Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor

Solvent exchange of 18O-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 equilibration technique. Exchange of buried waters with solvent water is complete before 10-15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Leukoencephalomalacia in a white-tailed deer from North Carolina

Leukoencephalomalacia similar to equine leukoencephalomalacia occurred in a white-tailed deer (Odocoileus virginianus) from North Carolina. The deer was recumbent and stuporous when presented for examination. It was believed that the disease resulted from ingestion of moldy field corn, infected with Fusarium moniliforme.     

Plantarflexor muscle function in young and elderly women

Contractile properties of the ankle plantarflexor muscles were compared between groups of young (means = 26 y) and elderly (means = 82 y) women. The H-reflex muscle contraction in the elderly group was characterized by a significant slowing of torque generation, as compared to the young women (means for average rate of torque development were young = 0.16 Nm ms-1 +/- 0.02 (SE), elderly = 0.09 Nm ms-1 +/- 0.02, P less than 0.05). However, the proportion of the total motor unit pool activated by the reflex was similar for the young and elderly groups at 63% and 70%, respectively. Maximal voluntary isometric torques were significantly lower (71%) in the elderly (young means = 135.3 Nm +/- 9.3, elderly means = 39.2 Nm +/- 2.9, P less than 0.01). These results are consistent with, and extend, previous reports showing decreased strength and speed of contraction in elderly muscle. Given that the average body weight was similar for the young and elderly groups, it was concluded that the aged plantarflexor muscles exhibited considerable impairment in ability to generate stabilizing torques about the ankle joint.     

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations.

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.