The biology and morphology of the marine harpacticoid copepod Heteropsyllus nunni Coull, during encystment diapause

Heteropsyllus nunni Coull, a meiobenthic harpacticoid copepod is the marine crustacean to undergo a state of diapause within a cyst. A 12 month field study indicated H. nunni adults reached peak population densities in winter, with nauplii maturing in the spring, becoming adults by April or May.At the last stage of development, a mature but unmated adult, they begin to prepare for encystment diapause. The copepods remain within their cyst in a state of diapause for 3–4 months during the summer only. Studies on the effects of temperature and photoperiod suggested that these two environmental cues are not crucial for induction or termination of diapause. Low temperature delayed development and time to encystment, while high temperatures accelerated development, making the time to encystment shorter. There were males than females in the cysts in laboratory experiments. Upon excystment, the copepods mate, and females begin egg production within one week. Adults that have excysted and mated die after a few weeks of active reproductive effort. Nauplii go on to mature and begin the univoltine diapause/reproductive cycle.The copepods prepare for dormancy in two ways: they begin to produce and store two types of secretory products to be used in cyst construction; then they produce large quantities of lipid to be used as a nutrient supply throughout diapause. Histochemistry of the cyst-building material indicated the lower urosome is full of two chemically different products. Dorsally, there is a storage sac of proteinaceous material. The ventral sac of secretory product is a mucopolysaccharide. The copepod builds the spherical cysts in a matrix of small and large sand grains. The cysts fit tightly around the ventral portion of the animal in the its flexed position: however, there is a large space between the cyst and the sides of the copepod.Biochemical analysis of the cyst showed it is composed of an amino acid complex similar to collagenous material. Scanning electron microscopy revealed a complex of large cuticular pores located in the lower urosome and caudal rami. There are specific pores for secretion of the two cyst-building products.     

Long-Term Effect of Forskolin on the Activation of Adenylyl Cyclase in Astrocytes

Abstract: Long-term (48-h) forskolin treatment of rat astroglial cells led to a slight decrease (30–40%) in the response to isoproterenol, vasoactive-intestinal peptide, guanyl 5'-(βγ-imido)diphosphate, guanosine 5'- O -(3-thiotriphosphate) [GTP(S)], and AIF₄⁻ in crude membrane fractions. In contrast, the acute stimulatory effect of forskolin was increased by 1.25–1.5-fold. These two opposite effects of forskolin were mediated by a cyclic AMP-dependent mechanism. No changes in G_sα, G_iα, or Gβ protein levels could be determined by immunoblotting using specific antisera. No significant differences were observed in the ability of G proteins extracted from control and forskolin-treated cells to reconstitute a full adenylyl cyclase activity in membranes from S49 cyc⁻ cells, lacking G_sα protein. G_sα proteins were detected in two pools of membranes, one in the heavy sucrose fractions and the other in light sucrose fractions. Forskolin treatment of the cells shifted G_sα protein toward the light-density membranes. We did not find any significant change in the distribution of adenylyl cyclase. In contrast to the decreased stimulation of adenylyl cyclase activity by agonists acting via G_sα, observed in the crude membrane fraction, the responses of adenylyl cyclase to forskolin as well as to GTP(S) were increased in the purified plasma membrane fractions. These results may indicate that sensitization of the catalyst appears to be the dominant component in the astroglial cell response to long-term treatment by forskolin.     

Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoanParamecium tetraurelia

We have cloned the first bifunctional gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) from a free-living, ciliated protozoan,Paramecium tetraurelia, and determined its macronuclear sequence using a modified ligation-mediated polymerase chain reaction (PCR) that can be of general use in cloning strategies, especially where cDNA libraries are limiting. While bifunctional enzyme sequences are known from parasitic protozoa, none had previously been found in free-living protozoa. The AT-rich (68%) coding region spanning 1386 bp appears to lack introns. DHFR-TS localizes to a 500 kb macronuclear chromosome and is transcribed as an mRNA of 1.66 kb, predicted to encode a 53 kDa protein of 462 residues. The N-terminal one-third of the protein is encoded by DHFR, which is joined by a short junctional peptide of 12 amino acids to the highly conserved C-terminal TS domain. Among known DHFR-TS sequences, theP. tetraurelia gene is most similar to that fromToxoplasma gondii, based on primary sequence and parsimony analyses. The predicted secondary protein structure is similar to those of previously crystallized monofunctional sequences.     

Molecular Cloning, Expression Pattern, and Chromosomal Localization of the Human Na–Cl Thiazide-Sensitive Cotransporter (SLC12A3)

Electrolyte homeostasis is maintained by several ion transport systems. Na–(K)–Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na–(K)–Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na–Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na–(K)–Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The ex- pression pattern of the human Na–Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescencein situhybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.     

Uptake and transformation of metals and metalloids by microbial mats and their use in bioremediation

Summary Constructed microbial mats, used for studies on the removal and transformation of metals and metalloids, are made by combining cyanobacteria inoculum with a sediment inoculum from a metal-contaminated site. These mats are a heterotrophic and autotrophic community dominated by cyanobacteria and held together by slimy secretions produced by various microbial groups. When contaminated water containing high concentrations of metals is passed over microbial mats immobilized on glass wool, there is rapid removal of the metals from the water. The mats are tolerant of high concentrations of toxic metals and metalloids, such as cadmium, lead, chromium, selenium and arsenic (up to 350 mg L^–1). This tolerance may be due to a number of mechanisms at the molecular, cellular and community levels. Management of toxic metals by the mats is related to deposition of metal compounds outside the cell surfaces as well as chemical modification of the aqueous environment surrounding the mats. The location of metal deposition is determined by factors such as redox gradients, cell surface micro-environments and secretion of extra-cellular bioflocculents. Metal-binding flocculents (polyanionic polysaccharides) are produced in large quantities by the cyanobacterial component of the mat. Steep gradients of redox and oxygen exist from the surface through the laminated strata of microbes. These are produced by photosynthetic oxygen production at the surface and heterotrophic consumption in the deeper regions. Additionally, sulfur-reducing bacteria colonize the lower strata, removing and utilizing the reducing H₂S, rather than water, for photosynthesis. Thus, depending on the chemical character of the microzone of the mat, the sequestered metals or metalloids can be oxidized, reduced and precipitated as sulfides or oxides. For example precipitates of red amorphous elemental selenium were identified in mats exposed to selenate (Se-VI) and insoluble precipitates of manganese, chromium, cadmium, cobalt, and lead were found in mats exposed to soluble salts of these metals. Constructed microbial mats offer several advantages for use in the bioremediation of metal-contaminated sites. These include low cost, durability, ability to function in both fresh and salt water, tolerance to high concentrations of metals and metalloids and the unique capacity of mats to form associations with new microbial species. Thus one or several desired microbial species might be integrated into mats in order to design the community for specific bioremediation applications.     

Maintenance of Weight Loss: A Needs Assessment

This study identified facilitators and obstacles to maintenance of weight loss following a very-low-calorie-diet and behavior modification program. A survey was mailed to a random sample of 178 program completers and received a 61% response rate; the most frequent follow-up period was more than 2 years. Twenty-nine percent reported weighing the same (within 10 lbs) or less than the end of their participation in the treatment program (maintainers), while 71% reported their present weight was a mean of 65% higher than their initial weight loss (regainers). Maintainers were significantly more likely to report engaging in regular aerobic exercise, attending a maintenance support group, and confidence in their ability to manage their weight in the future, while regainers were more likely to report stress and motivation as frequent weight management obstacles. Respondents consistently identified the need for low/no cost ongoing support. Maintainers and relapsers reported similar challenges in managing their weight, yet with different results, suggesting the need to identify subgroups for which different post-treatment support options could be applied.     

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry

Summary The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90°) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40–60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained as control cells progress through the cell cycle. At later times postinfection (>42 h), total protein decreased due to cellular changes resulting from viral replication and cell death.