Excitation of skinned muscle fibers by imposed ion gradients. I. Stimulation of 45Ca efflux at constant [K][Cl] product

45Ca efflux from skinned muscle fibers is stimulated transiently, by a highly Ca2+-dependent mechanism, by KCl replacement of K propionate. In the present studies, Cl replaced the much less permeant anion methanesulfonate (Mes) either (a) at constant [K], in which increased [K][Cl] permits net KCl and water flux across internal membranes, or (b) at constant [K][Cl] (choline substitution), in which the imposed gradients and diffusion potentials should dissipate slowly. 45Ca efflux and isometric force were measured simultaneously on segments of frog semitendinosus fibers skinned by microdissection. EGTA was applied to chelate released 45Ca either (a) shortly after high [Cl] (interrupted response), to minimize reaccumulation, (b) before high [Cl] (pretreated response), to evaluate Ca2+ dependence, or (c) under control conditions in KMes. KCl replacement of KMes stimulated release of 65% fiber 45Ca within 1 min in interrupted responses; EGTA pretreatment was only moderately inhibitory with substantial residual stimulation. In contrast, choline Cl replacement of KMes induced release of 26-35% fiber 45Ca in interrupted responses; EGTA pretreatment was strongly inhibitory, but release significantly exceeded control with a small, sustained increase in Ca2+-insensitive efflux. These differences in 45Ca release and EGTA inhibition suggest that Cl replacement of Mes at constant [K] stimulates efflux by osmotic effects as well as imposed diffusion potentials; at least half the stimulated 45Ca loss (above control) in interrupted KCl responses is attributable to an osmotic component with low Ca2+ sensitivity. In the highly Ca2+-sensitive stimulation at constant [K][Cl], 45Ca release (above control) in interrupted responses correlated well with that in the pretreated responses of segments from the same fiber, with a slope of 8.4. This relationship suggests that imposed diffusion potentials stimulate a small Ca2+-insensitive component that gradates a much larger Ca2+-dependent efflux. The Ca2+-insensitive component apparently reflects intermediate steps in the excitation-contraction coupling that require positive feedback to result in sufficient Ca release for contraction.     

Proline excretion by Escherichia coli K12

Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion.     

Biotechnology for phosphorus removal during wastewater treatment

Advanced biological wastewater treatment for the removal of phosphorus in excess of the normal metabolic requirements of activated sludge type processes has been developed as an alternative to chemical addition. Current laboratory and pilot plant investigations have confirmed that a preliminary anaerobic zone and plug-flow type configuration are necessary for good enhanced biological phosphorus removal. Nitrate in the anaerobic stage inhibits the process whereas acetate enhances phosphorus uptake. The bacteria probably responsible are of the Acinetobacter genus and the presence of stored polyphosphate within these bacteria has been demonstrated. It has also been shown that pure cultures of Acinetobacter do not necessarily take up soluble substrate as phosphate is released during the anaerobic phase, in contrast to the current proposed mechanism, and that in certain cases natural chemical precipitation could make a significant contribution towards overall phosphorus removal. Several studies of pilot and full-scale plants have been reported.     

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.     

Overlapping of normal and malignant mouse cells in pure and mixed cultures

Cultured malignant mouse melanoma cells and mouse ascites carcinoma cells, after 24 h incubation with normal mouse cells or with each other, had much higher homologous overlap indices than those recorded from pure cultures of each cell type. The effect was particularly evident in ascites cells. Normal cells of mice of different ages showed varying responses in overlap frequency when incubated with malignant cells. Conditioned medium from each malignant cell type invoked increased overlapping of cells of the heterologous malignant type but did not affect homologous cells. The effects of whole conditioned medium on heterologous malignant cells showed a graded reduction after dilution of the medium and also occurred after dialysis. A dialysable chemical factor or factors, produced by the cells, is postulated.     

Concentrating Engines and the Kidney: II. Multisolute Central Core Systems

The analysis of the central core model of the renal medulla is extended to multisolute systems. It is shown that total solute concentration obeys the same differential equations for core and ascending limb as in a single solute system. Equations are derived for the concentration of individual solutes. Application of these equations to a two solute system shows that a central core system can concentrate with all transport being down a concentration gradient. This analysis applied to the renal medulla shows that mixing of urea from the collecting duct (CD) and salt from the loop of Henle in the central core of the inner medulla contributes to the concentration of urine during antidiuresis. It also sets criteria for completely passive function of the loop in the inner medulla, but whether these are satisfied cannot be determined from present experimental data.     

Volume-activated chloride permeability can mediate cell volume regulation in a mathematical model of a tight epithelium

Cell volume regulation during anisotonic challenge is investigated in a mathematical model of a tight epithelium. The epithelium is represented as compliant cellular and paracellular compartments bounded by mucosal and serosal bathing media. Model variables include the concentrations of Na, K, and Cl, hydrostatic pressure, and electrical potential, and the mass conservation equations have been formulated for both steady-state and time-dependent problems. Ionic conductance is represented by the Goldman constant field equation (Civan, M.M., and R.J. Bookman. 1982. Journal of Membrane Biology. 65:63-80). A basolateral cotransporter of Na, K, and Cl with 1:1:2 stoichiometry (Geck, P., and E. Heinz. 1980. Annals of the New York Academy of Sciences. 341:57-62.) and volume-activated basolateral ion permeabilities are incorporated in the model. MacRobbie and Ussing (1961. Acta Physiologica Scandinavica. 53:348-365.) reported that the cells of frog skin exhibit osmotic swelling followed by a volume regulatory decrease (VRD) when the serosal bath is diluted to half the initial osmolality. Similar regulation is achieved in the model epithelium when both a basolateral cotransporter and a volume-activated Cl permeation path are included. The observed transepithelial potential changes could only be simulated by allowing volume activation of the basolateral K permeation path. The fractional VRD, or shrinkage as percent of initial swelling, is examined as a function of the hypotonic challenge. The fractional VRD increases with increasing osmotic challenge, but eventually declines under the most severe circumstances. This analysis demonstrates that the VRD response depends on the presence of adequate intracellular chloride stores and the volume sensitivity of the chloride channel.     

Human monocytes selectively bind to cells expressing the tumorigenic phenotype

Summary The characteristics of the binding of human monocytes to tumor cells were studied by a newly developed microassay. First, we determined the kinetics and optimal conditions of the binding. Monocytes recognized and bound to tumor cells very rapidly within 10–20 min of cellular interaction. Binding was also more efficient at 37°C suggesting that active metabolism of monocytes is required. Second, we determined that selective binding of monocytes to cells with tumorigenic phenotypes occurs. For this purpose, lymphocytic leukemia cell lines versus normal lymphocytes, and tumorigenic versus nontumorigenic hybrids from the same parental lines were compared as the targets of the binding assay. In both cases, neoplastic cells were selectively bound by monocytes. Although tumor cells were bound rapidly and selectively by monocytes, initial recognition and binding did not necessarily lead to subsequent tumor cell lysis. This is based on the observation that some tumorigenic parental and hybrid lines were avidly bound by monocytes yet not subsequently killed in a cytotoxicity assay.This work was supported in part by a grant from the National Institutes of Health CA42992 and a grant from the Kleberg foundation Abbreviations used: [¹²⁵I]IdUrd [¹²⁵I]iododeoxyuridine; rIFN-, recombinant human interferon ; IL-1, interleukin 1; rTNF, recombinant human tumor necrosis factor     

Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.