Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125. Cloning, expression, properties, and molecular modelling.

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 degrees C (half-life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is approximately 64 degrees C, which is some 11 degrees C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at approximately 50 degrees C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold-loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both kcat and kcat/Km of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient. The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decreases with increasing temperature. However, binding of maleate and 2-methyl-L-aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine. A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed.     

Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Hypoglycemia and the Origin of Hypoxia-Induced Reduction in Human Fetal Growth

Background

The most well known reproductive consequence of residence at high altitude (HA >2700 m) is reduction in fetal growth. Reduced fetoplacental oxygenation is an underlying cause of pregnancy pathologies, including intrauterine growth restriction and preeclampsia, which are more common at HA. Therefore, altitude is a natural experimental model to study the etiology of pregnancy pathophysiologies. We have shown that the proximate cause of decreased fetal growth is not reduced oxygen availability, delivery, or consumption. We therefore asked whether glucose, the primary substrate for fetal growth, might be decreased and/or whether altered fetoplacental glucose metabolism might account for reduced fetal growth at HA.

Methods

Doppler and ultrasound were used to measure maternal uterine and fetal umbilical blood flows in 69 and 58 residents of 400 vs 3600 m. Arterial and venous blood samples from mother and fetus were collected at elective cesarean delivery and analyzed for glucose, lactate and insulin. Maternal delivery and fetal uptakes for oxygen and glucose were calculated.

Principal Findings

The maternal arterial – venous glucose concentration difference was greater at HA. However, umbilical venous and arterial glucose concentrations were markedly decreased, resulting in lower glucose delivery at 3600 m. Fetal glucose consumption was reduced by >28%, but strongly correlated with glucose delivery, highlighting the relevance of glucose concentration to fetal uptake. At altitude, fetal lactate levels were increased, insulin concentrations decreased, and the expression of GLUT1 glucose transporter protein in the placental basal membrane was reduced.

Conclusion/Significance

Our results support that preferential anaerobic consumption of glucose by the placenta at high altitude spares oxygen for fetal use, but limits glucose availability for fetal growth. Thus reduced fetal growth at high altitude is associated with fetal hypoglycemia, hypoinsulinemia and a trend towards lactacidemia. Our data support that placentally-mediated reduction in glucose transport is an initiating factor for reduced fetal growth under conditions of chronic hypoxemia.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Reduced fructosamine-3-kinase activity and its mRNA in human distal colorectal carcinoma

Fructosamine-3-Kinase (FN3K) is an enzyme phosphorilating fructoselysine (FL) residues on glycated proteins, resulting in the production of protein-bound FL-3-phosphate. The pathological role of the non-enzymatic modification of proteins by reducing sugars has become increasingly evident in various types of disorders, including the cancer. In this study, our aim was to study FN3K enzyme activity, as well as its mRNA in human colorectal cancer (CRC). Thirty consecutive CRC patients undergoing surgery of the colon were enrolled in the study. FN3K enzymatic activity and gene expression were analyzed using a radiometric assay and quantitative RT-PCR, respectively. FN3K is a functionally active enzyme in human colon tissue, without significant differences between normal mucosa and cancer. The mean level of FN3K mRNA was significantly lower in cancer than in the corresponding normal colorectal mucosa The colorectal tumors located on the left side showed lower levels of both enzymatic activity and mRNA FN3K than tumors located in the right side of colon. This paper is the first studying FN3K enzyme activity in human CRC, showing a significant relationship between enzymatic activity, its mRNA and tumor side.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

Molecular characterization of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp. TA144

The Antarctic Gram-negative bacterium Psychrobacter sp. TA144 contains two small cryptic plasmids, called pTAUp and pTADw. pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway. Several attempts to produce the wild-type biologically active PsyRep in Escherichia coli failed, possibly due to auto-regulation of the protein population. However, the serendipitous occurrence of a frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form. The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization. The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori+ sequence.