Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Role of light, carbon dioxide and nitrogen in regulation of buoyancy, growth and bloom formation of Anabaena flos-aquae

The availability of light, CO₂ and NH₄-N interacted to controlbuoyancy and growth of the gas vacuolate blue-green alga, Anabaenaflos-aquae. At high light intensities algal growth rates werehigh; however, the alga was non-buoyant regardless of the availabilityof CO₂ or NH₄-N. The mechanism for buoyancy loss involved increasedcell turgor pressures at higher light intensities which resultedin collapse of gas vacuoles. At lower light intensities algalgrowth rates and cell turgor pressures were reduced and buoyancywas controlled by the availability of CO₂ and inorganic nitrogen.Carbon dioxide limitation increased buoyancy, while reducedinorganic nitrogen availability reduced buoyancy. Mechanismsfor buoyancy regulation at low light intensities involved changesin cellular C/N ratios which appeared to affect the rate ofsynthesis and accumulation of protein-rich gas vacuoles. Algalspecific growth rates were combined with buoyancy data to forma single index (µ_bloom) to the rate of surface bloom formationof A.flos-aquae as a function of the availability of light,CO₂ and NH₄-N. The bloom formation index was enhanced with decreasedavailability of light and CO₂, and increased availability ofNH₄-N.     

Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

Testing of Arg-8-gonadotropin-releasing hormone-directed antisera by immunological and immunocytochemical methods for use in comparative studies

Three polyclonal antisera raised in rabbits against the mammalian molecular form of gonadotropin-releasing hormone (GnRH) were tested in enzyme-linked immunosorbent assays for crossreactivity with naturally occurring GnRHs and with GnRH analogues. Antisera were then tested immunocytochemically in order (i) to identify amino acids essential for the binding of each antiserum, and (ii) to evaluate the specificity of the immunocytochemical reaction in brain sections from various species of cyclostomes, amphibians, reptiles, and birds. Antiserum GnRH 80/1, recognizing mainly a discontinuous determinant including the NH2- and COOH-termini, crossreacts with GnRHs the molecular bending of which enables the spatial approach of both terminal amino acid residues. Antiserum GnRH 80/2, by requiring the COOH-terminus for binding and not tolerating substitutions by aromatic amino acids in the middle region of the molecule, recognizes chicken I GnRH, however, not the salmon form. The use of this antiserum is appropriate in species synthesizing the mammalian and/or the chicken I form of GnRH. GnRH antiserum 81/1 is specific mostly for mammalian GnRH. The results obtained by ELISAs are confirmed by immunocytochemical studies. A comparison between the results obtained in ELISA and in immunocytochemistry involving mammalian-, chicken I-, chicken II-, salmon-, and lamprey-directed GnRH antisera resulted in the following conclusions: (1) An antiserum recognizing the discontinuous antigen determinant including both NH2- and COOH-termini may be reactive in most vertebrate brain sections thus being appropriate for phylogenetically directed immunocytochemical studies. (2) Moreover, this discontinuous determinant seems to be immunocytochemically reactive in all parts of the neurons in the GnRH system, whereas, in some species, determinants located in the middle region of the molecule(s) tend to become reactive only during the axonal transport. (3) A crossreaction between tissue-bound antigen and antibodies recognizing the above cited discontinuous determinant indicates an appropriate bending of the molecule even in case of severe molecular differences, e.g., in lamprey form of GnRH. (4) It follows that in phylogenetic studies, an immunologically well characterized antiserum can be substituted for a species-directed antiserum.     

The basalis of the primate endometrium: a bifunctional germinal compartment

Radioautographic analysis of epithelial and stromal cell proliferation in the primate endometrial functionalis and basalis (rhesus monkey) has identified horizontal zonal patterns of mitotic activation and inhibition during natural menstrual cycles. At 1 h after a single i.v. injection of [3H]thymidine, mitotic activity in endometrial biopsies (hysterotomy) was determined on 9 days from the late proliferative to the late luteal phase (-2 days to + 14 days relative to the estrogen [E2]peak). Labeling indices (LIs) were determined within glandular segments of the 4 horizontal endometrial zones: Transient functionalis Zone I (luminal epithelium) and Zone II (uppermost gland); Germinal basalis: Zone III (middle gland) and Zone IV (basal gland). The size of the dividing epithelial populations (LI) differed zonally. During E2 dominance (-2 days to +3 days), the epithelial LIs of functionalis I (10 +/- 0.3%) and II (9.8 +/- 1.0%) were greater than those of basalis III (5.8 +/- 0.2%) and basalis IV (3.7 +/- 0.8%). During progesterone (P) dominance (+5 days to +14 days), epithelial mitosis was strongly inhibited in functionalis I (4.3 +/- 1.9%), functionalis II (0.8 +/- 0.2%), and basalis III (1.4 +/- 0.5%). Thus germinal basalis III was linked functionally with transient functionalis I and II by periovulatory uniformity in epithelial proliferation and postovulatory mitotic inhibition. A unique mitotic pattern set basalis IV apart from other zones by a steady rise in LI from 1% (-2 days) to 11% (+10 days). The LIs for stromal fibroblasts remained quite uniform in basalis IV but varied in other zones. Thus the postovulatory primate basalis was a distinct bipartite compartment in which the mitotic rate in basalis IV glandular epithelium increased steadily whereas that of basalis III was strongly inhibited. The remarkable enhancement of epithelial mitotic activity in basalis IV may reflect expansion of the stem-progenitor cell population for gestational growth or for post-menstrual regeneration.     

Actions of extracellular matrix on Sertoli cell morphology and function

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.     

The role of mucus gel viscosity, spinnability, and adhesive properties in clearance by simulated cough

We investigated the role of the viscoelastic and adhesive properties of mucus gel simulants on the clearance of mucus by simulated cough. Mucus-like gels with widely varying viscoelastic properties were prepared from polysaccharides crosslinked with sodium borate. Cough was simulated by opening a solenoid valve connecting a model trachea to a pressurized tank. The clearance of gels lining the model trachea was quantified by observing marker particle transport. Viscosity elastic modulus, relaxation time and yield stress were measured with a steady-shear viscoelastometer. Spinnability (thread formation) was determined with a filancemeter. Adhesivity (surface tension) was measured by the platinum ring technique. The viscoelastic and adhesive properties of the mucus gel simulants spanned the ranges observed for bronchial secretions from patients with COPD. The relationship between simulated cough clearance and the viscoelastic and adhesive properties of the gels was analyzed by stepwise linear regression of the non-zero data matrix. The major independent variable relating to clearance was viscosity. Secondary, but highly significant dependences, were also found for spinnability and adhesivity. Elastic modulus, relaxation time and yield stress had no independent effect on cough clearance over the investigated range. The results indicate that, in the absence of airway surface liquid, cough-type clearance relates primarily with mucus gel viscosity. For a given viscosity, clearance is also impaired by spinnability, i.e. the capacity of the mucus to form threads. At constant viscosity and spinnability, clearance is further impaired by an increase in the adhesivity of the mucus. The negative dependence of each of these physical factors can be rationalized in terms of their inhibitory effect on wave formation in the mucus lining layer during high velocity airflow interaction.     

Inhibition and active-site modelling of prolidase

Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions.     

Oncogenes and human breast cancer.

The role of oncogenes in breast tumorigenesis is unclear. Alterations and/or amplification of several oncogene sequences have been observed in primary human breast tumors, in breast tumor cell lines, and in mammary tumors in model systems. In principle, such alterations could be sites of primary lesions for human breast cancer, causes of tumor progression or metastasis, or simply secondary lesions of highly aberrant tumor genomes. The present study tested genetic linkage of breast cancer susceptibility to nine oncogenes in 12 extended families including 87 affected individuals. Lod scores for close linkage of each candidate sequence to breast cancer were -19.6 for HRAS, -12.3 for KRAS2, -1.0 for NRAS, -6.0 for MYC, -6.1 for MYB, -8.2 for ERBA2, -7.9 for INT2, and -5.1 for RAF1. Regions of chromosome 11p associated with tumor homozygosity and the region of 3p carrying the gene for Von Hippel-Lindau disease could also be excluded from linkage to human breast cancer. The 5-kb allele of the MOS oncogene, previously proposed to be associated with breast cancer, was absent in these families, suggesting that polymorphism at this locus is not associated with inherited susceptibility. These results strongly suggest that oncogenes are not the sites of primary alterations leading to breast cancer. On the other hand, alterations in one or more of these sequences may be associated with tumor progression.