Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction.

We isolated cDNAs encoding a 115 kd human atrial natriuretic peptide (alpha ANP) receptor (ANP-A receptor) that possesses guanylate cyclase activity, by low-stringency hybridization with sea urchin Arbacia punctulata membrane guanylate cyclase probes. The human ANP-A receptor has a 32 residue signal sequence followed by a 441 residue extracellular domain homologous to the 60 kd ANP-C receptor. A 21 residue transmembrane domain precedes a 568 residue cytoplasmic domain with homology to the protein kinase family and to a subunit of the soluble guanylate cyclase. COS-7 cells transfected with an ANP-A receptor expression vector displayed specific [125I]alpha ANP binding, and exhibited alpha ANP stimulated cGMP production. These data demonstrate a new paradigm of cellular signal transduction where extracellular ligand binding allosterically regulates cyclic nucleotide second-messenger production by a receptor cytoplasmic catalytic domain.     

Benthic algal response to N and P enrichment along a pH gradient

Nutrient enrichment and its effect on benthic algal growth, community composition, and average cell size was assessed across two sites of differing pH within a single habitat. Nutrients were added using in situ substrata, which released either N, P, or no additional nutrients (controls) at each site for 21 days. Upon collection, chlorophyll and biovolume standing stocks of the attached algal microflora were measured. Chlorophyll concentration was different among all treatments, accumulating greatest on P, followed by N, and the least on C substrata (P < 0.001) and was highest at site-2 (P < 0.001), while total algal biovolume was highest on P compared to both N and C substrata (P < 0.05) and did not vary between sites. Increased growth on P substrata was due to the enhanced biovolume of filamentous green algae, although the affected taxa varied between sites. Biovolume to cell density ratios (as a measure of average cell size) were highest on P substrata over both N-enriched and control substrata (P < 0.05) and this pattern was similar between sites. Progression towards a community composed of larger cells following P enrichment observed along this pH gradient, seems to be related to the dominance of larger celled filamentous green algae. Thus, nutrients exhibited greater control on benthic algal growth than did changes in hydrogen ion concentration.Contribution number 581, Great Lakes Environmental Research LaboratoryContribution number 581, Great Lakes Environmental Research Laboratory     

Chromosomal distribution of three members of the human natriuretic peptide receptor/guanylyl cyclase gene family

Chromosomal localization of the genes encoding three homologous human proteins, the ANPRA, ANPRB, and ANPRC cell surface receptors, was determined by polymerase chain reaction (PCR) analysis of genomic DNA from somatic cell hybrids. The ANPRA gene was assigned to 1q12----qter by intron-specific PCR. The ANPRB gene was assigned to 9p11----p22 using species-specific length variation in PCR fragments. The ANPRC gene was assigned to chromosome 5 using human-specific PCR primers identified by screening a human primer panel on parental DNA samples (shotgun primer screening). Chromosomal assignments based on PCR analysis were confirmed and the genes further sublocalized by in situ hybridization of cloned cDNA probes to human metaphase chromosomes. The ANPRA gene was sublocalized to 1q21----q22, the ANPRB gene to 9p12----p21, and the ANPRC gene to 5p13----p14.     

Microbial cell responses to a non-ionic surfactant

Summary The effects of a non-ionic surfactant, Pluronic F-68, on growth of microbial cell cultures have been studied. Growth ofSaccharomyces cerevisiae at 30°C or 37°C as measured by viable cell counts was unaffected by culture with pluronic. However, corresponding absorbance measurements forS. cerevisiae incubated with 5–10% pluronic were lower than controls at both temperatures. Absorbance ofE. coli cultures was also significantly reduced by incubation with 5.0–10.0% pluronic at both temperatures although viable counts again revealed no significant inhibition of growth.     

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC- 12 cells

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen.

Five different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4 degrees C were 1) storage in buffer, 2) storage in 50% methanol, and 3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82-0.91, P less than 0.001 for all TAAs). The cell suspensions preserved at 4 degrees C all showed a significant increase in background fluorescence (P less than 0.05) and a reduction in the TAA specific fluorescence (P less than 0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week.