably, an increase of hepatic ALA synthetase allows the delivery of a higher substrate concentration to the enzyme at the level of the block, thus raising the rate of the synthesis of end product toward normal. Using a simplified Michaelis-Menten model of an irreversible pathway in a homogeneous system, quantitative relationships between the degree of block and the magnitude of induction of the first enzyme necessary to return the steady state rate of the pathway to normal have been developed. This is intended as a point of departure for refinements which may ultimately lead to more accurate quantitative relationships.Despite the fact that various forms of experimental porphyria do not produce the specific enzyme decrease of acute intermittent porphyria, they have provided the basis for a number of discoveries which have direct application to this disease.Reprint requests should be sent to this address.     

A steady state model of sequential irreversible enzyme reactions

Summary One of the questions which arises in the study of certain inborn errors of metabolism as well as in the field of enzyme kinetics is: what are the quantitative relationships between parameters of enzyme activity and substrate pool sizes in a metabolic pathway? A steady state model has been devised to answer this question for a homogeneous system of non-branched sequential irreversible enzyme reactions which follow Michaelis-Menten kinetics. The concentration of a substrate in such a pathway, [S_i], is a function of 5 variables: (a) the K_M of the enzyme which forms the substrate (K_{M (i–1)}), (b) the K_M of the enzyme which utilizes the substrate (K_{M i}), (c) the V_max of the enzyme which forms the substrate (V_{m (i–1)}), (d) the V_max of the enzyme which utilizes the substrate (V_{m i}) and (e) the immediate precursor concentration [S_(i–1)] where [S_i] = K_{M i} V_{m (i–1)} [S_(i–1)]/[S_(i–1)] (V_mi -V_{m (i–1)}) + K_{M (i–1)}) V_mi The model introduces and defines the concept of and conditions for amplification. An input in the form of a steady state concentration of precursor [S_(i–1)] may be amplified as an output in the form of an increased steady state concentration of product [S_i]. The model also defines the values of the above 5 parameters which do not allow attainment of a steady state for the type of pathway considered.From the Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Bacillus subtilis as potential producer for polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by microbes to overcome environmental stress. Commercial production of PHAs is limited by the high cost of production compared to conventional plastics. Another hindrance is the brittle nature and low strength of polyhydroxybutyrate (PHB), the most widely studied PHA. The needs are to produce PHAs, which have better elastomeric properties suitable for biomedical applications, preferably from inexpensive renewable sources to reduce cost. Certain unique properties of Bacillus subtilis such as lack of the toxic lipo-polysaccharides, expression of self-lysing genes on completion of PHA biosynthetic process – for easy and timely recovery, usage of biowastes as feed enable it to compete as potential candidate for commercial production of PHA.     

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp,Ctenopharyngodon idellus

Background  

Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.