The structure and dimensions of redds and egg pockets of the endangered salmonid, Sakhalin taimen

Each redd of the Sakhalin taimen Hucho perryi had a V–shaped pot that is specific to this species. The shape of each multiple–egg–pocket redd was a combination of one–egg–pocket redds, and the number of egg pockets in the redd could be estimated from the shape. False redds were small and did not have any V–shaped pots. The factors affecting redd size were examined numerically; consequently, numbers of egg pockets and eggs and female fork length were correlated significantly with tail length. Each egg pocket contained 11–920 eggs (mean: 546·7±95% CL 81·9) and the egg pocket depth was correlated significantly with female fork length. Mean number of redds per female was three, and the number of females could be estimated as one third of the redds.     

Weight Loss,Weight Maintenance,and Improved Cardiovascular Risk Factors after 2 Years Treatment with Orlistat for Obesity

Objective: To determine the effect of orlistat, a new lipase inhibitor, on long‐term weight loss, to determine the extent to which orlistat treatment minimizes weight regain in a second year of treatment, and to assess the effects of orlistat on obesity‐related risk factors. Research Methods and Procedures: This was a 2‐year, multicenter, randomized, double‐blind, placebo‐controlled study. Obese patients (body mass index 28 to 43 kg/m²) were randomized to placebo or orlistat (60 or 120 mg) three times a day, combined with a hypocaloric diet during the first year and a weight maintenance diet in the second year of treatment to prevent weight regain. Changes in body weight, lipid profile, glycemic control, blood pressure, quality of life, safety, and tolerability were measured. Results: Orlistat‐treated patients lost significantly more weight (p < 0.001) than placebo‐treated patients after Year 1 (6.6%, 8.6%, and 9.7% for the placebo, and orlistat 60 mg and 120 mg groups, respectively). During the second year, orlistat therapy produced less weight regain than placebo (p = 0.005 for orlistat 60 mg; p < 0.001 for orlistat 120 mg). Several obesity‐related risk factors improved significantly more with orlistat treatment than with placebo. Orlistat was generally well tolerated and only 6% of orlistat‐treated patients withdrew because of adverse events. Orlistat leads to predictable gastrointestinal effects related to its mode of action, which were generally mild, transient, and self‐limiting and usually occurred early during treatment. Discussion: Orlistat administered for 2 years promotes weight loss and minimizes weight regain. Additionally, orlistat therapy improves lipid profile, blood pressure, and quality of life.     

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pH_c) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pH_c and the central nutrient signalling cAMP‐Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pH_c correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pH_c showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.     

Decreased Affinity of Recombinant Human Tumor Necrosis Factor-related Apoptosis-inducing Ligand (rhTRAIL) D269H/E195R to Osteoprotegerin (OPG) Overcomes TRAIL Resistance Mediated by the Bone Microenvironment

The bone marrow microenvironment provides important signals for the survival and proliferation of hematopoietic and malignant cells. In multiple myeloma, plasma cells are surrounded by stromal cells including osteoblasts. These stromal cells protect multiple myeloma cells from apoptosis induced by chemotherapeutic agents. Osteoprotegerin (OPG), a soluble receptor of the cytokine TNF-related apoptosis-inducing ligand (TRAIL), is secreted by osteoblasts and has been implicated in the prevention of cell death induced by TRAIL in malignant cells. Previously, we have designed death receptor-specific TRAIL variants that induce apoptosis exclusively via one of its death receptors. Here, we have studied in detail the interaction between recombinant human (rhTRAIL) variants and OPG. We show that a DR5-specific variant (rhTRAIL D269H/E195R) displays a significantly decreased affinity to OPG. Furthermore, this rhTRAIL variant shows a much higher activity when compared with rhTRAIL WT and retains its effectiveness in inducing cell death in multiple myeloma cell lines, in the presence of OPG secreted by stromal cells. We also demonstrate that stromal cells are largely insensitive to high concentrations of this rhTRAIL variant. In conclusion, rhTRAIL D269H/E195R is a potential therapy for multiple myeloma due to its high effectiveness and diminished binding to OPG.     

Phosphatidic Acid (PA)-preferring Phospholipase A1 Regulates Mitochondrial Dynamics

Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A₁ (PA-PLA₁)/DDHD1 is the first identified intracellular phospholipase A₁ and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA₁ regulates mitochondrial dynamics. PA-PLA₁, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA₁ on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA₁ in testis, PA-PLA₁ knock-out mice have a defect in sperm formation. In PA-PLA₁-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA₁ in the organization of mitochondria during spermiogenesis.     

Retention of estradiol negative feedback relationship to LH predicts ovulation in response to caloric restriction and weight loss in obese patients with polycystic ovary syndrome

The present study tests the hypothesis that specific endocrine, metabolic, and anthropometric features distinguish obese women with polycystic ovary syndrome (PCOS) who resume ovulation in response to calorie restriction and weight loss from those who do not. Fifteen obese (body mass index 39 +/- 7 kg/m(2)) hyperandrogenemic oligoovulatory patients undertook a very low calorie diet (VLCD), wherein each lost > or =10% of body weight over a mean of 6.25 mo. Body fat distribution was quantitated by magnetic resonance imaging. Hormones were measured in the morning at baseline, after 1 wk of VLCD, and after 10% weight loss. To monitor LH release, blood was sampled for 24 h at 10-min intervals before intervention and after 7 days of VLCD. Responders were defined a priori as individuals exhibiting two or more ovulatory cycles in the course of intervention, as corroborated by serum progesterone concentrations > or =18 nmol/l followed by vaginal bleeding. At baseline, responders had a higher sex hormone-binding globulin (SHBG) concentration but were otherwise indistinguishable from nonresponders. Body weight, the size of body fat depots, and plasma insulin levels declined to a similar extent in responders and nonresponders. Also, SHBG increased, and the free testosterone index decreased comparably. However, responders exhibited a significant decline of circulating estradiol concentrations (from 191 +/- 82 to 158 +/- 77 pmol/l, means +/- SD, P = 0.037) and a concurrent increase in LH secretion (from 104 +/- 42 to 140 +/- 5 U.l(-1).day(-1), P = 0.006) in response to 7 days of VLCD, whereas neither parameter changed significantly in nonresponders. We infer that evidence of retention of estradiol-dependent negative feedback on LH secretion may forecast follicle maturation and ovulation in obese patients with PCOS under dietary restriction.     

Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell–B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca²⁺ levels, and subsequently in the activation of Ca²⁺/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 · N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Δ1–65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Δ1–65)- or Rac1 · V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation of CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Δ1–65)-and Rac1 · V12-mediated signals. We propose a model for the CD5-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.