Cloning of a cDNA encoding an Artemia franciscana Na/K ATPase alpha-subunit.

Clones of cDNA that code for an isoform of the Artemia franciscana Na/K ATPase alpha subunit (NaKA alpha) have been isolated. The sequence of the longest of these clones (pArATNa136) is 3595 nucleotides; it codes for a 1004-amino acid protein whose sequence is identical to that of two previously sequenced Artemia NaKA alpha peptides. The encoded protein is over 73% identical to Drosophila melanogaster and vertebrate NaKA alpha s, and 73.8% identical to another Artemia NaKA alpha isoform previously described (named alpha 2850 in this article). The two Artemia cDNA clones code for mRNAs of different size; the clone pArATNa136 codes for a 4.5-kb mRNA while the alpha 2850 clone codes for a 3.6-kb mRNA. The degree of homology and the different size of the mRNAs encoded by both cDNAs suggest that they code for two different isoforms of the protein.     

Effects of progesterone-impregnated sponge treatment on peripheral plasma hormone levels and fertility in the cyclic ewe

Cyclic ewes were treated with 500-mg progesterone-impregnated sponges for the synchronization of oestrus. In the first experiment, the sponges were removed from 79 ewes at intervals over a 17-day treatment and the residual amount of progesterone was measured in order to assess the rate of absorption of the hormone from the sponges by the animals. The residual progesterone was found to decrease linearly with the duration of sponge insertion but there was also a significant quadratic component indicative of a slowing down in the rate of progesterone absorption towards the end of the treatment period. In the second experiment, 13 cyclic ewes were treated with 500-mg progesterone sponges for 17 days and the eight ewes in oestrus following spongewithdrawal were mated. The peripheral plasma progesterone was assayed at intervals during sponge insertion and at weekly intervals after sponge withdrawal. The residual progesterone levels on the sponges and the plasma progesterone levels of the treated ewes were examined in relation to their oestrous response and fertility. There was a significantly higher residual level of hormone remaining on sponges from ewes that mated than on sponges from those that did not (P < 0.01). The 13 ewes exhibited luteal phase levels of plasma progesterone when assayed during the period of sponge insertion regardless of their response to treatment. The mated, fertile ewes had significantly higher plasma hormone levels than the non-mated and the mated infertile ewes, after sponge withdrawal.     

Investigation of the regulation of pigmentation in alpha-melanocyte-stimulating hormone responsive and unresponsive cultured B16 melanoma cells

In vitro melanocyte-stimulating hormone (MSH) stimulates melanogenesis in some, but not all, melanocytes and melanoma cells. In an attempt to explain this variation in response to alpha MSH, we examined cyclic adenosine monophosphate (cAMP) accumulation, tyrosinase activity, and melanin production in primary (1 degree) murine B16 melanoma cells and in two B16 cell lines (B16 F1 and B16 F10) that are known to respond to alpha MSH. In vivo all three B16 melanoma cell types produced pigmented tumours. In vitro alpha MSH increased tyrosinase activity and melanin content in the F1 and F10 cells but not in the B16 1 degree cells. alpha MSH, however, increased cAMP production in all three cell types, confirming that the inability of B16 1 degree cells to produce melanin in response to alpha MSH is not due to a lack of alpha MSH receptors or cAMP response to alpha MSH. Further, we present evidence for a separate pathway of melanogenesis that is independent of cAMP as calmodulin antagonists, which do not elevate cAMP, increased tyrosinase activity, and melanin production in both 1 degree and F1 cells.     

Can the biotic nestedness matrix be used predictively?

The biotas of a suite of neighboring patches of remnant vegetation often form a series of nested sub-sets, in which the species present in species-poor patches are non-random sub-sets of those present in richer patches. There has been recent interest in ways in which this knowledge may be used to aid conservation. We focus here on whether nested patterns can be used predictively. If nestedness in a fragmented system increases over time through biotic relaxation, locations where particular species may become extinct or are likely to colonize might be predictable and this could be useful in threatened-species management. We used the Temperature Calculator of Atmar and Patterson (1995) to arrange a matrix of bird species' occurrences in a series of buloke Allocasuarina leuhmannii woodland remnants so that nestedness was maximized. Probability bands generated by the calculator were used to predict possible colonization and extinction events. We then re-surveyed the avifauna of the fragments after a seven-year interval to test these predictions. Although nestedness increased between the two survey periods, there was no linear relationship between the generated probability of extinctions or colonizations and the accuracy of the predictions. The predictions derived from the calculator were no more accurate than a second set of predictions generated by use of a simple non-nested model. Despite the increase in nestedness, the arrangement of sites in each of the two maximally packed matrices was substantially different. For the nestedness matrix to generate accurate predictions, an increase in nestedness must be due to a minimization of unexpected species presences and absences rather than an extensive redistribution of species among remnants, as we found. The potential utility of nested patterns in predicting systematic colonization and extinction events should be further evaluated in other, less dynamic, fragmented systems such as those undergoing biotic relaxation.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

Automatic gas meter for laboratory fermenters

Summary A versatile, cheap and automated system is designed for the measurement of small gas flows produced in laboratory-scale fermentation processes. An automatic sampling device for programmed times is linked to the flow meter. The displacement of a liquid by the gas being measured is the principle on which both the meter and the sampling device are based. The operation of the system is controlled by a simple electronic circuit.     

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

Secretion of melanophore-stimulating hormone (MSH) in long-term cultures of pituitary neurointermediate lobes

Summary Neurointermediate lobes from pituitaries of the frog, Rana berlandieri forreri (Rana pipiens, sensu lato), were maintained in organ culture in media with and without serum for up to six months. The cultured tissues were examined periodically by light microscopy and transmission electron microscopy and by bioassay of the melanophore-stimulating hormone (MSH) secreted and present in the culture media. Light-microscopic observations revealed a high degree of preservation of the pars intermedia at four weeks with isolated areas of some glands maintaining histological integrity for the entire six months. Similarly, at the ultrastructural level the cells appeared morphologically intact and to be actively synthesizing and secreting hormone. Bioassays showed the glands to be continuously secreting MSH; however, larger yields of hormone were obtained in media lacking serum. No significant ultrastructural differences between cells grown in the presence or absence of serum were detected. The difference in concentration of MSH between the two groups therefore apparently results from enzymatic degradation of the hormone by the serum. Organ culture of the vertebrate neurointermediate lobe may provide a unique method for the production of large quantities of MSH and for the study of other melanotropic and opiate peptides as they may be synthesized and secreted by the pars intermedia.