Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.     

Sources,fates, and impacts of nitrogen inputs to terrestrial ecosystems: review and synthesis

The relative importance of nitrogen inputs from atmospheric deposition and biological fixation is reviewed in a number of diverse, non-agricultural terrestrial ecosystems. Bulk precipitation inputs of N (l–l2 kg N ha^–1 yr^–1) are the same order of magnitude as, or frequently larger than, the usual range of inputs from nonsymbiotic fixation (< 1=" –=" 5=" kg=" n=">^–1 yr^–1), especially in areas influenced by industrial activity. Bulk precipitation measurements may underestimate total atmospheric deposition by 30–40% because they generally do not include all forms of wet and dry deposition. Symbiotic fixation generally ranges from 10–160 kg N ha^–1 yr^–1) in ecosystems where N-fixing species are present during early successional stages, and may exceed the range under unusual conditions.Rates of both symbiotic and nonsymbiotic fixation appear to be greater during early successional stages of forest development, where they have major impacts on nitrogen dynamics and ecosystem productivity. Fates and impacts of these nitrogen inputs are important considerations that are inadequately understood. These input processes are highly variable in space and time, and few sites have adequate comparative information on both nitrogen deposition and fixation.

The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The -crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the -crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat -crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [³⁵S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS PAGE of the treated cells showed incorporation of label into a broad 97–121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97–121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5′-O-(3-thiotriphosphate). The 97–121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5′-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteoglycans.

The results demonstrate that chromaffin vesicle matrix proteins are rapidly and intensely thiophosphorylated in cultured chromaffin cells but not in isolated vesicles. The data suggest that phosphorylation must play an important role in the normal function of these vesicle proteins.     

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice

Summary A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.     

Elevated temperature alters the ionic dependence of amine-induced pacemaker activity in a conditional burster neuron

Summary The anterior burster neuron of the lobster (Panulirus interruptus) stomatogastric ganglion is a conditional burster that functions as the primary pacemaker for the pyloric motor network. When modulatory inputs to this cell are blocked, it loses its bursting properties and becomes quiescent. Applications of the monoamines, dopamine, octopamine or serotonin restore rhythmic bursting in this cell (Flamm and Harris-Warrick 1986). At 15 °C, serotonin- and octopamine-induced oscillations depend critically upon sodium entry (blocked by low sodium saline or tetrodotoxin); dopamine-induced oscillations depend upon calcium entry (blocked by reduced extracellular calcium; Harris-Warrick and Flamm 1987). We show here that the ionic dependence of amine-induced oscillations in the anterior burster cell differs at 15 and 21 °C. At 21 °C, all amines have the potential to induce rhythmic oscillations in saline containing tetrodotoxin. At the elevated temperature and in tetrodotoxin, both calcium and sodium currents are essential for the maintenance of dopamine-induced oscillaions; serotonin-induced oscillations do not depend upon either calcium or sodium alone; octopamine-induced oscillations do not depend upon calcium and show a variable dependence upon sodium. Thus, multiple ionic mechanisms, which vary with both the modulator and the ambient temperature, can be recruited to support rhythmic activity in a conditional burster neuron.Abbreviations AB anterior burster - PD pyloric dilator - PY pyloric constrictor - DA dopamine - 5HT serotonin - Oct octopamine - STG stomatogastric ganglion - TTX tetrodotoxin - GSP graded synaptic potential     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.