Effects of cold on vascular permeability and edema formation in the isolated cat limb.

We investigated the effects of cold temperatures on microvascular protein permeability in the isolated constant-flow perfused cat hindlimb. The perfusates were 20% cat plasma-80% albumin-electrolyte solution (low-viscosity perfusate, approximately 1 cP) or whole blood (high-viscosity perfusate, approximately 4 cP). The time at low temperature (less than 10 degrees C) was less than 3 h (short term) or greater than 5 h (long term). Decreases in the solvent drag reflection coefficient (sigma f) indicated increases in permeability. The sigma f's were determined with the integral-mass balance method from measurement of changes in protein concentration and hematocrit induced by fluid filtration into the tissues. Short-term cold exposure did not increase permeability with either a low- or a high-viscosity perfusate, whereas long-term exposure with limb temperatures of approximately 5 degrees C significantly increased permeability when the perfusate was whole blood. In addition, we verified our previous prediction that flow had to be reduced to 6-8 ml.min-1.100 g-1 to avoid the hydrostatic edema caused by short-term perfusion with whole blood at approximately 5 degrees C. Also, we found that at approximately 3 degrees C histamine's permeability-increasing effect was totally abolished, whereas at approximately 20 degrees C this effect was partially inhibited. Hence, constant-flow perfusion at low temperature with whole blood can cause edema by a pressure-dependent mechanism, whereas long-term perfusion with this perfusate at low temperatures can cause a permeability increase that further compounds edema formation. Histamine is not responsible for this permeability increase.     

Genetic and Molecular Organization of Ribosomal DNA (Rdna) Variants in Wild and Cultivated Barley

Twenty rDNA spacer-length variants (slvs) have been identified in barley. These slvs form a ladder in which each variant (with one exception) differs from its immediate neighbors by a 115-bp subrepeat. The 20 slvs are organized in two families, one forming an eight-step ladder (slvs 100-107) in the nucleolus organizer region (NOR) of chromosome 7 and the other a 12-step ladder (slvs 108a-118) in the NOR of chromosome 6. The eight shorter slvs (100-107) segregate and serve as markers of eight alleles of Mendelian locus Rrn2 and the 12 longer slvs (108a-118) segregate and serve as markers of 12 alleles of Mendelian locus Rrn1. Most barley plants (90%) are homozygous for two alleles, including one from each the 100-107 and the 108a-118 series. Two types of departures from this typical pattern of molecular and genetic organization were identified, one featuring compound alleles marked by two slvs of Rrn1 or of Rrn2, and the other featuring presence in Rrn1 of alleles normally found in Rrn2, and vice versa. The individual and joint effects on adaptedness of the rDNA alleles are discussed. It was concluded that selection acting on specific genotypes plays a major role in molding the strikingly different allelic and genotypic frequency distributions seen in populations of wild and cultivated barley from different ecogeographical regions.     

Desensitization of Bacillus subtilis aspartokinase I to allosteric inhibition by meso-diaminopimelate allows aspartokinase I to function in amino acid biosynthesis during exponential growth.

Strains of Bacillus subtilis deficient in aspartokinases II and III are unable to grow in the absence of lysine, methionine, and threonine, although they have normal levels of aspartokinase I (J.J. Zhang, F.M. Hu, N.Y. Chen, and H. Paulus, J. Bacteriol. 172:701-708, 1990). Revertants with the ability to grow in the absence of lysine and methionine had an altered aspartokinase I, which was insensitive to feedback inhibition by meso-diaminopimelate. This suggests that inhibition by meso-diaminopimelate limits the ability of aspartokinase I to function in amino acid biosynthesis.     

Post-translational modification of a monocyte-specific chemoattractant synthesized by glioma, osteosarcoma, and vascular smooth muscle cells

Chemotaxis is an important step in monocyte recruitment in inflammation, wound healing, and tumor growth. We reported previously that monocyte chemotactic activity secreted by malignant cells and normal smooth muscle cells is associated with a protein or family of proteins that are related to the monocyte-specific smooth muscle cell-derived chemotactic factor (SMC-CF) (Graves, D. T., Jiang, Y. L., Williamson, M. J., and Valente, A. J. (1989) Science 245, 1490-1493). Similar monocyte chemotactic proteins (MCP-1) produced by U-105MG human glioma cells have also been identified (Yoshimura, T., Robinson, E. A., Tanaka, S., Appella, E., Kuratsu, J., and Leonard, E. J. (1989) J. Exp. Med. 169, 1449-1459). We now report that the MCP-1 gene is expressed in MG-63 human osteosarcoma and vascular smooth muscle cells and that SMC-CF antiserum specifically immunoprecipitates proteins synthesized by U-105MG glioma cells. Experiments were undertaken to elucidate the processing pathway of MCP-1/SMC-CF-like proteins in each of these cell types. These experiments demonstrate that larger MCP-1/SMC-CF-like proteins are derived from a Mr = 9000 precursor. Post-translational modification involves the addition of O-linked carbohydrates and sialic acid residues. Differences in carbohydrate processing account for the heterogeneity in MCP-1/SMC-CF-like proteins produced by different cell types. Secretion of these proteins occurs rapidly following processing events in the endoplasmic reticulum-Golgi compartment.     

Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species

Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.     

Solution structure of the nogalamycin-DNA complex

The nogalamycin-d(A-G-C-A-T-G-C-T) complex (two drugs per duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. Two equivalents of nogalamycin binds to the self-complementary octanucleotide duplex with retention of 2-fold symmetry in solution. We have assigned the proton resonances of nogalamycin and the d(A1-G2-C3-A4-T5-G6-C7-T8) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site on duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the nogalamycin and the DNA in the complex. The molecular dynamics calculations were guided by 274 intramolecular nucleic acid distance constraints, 90 intramolecular nogalamycin distance constraints, and 104 intermolecular distance constraints between nogalamycin and the nucleic acid protons in the complex. The aglycon chromophore intercalates at (C-A).(T-G) steps with the long axis of the aglycon approximately perpendicular to the long axis of the flanking C3.G6 and A4.T5 base pairs. The aglycon selectively stacks over T5 and G6 on the T5-G6-containing strand with the aglycon edge containing OH-4 and OH-6 substituents directed toward the C3-A4-containing strand. The C3.G6 and A4.T5 base pairs are intact but buckled at the intercalation site with a wedge-shaped alignment of C3 and A4 on the C3-A4 strand compared to the parallel alignment of T5 and G6 on the T5-G6 strand in the complex. The nogalose sugar in a chair conformation, the aglycon ring A in a half-chair conformation, and the COOCH3-10 side chain form a continuous domain that is sandwiched within the walls of the minor groove and spans the three base pair (G2-C3-A4).(T5-G6-C7) segment. The nogalose ring is positioned in the minor groove such that its nonpolar face is directed toward the G6-C7 sugar-phosphate backbone while its polar face containing OCH3 groups is directed toward the G2-C3 sugar-phosphate backbone in the complex. The intermolecular contacts include a nonpolar patch of aglycon (CH3-9) and nogalose (CH3-3') methyl groups forming van der Waals contacts with the base-sugar residues in the minor groove and intermolecular hydrogen bonds involving the amino groups of G2 and G6 with the ether oxygens OCH3-3' and O7, respectively, on the nogalose sugar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density

Influenza virus gains access to the cytoplasm of its host cell by means of a fusion event between viral and host cell membrane. Fusion is mediated by the envelope glycoprotein hemagglutinin (HA) and is triggered by low pH. To learn how many hemagglutinin trimers are necessary to cause membrane fusion, we have used two NIH 3T3 fibroblast cell lines that express HA protein at different surface densities. On the basis of quantitations of the number of HA trimers per cell and the relative surface areas of the two cell lines, the HAb-2 cells have a 1.9-fold higher plasma membrane surface density than the GP4F cells. The membrane lateral diffusion coefficient and the mobile fraction for HA is the same for both cell lines. A Scatchard analysis of the binding of glycophorin-bearing liposomes to the cells showed 1700 binding sites for the GP4F cells and 3750 binding sites for the HAb-2 cells, with effectively the same liposome-cell binding constant, about 7 x 10(10) M-1. Binding was specific for glycophorin on the liposomes and HA expressed on the cells. A competition experiment employing toxin-containing and empty liposomes allowed us to quantitate the number of liposomes that fused per cell, which was a small constant fraction of the number of bound liposomes. For the HAb-2 cells, about 1 in every 70 bound liposomes fused and for the GP4F cells about 1 in every 300 bound liposomes fused. Hence, the HAb-2 cells showed 4.4 times more fusion per bound liposome, even though the surface density of HA was only 1.9 times greater. We conclude the following: (i) One HA trimer is not sufficient to induce fusion. (ii) The HA bound to glycophorin is not the HA that induces fusion. That is, even though each HA has a binding and a fusion function, those functions are not performed by the same HA trimer.     

Group IIIA-metal hydroxides indirectly neutralize the voltage sensor of the voltage-dependent mitochondrial channel, VDAC, by interacting with a dynamic binding site.

The voltage-dependent, anion-selective mitochondrial channel, VDAC, undergoes two different conformational changes from the open to a closed state under positive and negative applied electric fields. Micromolar quantities of aluminum hydroxide and other metal trihydroxides have recently been shown to be able to inhibit this voltage-dependent closure (Dill et al. (1987) J. Membr. Biol. 99, 187-196; Zhang and Colombini (1989) Biochim. Biophys. Acta 991, 68-78). It was suggested that the inhibition results from the neutralization of the positively charged voltage sensors by the metal species. In the present study, the dynamics of the metal-binding site accompanying channel closure was investigated by adding In(OH)3 to only one side of the membrane and examining its effect on the channel's gating processes. Indium added to open channels inhibited channel closure only when the metal-containing side was on the lower potential side of the applied field. If indium was added only to the higher-potential side, the channels closed and tended to remain closed after the field was abolished. The addition of metal hydroxide after closing the channels with a negative potential on the metal side did not result in channel re-opening as would be expected for sensor neutralization. Inhibition occurred immediately, however, if the channels were first allowed to open briefly. The closed-state selectivity seemed to be very similar in the absence or presence of the metal, indicating that the metal-binding sites are not located within the pore of the channel in the closed conformation. The results are consistent with a voltage-dependent translocation across the membrane of each of two metal-binding sites on VDAC. This translocation is tightly coupled with channel opening and closing.     

Effects of heavy metal on rat liver microsomal Ca2(+)-ATPase and Ca2+ sequestering. Relation to SH groups.

In isolated hepatic microsomal vesicles the heavy metals Cd2+, Cu2+, and Zn2+ inhibit Ca2+ uptake and evoke a prompt efflux of Ca2+ from preloaded vesicles in a dose-dependent manner. N-Ethylmaleimide also inhibits Ca2+ uptake and causes Ca2+ release, but it is less effective in these respects than the heavy metals. Measurement of mannose-6-phosphatase activity indicate that the heavy metal-induced Ca2+ efflux is not caused by a general increase in membrane permeability. Heavy metals also inhibit the Ca2(+)-ATPase activity and the formation of the phosphorylated intermediate of the enzyme. In contrast, the sulfhydryl modifying reagent, N-ethylmaleimide inhibits the Ca2(+)-ATPase activity while it has a relatively small effect on Ca2+ release. Thus, the effects of these agents on Ca2+ sequestering and Ca2(+)-ATPase activity are not strictly proportional. The sulfhydryl group reducing agent dithiothreitol protects the microsomes from the effects of heavy metals, while glutathione is less protective. Addition of vanadate to vesicles, at a concentration which completely blocked the activity of the Ca2(+)-ATPase, resulted in a small and slow release of the accumulated Ca2+. Subsequent additions of heavy metals evoked a massive Ca2+ release. Thus, the effects of heavy metals on Ca2+ efflux cannot be due entirely to their inhibition of the Ca2+ pump. The heavy metal-induced Ca2+ efflux is not inhibited either by ruthenium red or tetracaine.