The role of calmodulin in the regulation of (Mg2+ + Ca2+)-ATPase activity in erythrocyte membranes of cystic fibrosis patients and controls

(Mg²⁺ + Ca²⁺)-ATPase activity has been found to be significantly reduced in EDTA-washed erythrocyte membrane preparations from cystic fibrosis patients compared to aged-matched controls. Calmodulin was found to be present in erythrocytes from cystic fibrosis patients and characterized similarly to calmodulin isolated from control preparations. Calmodulin from control erythrocyte preparations stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of EDTA-washed erythrocyte membranes derived from cystic fibrosis patients to the same extent as those membranes derived from controls. Similarly, calmodulin obtained from erythrocytes of cystic fibrosis patients stimulated the (Mg²⁺ + Ca²⁺)-ATPase activity of control and cystic fibrosis erythrocyte membrane preparations to a similar extent. These results indicate that this decrease in (Mg²⁺ + Ca²⁺)-ATPase activity in erythrocytes from cystic fibrosis patients is not due to an alteration in the regulatory function of calmodulin.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

The determination of lactate turnover in vivo with 3H- and 14C-labelled lactate. The significance of sites of tracer administration and sampling.

The D-ribulokinase and D-xylulokinase of Klebsiella aerogenes were purified to homogeneity from Escherichia coli K12 construct strains that synthesized these enzymes constitutively. The D-ribulokinase, which is encoded in the ribitol operon, is active as a dimer of 60 000 subunit mol.wt., whereas the D-xylulokinase, which is encoded in the D-arabitol operon, is active as a dimer of 54 000 subunit mol.wt. The amino acid compositions and N-terminal sequences of both pentulokinases are reported. The Kapp. values of the enzymes for their D-pentulose substrates were determined, and the D-ribulokinase was shown to have a low-affinity side-specificity for ribitol and D-arabitol. These results are discussed in the context of the evolution of the Klebsiella aerogenes pentitol operons.     

A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.     

Esterase activity in human breast cyst fluid: associations with steroid sulfates and cations.

Human breast cyst fluid (BCF) contains an esterase that on the basis of electrophoretic mobility and response to inhibitors differs from those found in the plasma. From a total of 384 BCF samples analyzed for esterase using p-nitrophenyl hexanoate as substrate, 149 (39%) showed significant activity. The samples had been analyzed for the concentrations of the sulfates of estrone, estriol, dehydroepiandrosterone, as well as the potassium and sodium cations (K+/Na+). The data were submitted to statistical analysis using the Spearman rank order test. The esterase-positive samples exhibited a significant positive association with each of the steroid sulfates and the K+/Na+ ratios. Except for protein concentration, there was no significant correlation between the esterase-positive and esterase-negative cysts. These observations may have physiological significance in that high K+/Na+ ratio cysts have been related to the histological status of the cyst.     

Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts.

The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.     

Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde.

Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.     

Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticulum protein structure and calcium release.

In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.     

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.