Development and characterization of monoclonal antibodies to human type III procollagen

Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.     

Phosphorylation of calcium adenosinetriphosphatase by inorganic phosphate: reversible inhibition at high magnesium ion concentrations

Magnesium stimulates phosphorylation of the calcium pump protein of the sarcoplasmic reticulum by inorganic phosphate, but the effect is reversed by high [Mg2+]. This reversal is readily explained in terms of the generally accepted existence of two conformational states of the enzyme, E1 and E2. E2 is the form of the enzyme that can be phosphorylated by Pi, and it has one binding site for Mg2+. E1 is the form of the enzyme that has two high-affinity Ca2+ binding sites, and it is phosphorylated by ATP when Ca2+ is bound. Mg2+ can bind weakly to the two Ca2+ sites and to a third site known to be present on E1; this stabilizes E1 at the expense of E2 when [Mg2+] is large. Stabilization of E1 at pH 6.2 and 25 degrees C was found to be a highly cooperative function of [Mg2+] and was not prevented by increasing [Pi]. The latter result requires the existence of a binding site for Pi on E1, with an affinity for Pi comparable to that of E2. Cooperativity with respect to [Mg2+] requires that E2 is the stable state of the enzyme in the absence of ligands, with an equilibrium constant [E2]/[E1] on the order of 10(3) or higher at pH 6.2 and 25 degrees C.     

Application of high-performance liquid chromatography to competitive labeling studies: The chemical properties of functional groups of glucaton

A competitive-labeling study of glucagon was carried out using [³H]- and [¹⁴C]-1-fluoro-2,4-dinitrobenzene to determine simultaneously the chemical properties of the α-amino and imidazole groups of the N-terminal histidine residue, and the lysine and tyrosine residues, under conditions where glucagon is in its physiologically active monomer form. The dinitrophenyl derivatives of these groups were purified by high-performance liquid chromatography which greatly simplified the separation steps of the procedure. The results showed the α-amino and tyrosine groups to have relatively normal behavior, with pK values of 7.98 and 10.22, respectively, while the lysine had a low pK of 8.46. The imidazole function had an apparent pK of 7.84, substantially higher than previous estimates. This difference may be accounted for by the effect of the charged form of the adjacent α-amino group on the nucleophilicity of the imidazole group.     

Characterization of the IS895 family of insertion sequences from the cyanobacterium Anabaena sp. strain PCC 7120.

A family of repetitive elements from the cyanobacterium Anabaena sp. strain PCC 7120 was identified through the proximity of one element to the psbAI gene. Four members of this seven-member family were isolated and shown to have structures characteristic of bacterial insertion sequences. Each element is approximately 1,200 bp in length, is delimited by a 30-bp inverted repeat, and contains two open reading frames in tandem on the same DNA strand. The four copies differ from each other by small insertions or deletions, some of which alter the open reading frames. By using a system designed to trap insertion elements, one of the elements, denoted IS895, was shown to be mobile. The target site was not duplicated upon insertion of the element. Two other filamentous cyanobacterial strains were also found to contain sequences homologous to IS895.     

Cleavage of a smooth muscle myosin heavy chain near its C terminus by alpha-chymotrypsin. Effect on the properties of myosin.

Limited proteolysis of gizzard myosin by alpha-chymotrypsin converted the heavy chain doublet pattern, seen by gel electrophoresis, to a single band. Light chain degradation was not observed and only minor cleavage occurred at other heavy chain sites. Using a polyclonal antibody raised against a unique sequence from the slower-migrating heavy chain (SM1) it was shown that this conversion was due to the loss of a peptide approximately 4000 daltons from the C terminus of SM1. The peptide was isolated and sequenced, and the cleavage site was identified between phenylalanine 1943 and alanine 1944. Addition of antibody before protease protected SM1 from cleavage. The following changes were observed (a) the Mg2(+)-dependence of actin-activated ATPase of digested phosphorylated myosin was altered and activity was relatively high at low Mg2+ levels, i.e. similar to phosphorylated heavy meromyosin; (b) the KCl dependence of Mg2(+)-ATPase of the digested myosin, particularly the phosphorylated form, showed an altered pattern consistent with the stabilization of the 6 S conformation; (c) the tendency for aggregation was increased by proteolysis of phosphorylated myosin. These results show that the C-terminal region of a gizzard myosin heavy chain can modify some of the properties of myosin. It is suggested that the observed modifications reflect an enhanced tendency of the digested myosin to aggregate.     

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.     

A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor

A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.     

Alveolar macrophage activation in experimental legionellosis.

Legionella pneumophila is a facultative intracellular parasite of alveolar macrophages. In vitro studies have shown that lymphokine-activated mononuclear phagocytes inhibit intracellular replication of L. pneumophila. To determine if recovery from legionellosis is associated with activation of alveolar macrophages in vivo to resist L. pneumophila, we studied an animal model of Legionnaires' disease. Rats were exposed to aerosolized L. pneumophila and alveolar macrophages were harvested during the recovery phase of infection. We compared these alveolar exudate macrophages with normal resident alveolar macrophages for the capacity to support or inhibit the intracellular growth of L. pneumophila. We also measured Ia expression as a marker of immunologic activation, and studied binding of bacteria, superoxide release, and the expression of transferrin receptors as potential mechanisms of resistance to L. pneumophila. For perspective on the specificity of these responses, we also studied alveolar exudate cells elicited by inhalation of heat-killed L. pneumophila, live Listeria monocytogenes, and live Escherichia coli. We found that alveolar exudate macrophages elicited by live L. pneumophila, but not heat-killed L. pneumophila, resisted the intracellular growth of L. pneumophila. Exudate macrophages in resolving legionellosis exhibited increased Ia expression, diminished superoxide production, and downregulation of transferrin receptors. Binding of L. pneumophila to exudate macrophages was indistinguishable from that to resident macrophages in the presence of normal serum, and augmented in the presence of immune serum. Alveolar exudate macrophages elicited by E. coli also inhibited growth of L. pneumophila, and exhibited a modest increase in Ia expression without change in transferrin receptors. Exudate cells induced by L. monocytogenes exhibited up-regulation of Ia without diminution of superoxide release. Alveolar cells harvested after inhalation of heat-killed L. pneumophila did not differ from resident alveolar macrophages in the expression of surface markers. These findings suggest that alveolar macrophages are immunologically activated in vivo to serve as effector cells in resolving legionellosis, and that live bacteria are required to induce this expression of immunity. The mechanism of resistance to parasitism by L. pneumophila may entail restriction of the intracellular availability of iron, but does not involve diminished bacterial binding or an augmented respiratory burst.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Chromosome recombination and defective genome segregation induced in Chinese hamster cells by the topoisomerase II inhibitor VM-26

We found that 4-demethylepipodophyllotoxinthenylidene--d-glucoside (VM-26; Teniposide), which specifically inhibits the enzyme DNA topoisomerase II, induces the formation of quadriradial chromosomes in Chinese hamster ovary cells. VM-26 traps topoisomerase II molecules when they are covalently integrated into DNA during their reaction. Quadriradial chromosomes are formed by reciprocal exchange of double-stranded DNA between single chromatids of two different chromosomes. Using synchronised cells, we found that they were formed after a single replication cycle in the presence of VM-26 at a low concentration (0.008M), which does not affect DNA replication, and occurred in 50% of the mitotic cells at a concentration of 0.16 M. They were also formed when VM-26 was present for only 1.5 h before mitosis, after the completion of S-phase DNA replication. Chromatids bearing a translocated segment of another chromatid, which were derived from recombined chromosomes, were observed in late metaphase cells. Segregation of the daughter genomes was defective in many mitotic cells, probably because chromatids with two or no centromeres and kinetochores, formed from chromosomes recombined between their centromeres, could not be segregated. In the light of evidence that topoisomerase II molecules covalently integrated in DNA are trapped and therefore more abundant in the presence of VM-26, and that this enzyme can effect recombination of double-stranded DNA in vitro, we interpret these observations as evidence that topoisomerase II can mediate chromosome recombination in vivo.by M. Trendelenburg