Characterization of atrial natriuretic peptide in urine from rats treated with a neutral endopeptidase inhibitor.

To explore the mechanisms for the natriuretic effects of a neutral endopeptidase inhibitor, candoxatril, the concentration of atrial natriuretic peptide (ANP) and its molecular forms in the urine of Dahl salt-sensitive (S) rats were examined. Candoxatril-induced natriuresis (+120%, p less than 0.05) was associated with a marked increase in the urinary ANP excretion (+1200%, p less than 0.05). Analysis by Sephadex G-50 gel filtration revealed that molecular weight of the major fraction of immunoreactive (ir-) ANP in the plasma of candoxatril-treated Dahl S rats was 3K, whereas that in the urine was 2.5 K. Further analysis by reverse phase high performance liquid chromatography showed that ir-ANP in the plasma of Dahl S rats was alpha-rANP (1-28), while that in the urine from rats treated with candoxatril was alpha-rANP (1-25). These results indicate that candoxatril inhibits the complete degradation of ANP in the kidney, thereby increasing the amount of biologically active ANP reaching the distal nephron and contributing to natriuresis.     

Initiation of DNA synthesis in the transfer origin region of RK2 by the plasmid-encoded primase: detection using defective M13 phage

The broad host range IncP (IncP1) plasmids of gram-negative bacteria encode DNA primases that are involved in conjugal DNA synthesis. The primase of RK2/RP4 is required for efficient DNA transfer to certain gram-negative bacteria, indicating that the enzyme primes complementary strand synthesis in the recipient. In vitro, the primase initiates synthesis of oligoribonucleotides at 3'-dGdT-5' dinucleotides on the template strand. In this report, replication-defective M13 phage are used to assay the ability of the RK2-encoded primase to initiate complementary strand synthesis in vivo on single-strand templates containing the RK2 origin of conjugal transfer (oriT) or the RK2 origin of vegetative replication (oriV). The results show that sequences from either strand of the oriT region serve as efficient substrates for the RK2 primase and can enhance the growth of the defective M13 vectors delta E101 and delta Elac to levels approaching wild-type. The primise-oriT interaction appeared specific, since neither the oriV sequence nor another RK2 region, trfB, significantly enhanced growth of the defective phage, either in the presence or in the absence of the primase. In contrast to ColEl and F, this study also shows that the oriV region of RK2 lacks sites that are recognized by the host-specified DNA priming systems. The results suggest that the oriT region contains sites on both DNA strands that are efficient substrates for the plasmid-encoded primase, facilitating initiation of complementary strand DNA synthesis in both donor and recipient during conjugation.     

Immunohistochemical Detection of DNA Replication in Mouse Uterine Cells by Bromodeoxyuridine Labeling of Wax- and Resin-Embedded Tissue Sections

TO apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stromata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cyctoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

A seed-specific Brassica napus oleosin promoter interacts with a G-box-specific protein and may be bi-directional

Corrigendum

Upstream regulatory sequences from two -conglycinin genes     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form the BiP bodies.

Immunofluorescence staining of yeast cells with anti-binding protein (BiP) antibodies shows uniform staining of the endoplasmic reticulum (ER). We have found that overproduction of Sec12p, an ER membrane protein, causes a change of BiP distribution within the cell. Upon induction of Sec12p by the GAL1 promoter, the staining pattern of BiP turns into bright dots scattering in the cell, whereas the staining of Sec12p remains to be the typical ER figure. Overproduction of other ER membrane proteins, HMG-CoA reductase or Sed4 protein, does not induce such relocalization of BiP. Pulse-chase experiments and electron microscopy have revealed that the overproduction of Sec12p inhibits protein transport from the ER to the Golgi apparatus. When the transport is arrested by one of the sec mutations that block the ER-to-Golgi step at the restrictive temperature, the BiP staining also changes into the punctate pattern. In contrast, the sec mutants that block later or earlier steps of the secretory pathway do not induce such change of BiP localization. These observations indicate that relocalization of BiP is caused by the inhibition of ER-to-Golgi transport. Using immunoelectron microscopy, we have found that the punctate staining is because of the accumulation of BiP in the restricted region of the ER, which we propose to call the "BiP body." This implicates existence of ER subdomains in yeast. A vacuolar protein, proteinase A, appears to colocalize in the BiP body when the ER-to-Golgi transport is blocked, suggesting that the BiP body may have a role as the site of accumulation of cargo molecules before exit from the ER.     

Histological studies on male sterility of hybrids between laboratory and wild mouse strains

In this study the cellular mechanisms of male sterility in F1 hybrids (BNF1) between BALB/c and wild-derived M.MUS-NJL (NJL) was investigated. Cell proliferation and differentiation in the sterile testis were examined by bromodeoxyuridine-labeling and use of germ cell stage-specific antibodies. In BNF1 testes, spermatogonia actively proliferated with a seminiferous epithelial cycle, and were retained in the basal layer of the tubules. However, preleptotene, leptotene and zygotene spermatocytes moved to the adluminal region. Immunohistological data with germ cell stage-specific antibodies indicated the presence of few, if any, pachytene spermatocytes in BNF1 testes. Thus, spermatogenesis seemed to be blocked at the zygotene stage. For examination of germ cell-Sertoli cell interactions, testes of aggregation chimeras between BNF1 and C3H/HeN were analyzed immunohistologically with C3H-specific antibody. Results showed that spermatogenesis of C3H-germ cells was normal, even when these cells in contact with BNF1-Sertoli cells. Differentiation of BNF1-germ cells progressed from zygotene to pachytene stage spermatocytes when these cells were surrounded by C3H-Sertoli cells, but never proceeded beyond the pachytene stage. These observations suggest that at least two different cellular factors may be involved in spermatogenesis, one acting in the germ cells and the other mediated by Sertoli cells. Furthermore, mating experiments revealed that the degree of spermatogenesis varied in different F1 hybrids, and that the major sterility factor was closely linked to the T -locus on chromosome 17.     

A model for feature linking via collective oscillations in the primary visual cortex

A neural network model for explaining experimentally observed neuronal responses in cat primary visual cortex is proposed. In our model, the basic functional unit is an orientation column which is represented by a large homogeneous population of neurons modeled as integrate-and-fire type excitable elements. The orientation column exhibits spontaneous collective oscillations in activity in response to suitable visual stimuli. Such oscillations are caused by mutual synchronization among the neurons within the column. Numerical simulation for various stimulus patterns shows that as a result of activity correlations between different columns, the amplitude and the phase of the oscillation in each column depend strongly on the global feature of the stimulus pattern. These results satisfactorily account for experimental observations.