Polyproline binding is an essential function of human profilin in yeast.

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1Delta. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.     

Functional Effects of Adiponectin on Endothelial Progenitor Cells

Adiponectin is an adipokine whose plasma levels are inversely correlated to metabolic syndrome components. Adiponectin protects against atherosclerosis and decreases risks in myocardial infarction. Endothelial progenitor cells (EPCs) are a heterogeneous population of circulating cells involved in vascular repair and neovascularization. EPCs number is reduced in patients with cardiovascular disease. We hypothesize that the positive effects of adiponectin against atherosclerosis are explained in part by its interactions with EPCs. Cells were obtained from healthy volunteers' blood by mononuclear cell isolation and plating on collagen‐coated dishes. Three sub‐populations of EPCs were identified and characterized using flow cytometry. EPCs' expression of adiponectin receptors, AdipoR1, and AdipoR2 was evaluated by quantitative PCR. The effects of recombinant adiponectin on EPCs' susceptibility to apoptosis were assessed. Finally, expression of neutrophil elastase by EPCs and activity of this enzyme on adiponectin processing were assessed. Quantitative PCR analysis of EPCs mRNAs showed that AdipoR1 mRNA is expressed at higher levels than AdipoR2. Expression of AdipoR1 protein was confirmed by western blot. Adiponectin significantly increased survival of two sub‐populations of EPCs in conditions of serum deprivation. Such effect could not be demonstrated in the third EPCs sub‐population. We also demonstrated that EPCs, particularly one sub‐population, express neutrophil elastase. Neutrophil elastase activity was confirmed in EPCs' conditioned media. Adiponectin protects some EPCs sub‐populations against apoptosis and therefore could modulate EPCs ability to induce repair of vascular damage. Neutrophil elastase activity of EPCs could locally modulate adiponectin activity by its involvement in the generation of the globular form of adiponectin.     

Comparison of different membranes for use in the colony-immunoblot technique

We compared five different supports (Whatman paper filters Nos. 1, 5, and 40, nitrocellulose, and Nylon 66) for their suitability in the colony-immunoblot (CIB) technique. Results indicate that Whatman No. 5 filter paper recovered 94-98% of the bacterial colonies tested, were more resistant to tearing than the other Whatman papers tested, and showed reduced cross-reactions as compared with nitrocellulose membranes. Whatman No. 5 filters are 20 times less expensive than the nitrocellulose membranes usually used in the CIB technique. We thus adopted the former for our ecological studies of the murine oral cavity.     

Action at a distance in Mu DNA transposition: an enhancer-like element is the site of action of supercoiling relief activity by integration host factor (IHF).

The first committed step in the in vitro strand transfer reaction of a mini-Mu donor molecule is the formation of a Type 1 complex in which the Mu ends are held together in a non-covalent protein-DNA complex. Efficient formation of this complex at high levels of donor supercoiling (sigma approximately -0.06) requires the Mu A and Escherichia coli HU proteins. At in vivo levels of supercoiling, efficient reaction also requires E. coli integration host factor (IHF). We demonstrate that this supercoiling relief activity of IHF is mediated through an IHF binding site in the Mu early promoter region. This site is part of a larger enhancer-like element which includes operator 1 (01) and part of operator 2 (02) with the IHF site in between. The enhancer-like element stimulates the initial rate of the in vitro reaction 100-fold and acts in a distance-independent fashion. Inversion of the orientation of the element results in a total loss of enhancer activity in the absence of IHF. However, a 10-fold stimulation in the initial rate of reaction is induced by the addition of IHF. Furthermore, correct helical phasing between 01 and 02 is required for maximal activity. The results indicate that a specific geometrical configuration of the enhancer-like element, which includes a sharp bend between 01 and 02, is required for optimal induction of synapsis.     

Dissociation of the inhibition of fast axonal transport by chlorimipramine from an effect on axonal microtubules

The effect of in vitro exposure of bullfrog spinal nerves to 0.2 mM chlorimipramine on the density of axonal microtubules was studied in an attempt to clarify the mechanism by which chlorimipramine inhibits fast axonal transport. A 17-h exposure to chlorimipramine reduced the density of microtubules in unmyelinated axons by only 18%; this microtubular loss does not reach the upper limit of the range of microtubule reduction associated with inhibition of fast axonal transport. A 23-h exposure to chlorimipramine, which had decreased microtubular density in unmyelinated axons by 40% in a previous study, did not decrease microtubular density in myelinated axons in the present study. These results rule out microtubular destruction as the mechanism responsible for inhibition of fast orthograde axonal transport by chlorimipramine, and greatly reduce the likelihood that microtubular destruction plays a significant role in the inhibition of fast retrograde transport by chlorimipramine.     

Measurement of liver adenine nucleotides and S-adenosyl amino acids by one-step high-performance liquid chromatography

A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs.     

Detection of false-positives among total and fecal coliform counts by factorial analysis of correspondence

Application of an analysis of correspondence to the biochemical characteristics of total and fecal coliforms isolated in the Ivory Coast permitted us to separate two small clusters of isolates different from the main clusters, which included isolates from human and animal feces. The isolates grouped in the small clusters were from water samples. An analysis of the biochemical characteristics which permitted the segregation of the "water-specific" isolates from the main clusters indicates that water-specific total coliforms were citrate positive, indole negative, and amygdaline positive. Water-specific fecal coliforms were either citrate positive, indole negative, amygdaline positive, and inositol negative or indole negative, amygdaline positive, and inositol positive. Any isolates not fitting the above patterns could be considered of fecal origin. If this observation is confirmed under temperate climates and for a greater number of isolates, these simple tests could be used to confirm the fecal origin of coliforms.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.