Germinal centres and the B-cell system. V

Summary Thoracic duct lymphocytes (TDL) were studied with respect to their capacity to give rise to germinal centres (GC) and to form primary antibody in an adoptive transfer system of the rat. Challenge with sheep erythrocytes (SRBC) 24h after lethal irradiation (900 rads) and syngeneic TDL reconstitution (10⁸) lead to conspicuous GC activity already 7 days after transfer. In contrast, using syngeneic bone marrow (BM) in the adoptive transfer system, no GC formation was observed over the period studied (14 days after reconstitution). Reconstitution experiments using in vivo-separated T-TDL (1–5 % s-Ig⁺) and B-TDL (>90% s-Ig⁺) subpopulations, either separately or in combination, indicated that GC originate from B-TDL but require T-TDL for induction.Abbreviations BM Bone Marrow - TDL Thoracic Duct Lymphocytes - GC Germinal Centre - SRBC Sheep Red Blood Cells - GCDC Germinal Centre-Derived Cells - GCPC Germinal Centre-Precursor Cells - DAB Mineral Salt Solution Dulbecco A + B - FCS Fetal Calf Serum - PALS Peri-Arteriolar Lymphocyte Sheath - AFCP Antibody-Forming Cell Precursors     

Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties.

Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia.     

Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

Differences in adaptive stabilization of structures in response to stress and hypoxia relate with the accumulation of hsp70 isoforms

The phenomenon of adaptive stabilization of structures (PhASS) develops during adaptation of the organism to intermittent restraint stress. The PhASS manifests itself in a considerably increased resistance of the heart to a broad spectrum of harmful factors. In the present work, the content of hsp70 and their role in the development of PhASS during adaptation to intermittent restraint stress and to intermittent hypoxia were studied. In adaptation to restraint stress, five hsp70 isoforms with pI ranging from 5.7 to 6.3 were accumulated in the myocardium. The heart simultaneously became strikingly resistant to reperfusion paradox and heat shock. In adaptation to hypoxia, only two hsp70 isoforms with pI about 5.8 were accumulated. The resistance to reperfusion paradox was not increased and the resistance to heat shock was increased only moderately. These data suggest a role of different hsp70 isoforms in the mechanism of PhASS as well as adaptive protection of the heart.Abbreviation hsp70 heat shock proteins - PhASS Phenomenon of Adaptive Stabilization of Structures - CK Creatine Kinase     

Localization of trehalase in vacuoles and of trehalose in the cytosol of yeast (Saccharomyces cerevisiae)

Protoplasts of Saccharomyces cerevisiae synthesized and degraded trehalose when they were incubated in a medium containing traces of glucose and acetate. Such protoplasts were gently lyzed by the polybase method and a particulate and soluble fraction was prepared. Trehalose was found in the soluble fraction and the trehalase activity mostly in the particulate fraction which also contained the vacuoles besides other cell organelles. Upon purification of the vacuoles, by density gradient centrifugation, the specific activity of trehalase increased parallel to the specific content of vacuolar markers. This indicates that trehalose is located in the cytosol and trehalase in the vacuole. It is suggested that trehalose, in addition to its role as a reserve may also function as a protective agent to maintain the cytosolic structure under conditions of stress.Non Standard Abbreviations AMPD 2-amino-2-methyl-1,3-propanediol - DTT dithiothreitol - MES 2-N-morpholinoethanesulfonic acid - PIPES piperazine-N,N-bis-2-ethanesulfonic acid - PMSF phenylmethylsulfonylfluoride     

Simultaneous measurement of prednisone, prednisolone and 6β-hydroxyprednisolone in urine by high-performance liquid chromatography coupled with a radioactivity detector

We describe the first method for routine measurement of prednisone, prednisolone and 6β-hydroxyprednisolone concomitantly in urine. Urine (3 ml) is extracted with ethyl acetate, washed with base and separated by high-performance liquid chromatography on a silica column with a solvent system of hexane—diethyl ether—ethanol—tetrahydrofuran—glacial acetic acid (59.9:31:2.3:6.5:0.3, v/v). The steroids are detected at 254 nm. Because no conventional internal standard was found, 6β-[³H]hydroxycortisol and [³H]prednisolone are added to urine prior to extraction; ³H is monitored by a radioactivity detector coupled with the chromatograph. The assay exhibits linearity from 200 to 7500 ng and an inter-day variability of < 11.4% (C.V.).     

Species boundaries in the messy middle—A genome‐scale validation of species delimitation in a recently diverged lineage of coastal fog desert lichen fungi

Species delimitation among closely related species is challenging because traditional phenotype‐based approaches, for example, using morphology, ecological, or chemical characteristics, may not coincide with natural groupings. With the advent of high‐throughput sequencing, it has become increasingly cost‐effective to acquire genome‐scale data which can resolve previously ambiguous species boundaries. As the availability of genome‐scale data has increased, numerous species delimitation analyses, such as BPP and SNAPP+Bayes factor delimitation (BFD*), have been developed to delimit species boundaries. However, even empirical molecular species delimitation approaches can be biased by confounding evolutionary factors, for example, hybridization/introgression and incomplete lineage sorting, and computational limitations. Here, we investigate species boundaries and the potential for micro‐endemism in a lineage of lichen‐forming fungi, Niebla Rundel & Bowler, in the family Ramalinaceae by analyzing single‐locus and genome‐scale data consisting of (a) single‐locus species delimitation analysis using ASAP, (b) maximum likelihood‐based phylogenetic tree inference, (c) genome‐scale species delimitation models, e.g., BPP and SNAPP+BFD, and (d) species validation using the genealogical divergence index (gdi). We specifically use these methods to cross‐validate results between genome‐scale and single‐locus datasets, differently sampled subsets of genomic data and to control for population‐level genetic divergence. Our species delimitation models tend to support more speciose groupings that were inconsistent with traditional taxonomy, supporting a hypothesis of micro‐endemism, which may include morphologically cryptic species. However, the models did not converge on robust, consistent species delimitations. While the results of our analysis are somewhat ambiguous in terms of species boundaries, they provide a valuable perspective on how to use these empirical species delimitation methods in a nonmodel system. This study thus highlights the challenges inherent in delimiting species, particularly in groups such as Niebla, with complex, relatively recent phylogeographic histories.