An influx of extracelluar calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid

The role of Ca²⁺ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura-2. Previous experiments have shown that a Sephadex G-75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107–121]. Using fura-2, we demonstrated that this Sephadex G-75 fraction also stimulates a rapid, transient increase in intracellular free Ca²⁺. This Ca²⁺ transient is blocked either by chelation of extracellular calcium or by addition of the Ca²⁺ antagonist La³⁺. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G-75 fraction, or ionomycin are all blocked by removal of extracellular Ca²⁺. These results strongly suggest that an influx of extracellular Ca²⁺ is responsible for intiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca²⁺ concomitant with the acrosome reaction.     

Oxidative Damage to DNA and Deoxyribose by β-Lactam Antibiotics in the Presence of Iron and Copper Salts

β-lactam antibiotics in the presence of certain metal ions damage deoxyribose and DNA with the release of thiobarbituric acid-reactive material. This damage can be substantially prevented by catalase, metal chelators and some scavengers of the hydroxyl radical. Ferric salts in the presence of certain β-lactam antibiotics were effective in degrading deoxyribose but they did not appear to damage DNA. In contrast copper salts and p-lactam antibiotics were extremely effective in damaging both DNA and deoxyribose.     

Opposing effects of heparin with TGF-beta or aFGF during repair of a mechanical wound of human endothelium. Influence of cAMP on cell migration.

The effects on vascular wound repair in vitro of aFGF and TGF-beta, growth factors having opposite influences on endothelial cell growth and angiogenesis, were studied using as a model a mechanical lesion of confluent endothelium. Modulation by heparin of the activities of these growth factors during the repair process was also examined. Whereas heparin alone inhibited repair by lowering both cell proliferation and cell migration, TGF-beta alone mainly inhibited cell proliferation. When added together, TGF-beta and heparin exerted a combined inhibitory effect resulting in a residual lesion 50% larger than in controls. aFGF alone accelerated lesion coverage and this effect was enhanced by 40% over control values when heparin was added with aFGF. This acceleration was slightly (less than 10%) but consistently diminished by TGF-beta. Cell density in confluent unwounded areas was increased by 40% in the presence of aFGF, but TGF-beta diminished cell density by 20%. A small (30%) increase in intracellular cAMP was measured whenever aFGF was present during the repair process. In comparison, intracellular cAMP inducing agents (forskolin, dbcAMP) accelerated cell migration by 20% during lesion recovery without affecting cell proliferation or density. The present results show that the inhibitory effects of TGF-beta during vascular wound repair are opposed by aFGF. Furthermore, heparin (or heparan sulfates in vivo) modulates growth factors having activating or inhibiting functions and thus plays a regulatory role during the repair process. cAMP-inducing substances other than growth factors are able to accelerate cell migration.     

Liver peroxisomes in newborns from clofibrate-treated rats. II. A biochemical study of the recovery period.

The fatty-acyl-CoA beta-oxidation (FAO) and catalase activities, as well as membrane fluidity of liver peroxisomes of newborns from normal and clofibrate-treated rats were studied during the recovery period, ie, throughout the first week of postnatal life. In the test animals the enzyme activities, which are significantly higher than controls at birth return to normal levels showing a somewhat different time course with FAO rapidly decreasing to control values within three days but with catalase still higher than controls at day 6. The half-life and degradation rate (Kd) of FAO are identical to those calculated by us for the whole organelles and to those reported by others for total catalase in normal or clofibrate-treated adult animals in the presence of catalase inhibitors. Soluble catalase shows turnover values which are similar though not identical to those of FAO, while total catalase has a very long half-life and a low Kd. Peroxisomal membrane fluidity, as determined by fluorescence anisotropy of 1-anilinonaphthalene-8-sulfonate (ANS) bound to purified peroxisomal fractions is higher in tests than in controls, recovering normal values within 6 days. Our results demonstrate that liver peroxisomes of rats prenatally exposed to clofibrate return to control conditions within about 1 week. The turnover parameters of enzymes and the membrane fluidity values are discussed in terms of disposal mechanism(s) for the excess of induced peroxisomes.     

Adaptive foraging by predators as a cause of predator-prey cycles

Summary When foraging has costs, it is generally adaptive for foragers to adjust their foraging effort in response to changes in the population density of their food. If effort decreases in response to increased food density, this can result in a type-2 functional response; intake rate increases in a negatively accelerated manner as prey density increases. Unlike other mechanisms for type-2 responses, adaptive foraging usually involves a timelag, because foraging behaviours do not often change instantaneously with changes in food density or risks. This paper investigates predator-prey models in which there are explicit dynamics for the rate of adaptive change. Models appropriate to both behavioural and evolutionary change are considered. Both types of change can produce cycles under similar circumstances, but under some evolutionary models there is not sufficient genetic variability for evolutionary change to produce cycles. If there is sufficient variability, the remaining conditions required for cycles are surprisingly insensitive to the nature of the adaptive process. A predator population that approaches the optimum foraging strategy very slowly usually produces cycles under similar conditions as does a very rapidly adapting population.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Target organ-specific inactivation of drug metabolizing enzymes in kidney of hamsters treated with estradiol

Chronic treatment of hamsters with estradiol for several months has previously been shown to decrease the specific content of cytochrome P450 in the kidney, a target of hormonal carcinogenesis, but not in liver. The reason for this decrease in metabolic enzyme activity is unknown and has been examined in this investigation. We now report that the decrease in specific content of renal cytochrome P450 by 73% in response to estradiol was not affected by co-treatment with tamoxifen for 1 month. The subcutaneous infusion of 250 μg/day estradiol for 7 days lowered renal cytochrome P450 by 71% from control values and was therefore used for further mechanistic studies. This treatment decreased renal activities of estradiol 2- or 4-hydroxylase by 77 to 80%, of 7-ethoxycoumarin-O-deethylase by 66% of control values, respectively, and completely eliminated aryl hydrocarbon hydroxylase activities, whereas liver enzymes remained unaffected. After 7 days of infusion of estradiol, fluorescent products of lipid peroxidation were more than doubled in hamster kidney but remained unchanged in liver. The possibility of enzyme destruction by binding of estradiol 2,3-quinone to metabolizing enzymes was investigatedin vitro. In the presence of 2-hydroxyestradiol, cumene hydroperoxide, and microsomes, conditions known to favor the oxidation of the steroid to quinone, the binding of catechol estrogen metabolite to microsomal protein increased 60 fold over control values in the absence of cofactor. Purified rat liver cytochrome P450c also oxidized 2-hydroxyestradiol to 2,3-estradiol quinone. The rate of oxidation was linear for the first 2–3 min, but thereafter decreased with time. Under these incubation conditions, irreversible binding of catechol estrogen metabolite to cytochrome P450c increased for the first 2–3 min and then remained at this plateau level. It was concluded that enzyme destruction by a reactive estrogen metabolite or by lipid peroxides may be a major reason for the organ-specific decrease in cytochrome P450 enzymes in kidneys of estrogen-treated hamsters.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.