Recurrent use of evolutionary importance for functional annotation of proteins based on local structural similarity

The annotation of protein function has not kept pace with the exponential growth of raw sequence and structure data. An emerging solution to this problem is to identify 3D motifs or templates in protein structures that are necessary and sufficient determinants of function. Here, we demonstrate the recurrent use of evolutionary trace information to construct such 3D templates for enzymes, search for them in other structures, and distinguish true from spurious matches. Serine protease templates built from evolutionarily important residues distinguish between proteases and other proteins nearly as well as the classic Ser-His-Asp catalytic triad. In 53 enzymes spanning 33 distinct functions, an automated pipeline identifies functionally related proteins with an average positive predictive power of 62%, including correct matches to proteins with the same function but with low sequence identity (the average identity for some templates is only 17%). Although these template building, searching, and match classification strategies are not yet optimized, their sequential implementation demonstrates a functional annotation pipeline which does not require experimental information, but only local molecular mimicry among a small number of evolutionarily important residues.     

Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization

Soluble factors that enhance maturation of murine B lineage precursor cells in vitro were partially purified from the serum of very young NZB mice and characterized biochemically and biologically. Activity was initially detected by induction of colony-forming activity and surface immunoglobulin (sIg) on normal sIg- marrow cells as well as responsiveness of a pre-B cell line. Pooled sera from 4- to 5-wk-old NZB mice were initially fractionated on Sephacryl S-300 and Sephadex G-100 superfine columns. Fractions with activity (corresponding to m.w. of 15,000 to 45,000) were pooled and further separated. The activity was eluted as a single peak by hydrophobic (phenyl-Sepharose, with 0.8 M (NH4)2SO4) and lentil lectin affinity chromatography but resolved into three distinct peaks in preparative isoelectric focusing (IEF), with pI values of 3.5, 7.8, and 8.4. The latter two merged into a single peak with a pI value of 8.8 when the sample was further treated with neuraminidase before IEF. These three IEF fractions, each of which were enriched at least 1000-fold in specific activity relative to starting serum, were then characterized. Each was stable at pH 2 but sensitive to trypsin, 10 M urea, and heat treatment (56 degrees C for 1 hr). In nonreduced SDS-poly-acrylamide gel electrophoresis, their mobilities corresponded to m.w. of 17,000 for peak I (pI 3.5), 15,000 for peak II (pI 7.8), and 15,000 for peak III (pI 8.4). Interleukin 1, interleukin 2, interleukin 3, colony-stimulating factor for granulocyte and macrophage progenitors, antiviral, or B cell growth factor type I-like activities were not demonstrable. Peaks II and III, but not peak I, induced Ig secretion of anti-stimulated B cells. Peak I was also less effective than peaks II and III in induction of sIg on an established pre-B cell line. However, all fractions were equally effective in enhancing maturation of normal sIg- B lineage cells. Thus, serum from 4- to 5-wk-old NZB mice contains at least two distinct soluble factors that can enhance the maturation of sIg- B lineage cells in vitro. The biologic and biochemical characteristics of these factors appear to differ from those of previously well-defined cytokines.     

Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana)

Summary Vascular adrenergic sensitivity to exogenous catecholamines was examined in tadpoles of the American bullfrog (Rana catesbeiana), ranging from stage III to XIV. Central arterial blood pressure was measured in decerebrate bullfrog tadpoles to determine a reasonable initial infusion pressure. Solutions of epinephrine and phenylephrine were infused into the vasculature of pithed tadpoles, and the resulting changes in vascular resistance (R _v) were used to construct log dose-response relationships. Epinephrine infusion produced a dose-dependent increase in R _v (EC₅₀=5.3·10^-7 M), which could be reversed by sodium nitroprusside (a smooth muscle relaxant) and blocked by phenoxybenzamine (an -adrenergic antagonist). Larval R _v also increased with infusion of the -agonist phenylephrine (EC₅₀=7.4·10⁸ M). Infusion of 10^-6 M isoproterenol (a -agonist) largely reversed the phenylephrine-induced increase in R _v. These results indicate that the capacity exists for both -mediated vasoconstriction and -mediated vasodilation early in bullfrog ontogeny. Neither initial R _v nor the responses to infused epinephrine or phenylephrine were significantly correlated to development over the range of larval stages used in this study.Abbreviations ECG electrocardiogram - EPI epinephrine - ISO isoproterenol - PHE phenylephrine - POB phenoxybenzamine - R _v vascular resistance - SNP sodium nitroprusside     

Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.     

Linkage of a Gene Causing High Bone Mass to Human Chromosome 11 (11q12-13)

The purpose of this paper is to report the linkage of a genetic locus (designated "HBM") in the human genome to a phenotype of very high spinal bone density, using a single extended pedigree. We measured spinal bone-mineral density, spinal Z(BMD), and collected blood from 22 members of this kindred. DNA was genotyped on an Applied Biosystems model 377 (ABI PRISM Linkage Mapping Sets; Perkin Elmer Applied Biosystems), by use of fluorescence-based marker sets that included 345 markers. Both two-point and multipoint linkage analyses were performed, by use of affected/unaffected and quantitative-trait models. Spinal Z(BMD) for affected individuals (N = 12) of the kindred was 5.54 +/- 1.40; and for unaffected individuals (N = 16) it was 0.41 +/- 0.81. The trait was present in affected individuals 18-86 years of age, suggesting that HBM influences peak bone mass. The only region of linkage was to a series of markers on chromosome 11 (11q12-13). The highest LOD score (5.21) obtained in two-point analysis, when a quantitative-trait model was used, was at D11S987. Multipoint analysis using a quantitative-trait model confirmed the linkage, with a LOD score of 5.74 near marker D11S987. HBM demonstrates the utility of spinal Z(BMD) as a quantitative bone phenotype that can be used for linkage analysis. Osteoporosis pseudoglioma syndrome also has been mapped to this region of chromosome 11. Identification of the causal gene for both traits will be required for determination of whether a single gene with different alleles that determine a wide range of peak bone densities exists in this region.     

A rapid plastic embedding technique for preparation of three-micron thick sections of decalcified hard tissue.

A 24 hour start-to-finish method is described for the preparation of three-micron-thick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerized by four hours heating at 42 C. Three-micron-thick sections are then easily prepared by using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

Further studies on a hybrid cell-surface antigen associated with human chromosome 11 using a monoclonal antibody

A monoclonal antibody has been obtained that recognizes an antigen encoded by human chromosome 11. We present evidence that this monoclonal antibody recognizes the same or a similar antigenic activity as that previously called a1. Genetic information necessary for a1 expression and recognition by the monoclonal antibody both map to 11p13 leads to 11pter. Mutants that have lost a1 are no longer recognized by the monoclonal antibody. The macroglycolipid fraction of human erythrocyte membranes which contains the a1 antigenic activity is able to convert antigen-negative Chinese hamster ovary cells into cells which are killed by the monoclonal antibody plus complement.     

A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals

A simple, rapid procedure for dual staining of cartilage and bone in rodents, particularly in late gestation, has been developed for routine use. The procedure involves rapid, complete skinning of fresh eviscerated specimens following a 30 sec immersion in a 70 C water bath. The unfixed specimen is stained in a mixture of 0.14% Alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Specimens are then macerated in 2% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin. Rapid staining of cartilage only is done in a mixture of 0.08% Alcian blue, glacial acetic acid, and ethanol, with subsequent maceration, clearing, and hardening as in the double staining procedure. Rapid staining of bone only, concurrent with maceration of soft tissue, can be done by placing fresh, unskinned specimens in a diluted mixture of alizarin red S in 2% KOH, with subsequent clearing and hardening in 1:1 distilled water and glycerin. Good quality fetal specimens can be prepared for examination by any of these procedures in a minimum of 11/2-2 days as compared to a minimum of 4-5 days for other procedures. Double stained specimens can be examined for abnormalities of the cartilage as well as bone.     

Glycosylation of vascular endothelial growth factor is not required for its mitogenic activity.

We have stably expressed the cDNA encoding the 165 amino-acid long form of human vascular endothelial growth factor (VEGF) in BHK-21 cells. VEGF was partially purified from the conditioned medium of transfected cells using heparin-sepharose affinity chromatography. The partially purified VEGF was mitogenic for various types of endothelial cells and inhibited the binding of pure [125I]VEGF to its receptors. Western blot analysis, using anti-VEGF antibodies, revealed a 47 kDa VEGF homodimer in the partially purified VEGF fraction. Preincubation of the transfected cells with the N-glycosylation inhibitor tunicamycin resulted in the conversion of the 47 kDa VEGF homodimer into a smaller, deglycosylated form of 42 kDa. Partially purified preparations of the deglycosylated VEGF displayed a mitogenic activity that was similar to that of the glycosylated form and efficiently inhibited the binding of native [125I]VEGF to the VEGF receptors of bovine aortic arch derived endothelial cells.