Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.     

Deficient fumarase activity in an infant with fumaricacidemia and its distribution between the different forms of the enzyme seen on isoelectric focusing.

A male infant, whose parents were first cousins, presented at 6 mo of age with hypotonia, microcephaly, and delayed development. He was found to have large amounts of fumaric and succinic acids present in the urine. In lysed cultured skin-fibroblast preparations, the activity of fumarase was found to be 22.7% of that in controls. Cell fractionation by homogenization and by digitonin treatment indicated that the residual activity in the cells of the patient was primarily located in the mitochondrial fraction rather than in the cytosolic fraction. Isoelectric focusing of fibroblast extracts showed that six bands of fumarase activity were discernible in control cell lines, two of them cytosolic with pI's of 5.53 and 5.60 and four of them mitochondrial with a pI of 5.65-6.8. In contrast, isoelectric focusing of fibroblast extracts from the fumarase-deficient patient showed only a single band of activity with a pI corresponding to the mitochondrial type seen in the controls. Immunoprecipitation of proteins with rabbit antifumarase antibody in (35S)-methionine-labeled fibroblasts indicated that a protein of correct size (Mr = 44,000 daltons) corresponding to fumarase was synthesized in similar amounts in both the patients and controls. It is proposed that in the patient's cells a single active species of fumarase that is mitochondrial in location is synthesized. Since it is known that mitochondrial and cytosolic fumarases are encoded by the same gene but differ slightly in amino acid sequence, it is possible that a point mutation might explain these findings.     

Effects of exogenous guanosine on chromatophore differentiation in the axolotl

Guanosine is shown to dramatically alter the pigment phenotype of axolotls by suppressing melanization and enhancing the biosynthesis and deposition of purine-derived pigments. Phenotypic changes caused by guanosine are manifested by altered chromatophore differentiation patterns such that few black pigment cells (melanophores) differentiate (and those that do are punctate and necrotic in appearance), whereas the development of yellow (xanthophore) and reflecting (iridophore) pigment cells is enhanced. Mechanisms for changes in chromatophore differentiation, and thus pattern formation, are discussed, including the possibility that pigment cells may undergo transdifferentiation in vivo.     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs.

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Alternative nonlinear model for estimating second-order rate coefficients for biodegradation.

A modification of the second-order model for biodegradation was derived, applied to an example data set, and shown to be superior for describing the anaerobic biodegradation of p-cresol by an enriched bacterial consortium. The modified model circumvents the no-growth assumption implicit in the use of the second-order rate equation, but still requires the assumption of first-order kinetics over the course of substrate depletion. Violation of the no-growth assumption is particularly important since overestimates of the pseudo-first-order rate coefficient lead to underestimates of the time required for the removal of a xenobiotic chemical from a contaminated environment. Our calculations show that the errors introduced into the pseudo-first-order rate coefficient (and the resulting estimates of the second-order rate coefficient) approach 100% if one doubling occurs in activity over the course of substrate depletion. For an exemplary data set, use of a first-order model resulted in a 100% overestimate of the first-order decay coefficient, which would in turn lead to a corresponding overestimate of the second-order rate coefficient. The modified model we describe is a potential alternative to the pseudo-first-order model for the routine estimation of second-order rate coefficients.     

Toxicity of enzymically-oxidized low-density lipoprotein

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.     

Simulation of double-stranded branch point migration.

A structural and dynamic model has been developed for the branch point formed when two DNA double helices exchange strands during genetic recombination. This model, which generalizes most previous structural models, maintains the twofold symmetry inherent in the covalent and hydrogen bonded structure, yet has three degrees of freedom about virtual bonds, constituting a simplified junction. Using this structural model, a three-step dynamic model for branch point migration has been developed: longitudinal diffusion about the virtual bonds to achieve a structure in which the helix axes are approximately parallel; opening of the base pairs; and rotary diffusion about the helix axis to effect a migratory event. The model, which includes the possible role of electrostatic interactions, solves problems inherent in previous treatments. We find that no significant electrostatic torques arise that promote branch point migration. The absence of a kinetic mechanism to circumvent thermodynamic barriers due to mispairing suggests that an energy source is used for those situations in living systems.     

The effect of predation on the structure and invasibility of assembled communities

Summary Fifty four microcosmic communities were assembled over 4 months from a 28-species source pool of phytoplankton using nine different invasion patterns each replicated six times. Three communities from each set of replicates then were invaded with a cladoceran that feeds on phytoplankton. All communities were then treated identically for an additional 4 months. In all nine invasion categories species richness was greater in predated communities. Predation opened communities to invasion by increasing the representation of infrequently sampled species at the expense of more common species. Invasion rate was four times more influential than predation and over eleven times more important than either invasion order or the timing pattern of interspecific arrivals in determining species richness in this system of communitites.     

Poly(gamma-glutamylcysteinyl)glycine Synthesis in Datura innoxia and Binding with Cadmium : Role in Cadmium Tolerance

The effects of Cd on poly(γ-glutamylcysteinyl)glycine [(γEC)_nG] biosynthesis and formation of (γEC)_nG:Cd complexes were measured in two cell lines of Datura innoxia with differing Cd tolerance. In addition, RNA synthesis, protein synthesis, and GSH concentrations were measured during a 48 hour exposure to Cd. Exposure to 250 micromolar CdCl₂ was toxic to the sensitive line, whereas the tolerant line survived and grew in its presence. Cd-sensitive cells synthesized the same amount of (γEC)_nG as tolerant cells during an initial 24 hour exposure to 250 micromolar CdCl₂. However, rates of (γEC)_nG:Cd complex formation differed between the two cell lines with the sensitive cells forming complexes later than tolerant cells. In addition, the complexes formed by sensitive cells were of lower molecular weight than those of tolerant cells and did not bind all of the cellular Cd. Pulse-labeling of cells with l-[³⁵S]cysteine resulted in equivalent rates of incorporation into the (γEC)_nG of both cell lines during the initial 24 hours after Cd. Rates of protein and RNA synthesis were similar for both cell lines during the initial 8 hours after Cd but thereafter declined rapidly in sensitive cells. This was reflected by a decline in viability of sensitive cells. The GSH content of both cell lines declined rapidly upon exposure to Cd but was higher in sensitive cells throughout the experiment. These results show that the biosynthetic pathway for (γEC)_nG synthesis in sensitive cells is operational and that relative overproduction of (γEC)_nG is not the mechanism of Cd-tolerance in a Cd-tolerant cell line of D. innoxia. Rapid formation of (γEC)_nG:Cd complexes that bind all of the cellular Cd within 24 hours appears to correlate with tolerance in these cells.     

Legumin antibodies recognize polypeptides in coated vesicles isolated from developing pea cotyledons

Summary A highly enriched coated vesicle fraction has been isolated from cotyledons of developing pea seeds. This, and coated vesicles isolated from bovine brain as well as from bean leaves were subjected to SDS-PAGE followed by Western blotting with legumin antibodies. A distinct cross reaction with two polypeptides at around 60 kDa was seen, but only with the coated vesicles isolated from peas. Since legumin is synthesized as a 60 kDa precursor, but occurs as 40 and 20 kDa polypeptides in the protein body, we interpret our results as giving support to the idea that reserve proteins, like lysosomal proteins, are transported via coated vesicles.Abbreviations CV coated vesicle - DTT dithiothreitol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis