Filipin-dependent Inhibition of Cholera Toxin: Evidence for Toxin Internalization and Activation through Caveolae-like Domains

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside G_M1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A₁ peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-β-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit–mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.     

Quasielastic light-scattering studies on human fibrinogen and fibrin. II. Fibrin polymerization

The shape of fibrin intermediate polymers as well as the rate of fibrinogen polymerization was studied using diffusion measured by quasielastic light scattering. After their length distribution was narrowed by gel filtration, the polymers yielded translational and rotational diffusion coefficients of 0.37 ± 0.05 × 10^?7 cm² sec^?1 and 142 ± 32 sec^?1, respectively. Theoretical considerations indicated the polymers to be rigid rods. The rate of polymerization of fibrinogen monitored by diffusion paralleled that provided by simulataneous intensity measurements. Both monitors indicated polymerization occurs most rapidly at 30°C.     

Rearing and release of Homichloda barkeri (Jacoby) (Coleoptera: Chrysomelidae: Alticinae) for the biological control of prickly acacia, Acacia nilotica ssp. indica (Mimosaceae) in Australia

Abstract Prickly acacia, Acacia nilotica ssp. indica , a major weed of the Mitchell Grass Downs of northern Queensland, has been the target of biological control projects since the 1980s. The striped leaf-feeding beetle, Homichloda barkeri (Coleoptera: Chrysomelidae: Alticinae), was the third insect approved for release for the control of this weed in Australia. However, mass rearing this insect under glasshouse conditions proved to be difficult and time consuming as there were problems associated with low egg-hatching rates and poor larval survival. Eggs in diapause were stimulated to hatch by repeated wetting and drying. Larvae were fed on potted prickly acacia plants in cages. Late-instar larvae were collected and transferred to a mix of sand, peat moss and vermiculite for pupation and adult emergence. Over 10 500 adults were released at 28 sites in north Queensland between November 1996 and December 1999. Initial releases of insects confined to gauze cages resulted in limited adult survival, oviposition and development of first-generation larvae. First-generation larvae were observed at two sites where cages were not used. However, later inspections of release sites, including those made in April and December 2000, April 2001 and late March 2002, failed to find any trace of the insect, which is assumed to have failed to establish. Possible reasons for this failure are discussed.     

Effects of simulated shoot and leaf herbivory on vegetative growth and plant defense in Acacia drepanolobium

Plants have considerable ability to respond to herbivory, both with (above-ground) regrowth and with increased defense. We simulated both leaf and shoot herbivory in controlled, replicated experiments on individuals of Acacia drepanolobium in Laikipia, Kenya. These experiments were carried out on individuals that had experienced different, experimentally controlled histories of large mammalian herbivory. Both forms of simulated herbivory were associated with compensatory regrowth. Branches whose shoots had been removed grew significantly more over the next year than paired control branches, fully compensating for the lost shoot length. Branches whose leaves were removed both grew faster and had more leaves one year later than did control branches. Shoot removal, but not leaf removal, increased the production of side shoots. However, because past herbivore pressure was negatively associated with net shoot growth, there may be a long-term cost of herbivory even when plants appear to fully compensate for herbivory in the short term. In contrast to the effects on growth, simulated herbivory did not significantly increase physical (spines) or chemical (tannins) defenses, and there were no significant negative correlations between compensatory growth and plant defense.     

The reactivity of alpha-hydroxyhaem and verdohaem bound to haem oxygenase-1 to dioxygen and sodium dithionite.

Recently we have shown that ferric alpha-hydroxyhaem bound to haem oxygenase-1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196-18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric alpha-hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous alpha-hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203-213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. alpha-hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of alpha-hydroxyhaem with haem oxygenase may be small and a substantial amount of free alpha-hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air-sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH-cytochrome P450 reductase.     

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.     

MALE STERILITY IN SOYBEAN-AN OVERVIEW

The most common type of reproductive mutations observed in the soybean [Glycine max (L.) Merr.] are those that induce male sterility. The high frequency of occurrence of male-sterile mutations indicates that a number of genes influence the processes of microgametogenesis and microsporogenesis. The current knowledge of these mutations is summarized. The origins of male-sterile mutations, their inheritance patterns, and known linkage relationships are detailed. The phenotypic expression of male-sterile mutations, including their effects on both male and female reproduction, is discussed. The influence of environment on the expressivity of male-sterile mutations, and the effects of male-sterile mutations on physiological processes (senescence and nitrogen fixation) are summarized. Male-sterile mutations have been useful in the study of soybean reproduction, genetic and cytogenetic investigations, and in evaluating the potential for commercial production of hybrid soybeans. These various applications of male-sterile mutations are presented.     

Metazoans from a sandy aquifer: dynamics across a physically and chemically heterogeneous groundwater system

We investigated the relationship between groundwater metazoans and their physical and chemical environment in a shallow Atlantic Coastal Plain aquifer adjacent to the Chesapeake Bay, Maryland, USA. Average abundance of the groundwater organisms over a 1 % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaaSGaaeaaca% aIXaaabaGaaGOmaaaaaaa!3776!\[{\raise0.7ex\hbox{$1$} \!\mathord{\left/ {\vphantom {1 2}}\right.\kern-\nulldelimiterspace}\!\lower0.7ex\hbox{$2$}}\] year period were large (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaWaa0aaaeaaca% WG4baaaaaa!36FB!\[\overline x \] = 471⁻¹; range = 0–10001⁻¹) and included a wide range of taxa (nematodes, rotifers, copepods, oligochaetes, and others). Highest meiofaunal abundances occurred in the summer and fall with considerable variation across a study site that spanned hundreds of meters. We found that over 70% of the variability in the abundance of total meiofauna at Wye Island could be explained by date, sampling location, and conductivity. Additional physical and chemical factors (e.g., dissolved oxygen, nitrate, dissolved organic carbon) which were significantly related to faunal abundances, differed among taxa. Nematode abundances were negatively related to nitrate concentrations. Copepod and oligochaete abundances were highest at intermediate pH values (4–6). Copepods also occurred in higher abundances at higher conductivity (> 0.25 dS m ⁻¹). Rotifers were most abundant at higher oxygen values (> 6 mg l⁻¹). The high faunal abundances found in this sandy aquifer, and the degree to which such habitats are understudied (especially in North America), suggest a great need for additional research to elucidate factors that control faunal dynamics.