Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

Immune unresponsiveness to secondary heterologous bacterial infection after sepsis induction is TRAIL dependent

Sepsis is the leading cause of death in most intensive care units, and patients who survive the hyperinflammation that develops early during sepsis later display severely compromised immunity. Not only is there apoptosis of lymphoid and myeloid cells during sepsis that depletes these critical cellular components of the immune system, but also the remaining immune cells show decreased function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and induction of sepsis-induced suppression of delayed-type hypersensitivity. The present study extends this earlier work to include a secondary heterologous bacterial infection (OVA(257)-expressing Listeria monocytogenes [LM-OVA]) subsequent to sepsis initiation to investigate sepsis-induced alterations in the control of this secondary infection and the associated naive Ag-specific CD8 T cell response. We found that CLP-treated wild-type (WT) mice had a reduced ability to control the LM-OVA infection, which was paralleled by suppressed T cell responses, versus sham-treated WT mice. In contrast, CLP-treated Trail(-/-) and Dr5(-/-) mice were better able to control the secondary bacterial infection, and the Ag-specific CD8 T cell response was similar to that seen in sham-treated mice. Importantly, administration of a blocking anti-TRAIL mAb to CLP-treated WT mice was able to restore the ability to control the LM-OVA infection and generate Ag-specific CD8 T cell responses like those seen in sham-treated mice. These data further implicate TRAIL-dependent immune suppression during sepsis and suggest TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.     

Marine reserves help coastal ecosystems cope with extreme weather

Natural ecosystems have experienced widespread degradation due to human activities. Consequently, enhancing resilience has become a primary objective for conservation. Nature reserves are a favored management tool, but we need clearer empirical tests of whether they can impart resilience. Catastrophic flooding in early 2011 impacted coastal ecosystems across eastern Australia. We demonstrate that marine reserves enhanced the capacity of coral reefs to withstand flood impacts. Reserve reefs resisted the impact of perturbation, whilst fished reefs did not. Changes on fished reefs were correlated with the magnitude of flood impact, whereas variation on reserve reefs was related to ecological variables. Herbivory and coral recruitment are critical ecological processes that underpin reef resilience, and were greater in reserves and further enhanced on reserve reefs near mangroves. The capacity of reserves to mitigate external disturbances and promote ecological resilience will be critical to resisting an increased frequency of climate‐related disturbance.     

Effect of gender on the development of hypocapnic apnea/hypopnea during NREM sleep.

We hypothesized that a decreased susceptibility to the development of hypocapnic central apnea during non-rapid eye movement (NREM) sleep in women compared with men could be an explanation for the gender difference in the sleep apnea/hypopnea syndrome. We studied eight men (age 25-35 yr) and eight women in the midluteal phase of the menstrual cycle (age 21-43 yr); we repeated studies in six women during the midfollicular phase. Hypocapnia was induced via nasal mechanical ventilation for 3 min, with respiratory frequency matched to eupneic frequency. Tidal volume (VT) was increased between 110 and 200% of eupneic control. Cessation of mechanical ventilation resulted in hypocapnic central apnea or hypopnea, depending on the magnitude of hypocapnia. Nadir minute ventilation in the recovery period was plotted against the change in end-tidal PCO(2) (PET(CO(2))) per trial; minute ventilation was given a value of 0 during central apnea. The apneic threshold was defined as the x-intercept of the linear regression line. In women, induction of a central apnea required an increase in VT to 155 +/- 29% (mean +/- SD) and a reduction of PET(CO(2)) by -4.72 +/- 0.57 Torr. In men, induction of a central apnea required an increase in VT to 142 +/- 13% and a reduction of PET(CO(2)) by -3.54 +/- 0.31 Torr (P = 0.002). There was no difference in the apneic threshold between the follicular and the luteal phase in women. Premenopausal women are less susceptible to hypocapnic disfacilitation during NREM sleep than men. This effect was not explained by progesterone. Preservation of ventilatory motor output during hypocapnia may explain the gender difference in sleep apnea.     

Intracellular polyamine biosynthesis is required for interleukin 2 responsiveness during lymphocyte mitogenesis

The objective of the present investigation was to define a more precise role for intracellular polyamine biosynthesis with respect to specific inducible events which regulate lymphocyte mitogenesis. In this regard, we have examined the effect of polyamine depletion on interleukin 2 (IL-2) production, receptor expression, and responsiveness in Con A stimulated mononuclear leukocytes (MNL). Polyamine depletion was achieved utilizing the specific irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO). Polyamine depletion of MNL augmented detectable levels of Con A-induced IL-2 activity. In contrast, the ability of polyamine depleted MNL to respond to saturating levels of IL-2 (100 U/ml) following 72 or 96 hr of Con A stimulation was reduced 100 and 81%, respectively. Nonetheless, polyamine depletion did not impair the induction of IL-2 receptor expression. High-affinity IL-2 receptor density in the polyamine depleted population was greater than control cells late in culture (96 hr). The expression of high-affinity IL-2 receptors did not correlate with an ability to respond to IL-2 in the polyamine depleted population. The results of this study demonstrate for the first time that intracellular polyamine biosynthesis is required for IL-2 responsiveness during a primary mitogenic lymphocyte response.     

Examination and characterization of distribution system biofilms

Investigations concerning the role of distribution system biofilms on water quality were conducted at a drinking water utility in New Jersey. The utility experienced long-term bacteriological problems in the distribution system, while treatment plant effluents were uniformly negative for coliform bacteria. Results of a monitoring program showed increased coliform levels as the water moved from the treatment plant through the distribution system. Increased coliform densities could not be accounted for by growth of the cells in the water column alone. Identification of coliform bacteria showed that species diversity increased as water flowed through the study area. All materials in the distribution system had high densities of heterotrophic plate count bacteria, while high levels of coliforms were detected only in iron tubercles. Coliform bacteria with the same biochemical profile were found both in distribution system biofilms and in the water column. Assimilable organic carbon determinations showed that carbon levels declined as water flowed through the study area. Maintenance of a 1.0-mg/liter free chlorine residual was insufficient to control coliform occurrences. Flushing and pigging the study area was not an effective control for coliform occurrences in that section. Because coliform bacteria growing in distribution system biofilms may mask the presence of indicator organisms resulting from a true breakdown of treatment barriers, the report recommends that efforts continue to find methods to control growth of coliform bacteria in pipeline biofilms.     

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2g/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM _r , ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.     

Forage variation in brood-rearing areas used by pacific black brant geese on the Yukon-Kuskokwim delta, Alaska

1 We investigated the effects of grazing by black brant geese on Carex subspathacea lawns on the Yukon-Kuskokwim delta, Alaska.
2 We compared variation in growth and forage quality in both grazed and temporarily exclosed sites to determine responses of C. subspathacea to grazing at landscape scales within two nesting colonies that had experienced different population dynamics over recent decades.
3 Landscapes differed in forage quality, grazing patterns, and in the effect grazing had on C. subspathacea forage characteristics. We found no effect of grazing on net above-ground primary productivity ( NAPP ) over a wide range of natural grazing intensities at the landscape scale.
4 No differences in forage quality, NAPP , or response of C. subspathacea growth rates to grazing pressures could be detected between colonies. This suggests that goose grazing does not have deleterious effects on C. subspathacea in this ecosystem.
5 It has been suggested that gosling growth rates are sensitive to seasonal declines in forage availability and quality. Spatial variation in forage quality and availability per sampled area exceeded seasonal variation in these characteristics and is likely to have dramatic effects on gosling growth and recruitment rates.