Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

Crystallization of a small fragment of an aminoacyl tRNA synthetase

Single crystals of an amino-terminal fragment of Escherichia coli alanine tRNA synthetase have been prepared by the vapor diffusion method. The fragment extends to amino acid residue 368 and catalyzes the synthesis of alanyl adenylate. The crystals grow in the presence of alanine as rhombic plates in space group P2(1)2(1)2(1) and with unit cell dimensions of a = 67.9 A, b = 98.5 A and c = 123.6 A (1 A = 0.1 nm). They diffract to better than 3 A resolution.     

Life History Studies on the Genus Trillium (Liliaceae) V. Reproductive Biology and Survivorship of Three Declinate-flowered Species

Absract Three pedicellate-declinate-flowered species of Trillium (Liliaceae), T. vaseyi and T. flexipes , were studied for their life history characteristics, e.g., stage class structures of natural populations and reproductive features, including energy allocation to reproductive activities. The populations structures of all three species showed similar depletion structures characterized by a conspicuous decrease of individuals in the small juvenile stages, as was also observed in pedicellate-erect-flowered Trillium species However, with respect to reproductive characteristics, these three declinate-flowered species showed different features from erect-flowered species, although they belong to the same pedicellate-flowered group. That is, these declinate-flowered Trillium species exhibited low seed setting rates of 30% in T. catesbaei , 45% in T. vaseyi and 34% in T. flexipes , suggesting that they possess different mating systems from erect-flowered species which showed high seed setting rates of 50–90%.     

Relocation and reorganization of germ plasm in Xenopus embryos after fertilization

In the unfertilized egg, germ plasm is widely distributed throughout the vegetal subcortex in small islets. Following fertilization or artificial activation, the location and organization changes, and by the 4- to 8-cell stage the germ plasm forms a small number of large patches overlying the vegetal pole. We distinguish three processes that produce these changes. The first of these is aggregation which involves the islets moving towards the vegetal pole to form large patches by coalescence. This phase requires microtubules but does not depend on cleavage or dynamic microfilaments. The second phase is ingression during which the patches of germ plasm move to the interior of the egg. The movement is due to a flow of cytoplasm from the vegetal pole internally and the cytoplasmic current does not require either microtubules or dynamic microfilaments. In the third phase, the germ plasm is trapped in the vegetal hemisphere by microtubular arrays--in normal development, the mitotic spindle.     

Bias in leaf area — sapwood area ratios and its impact on growth analysis in Pinus contorta

Summary Two alternative estimators of individual tree leaf area (A₁) area are used to derive estimates of leaf-area index (L) for 40 plots in Pinus contorta Dougl. stands. One estimator of A₁ is based on the common assumption of a constant ratio between A₁ and sapwood cross-sectional area at breast height (A_s). The second estimator of A₁ accounts for tree-to-tree variation in the relation between A₁ and A_s. The apparent relationship between stand growth and leaf-area index is strongly dependent on the way leaf area is estimated. When L is derived from a constant A₁A_s ratio, stand growth appears to be strongly correlated with L. However, when L is based on estmates of A₁ that account for tree-to-tree variation in the A₁ — A_s relation, stand growth is seen to be only weakly related to L. Stand structure, quantified as percent live-crown, accounts for a great deal of the observed variation in leaf-area efficiency. These contrasting relationships illustrate the importance of unbiased estimates of L in interpreting the link between stand-level processes and leaf area.Utah Aggriculural Experiment Station Journal Paper No. 3333     

Selling research, and it pays

While conceding that time has proven him wrong in doubting public acceptance of the idea of the "science shops," first sponsored by the Free University of Amsterdam to provide access to technical expertise for customers who could not afford more orthodox services, the author remains convinced that the somewhat analogous concept of "fee for service research," which offers experimental medical treatments for those who can afford to pay for them, is wrong. Such research has been justified as a private sector enterprise that provides patients who are unresponsive to standard forms of therapy with scientifically sound experimental options. However, the objections that such work might be kept secret and not contribute to medical knowledge, and that therapy would be available only to the rich, remain unanswered and pose an insurmountable obstacle to the support of this idea.     

Human renal clear cell carcinoma: Establishment and characterization of a new cell line (G-2101)

Summary A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simulataneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population. This work was supported in part by a grant from Triton Biosciences, Inc.     

Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin