Analysis of the composition of samples of Babesia bovis and the influence of different environmental conditions on genetically distinct subpopulations.

A recombinant DNA probe specific for a tandemly repeated sequence located within the BoVA1 gene of Babesia bovis was used to analyse 10 independent samples of B. bovis. Twelve different alleles of the BoVA1 gene and flanking regions were identified in the 18 different subpopulations analysed. Most samples of B. bovis originally derived from single animals contained more than one genetically distinct subpopulation. However, only one population of parasites was identified in samples of the Ka line used in Australia from 1979 until 1990 as the live attenuated vaccine strain. In contrast, the replacement attenuated vaccine line, Ta, contained two genetically distinct subpopulations of parasites. Changes in the ratios of subpopulations of parasites were identified during attenuation and under different culture conditions. Batch-to-batch variation in the composition of doses of the live attenuated vaccine may lead to differences in efficacy and in severity of the infection associated with vaccination.     

In vivo binding and clearance of circulating antigen by bispecific heteropolymer-mediated binding to primate erythrocyte complement receptor.

We have used the avidin/biotin system to construct soluble, cross-linked bispecific heteropolymers containing mAb to both the primate E C receptor and the DNP group. These heteropolymers facilitate in vitro binding of DNP-bovine gamma-globulin (DNP-BGG) to both human and squirrel monkey E. Intravenous injection in squirrel monkeys of DNP-BGG followed by heteropolymer leads to E binding and clearance from the circulation of a significant fraction of both heteropolymer and DNP-BGG, without lysis or clearance of the E. This methodology may potentially be used to treat a variety of infectious diseases and other syndromes associated with blood-borne pathogens.     

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies

Summary Cystic fibrosis (CF) is characterized by abnormal epithelial Cl^– conductance (G_Cl). In vitro studies that have shown that cAMP regulation is an intrinsic property of the CF-affected G_Cl(CF-G_Cl) have been carried out previously on cultured secretory cells and on nonepithelial cells. Even though G_Cl in absorption is defective in CF, a clear demonstration of cAMP regulation of CF-G_Cl in a purely absorptive tissue is lacking. We studied the cAMP regulation of CF-G_Cl in the microperfused intact human reabsorptive sweat duct. About 40% of the ducts responded to cAMP (responsive) while the remainder of the ducts did not. In responsive ducts, cAMP-elevating agents: -adrenergic agonist isoproterenol (IPR), CPT-cAMP, forskolin, theophylline or IBMX increased G _tby about 2.3-fold (n = no. of ducts = 8). Removal of media Cl^–, but not amiloride pretreatment (in the lumen), abolished the cAMP response, indicating exclusive activation of G_Cl. cAMP activated both apical and basolateral G_Cl. cAMP hyperpolarized gluconate: Cl^– (lumen: bath) transepithelial bionic potentials (V _t=–20.3±5.2 mV, mean ±se, n=9) and transepithelial 3 1 luminal NaCl dilution diffusion potentials (V _t=–8.8±2.9 mV, n=5). cAMP activated basolateral G_Cl as indicated by increased bi-ionic (gluconate: Cl^–, bath: lumen) diffusion potentials (by about 12 mV). The voltage divider ratio in symmetric NaCl solutions increased by 60%. Compared to responsive ducts, nonresponsive ducts were characterized by smaller spontaneous transepithelial potentials in symmetrical Ringer's solution (V _t=–6.9±0.8 mV, n=24, nonresponsive vs. –19.4±1.8 mV, n=22, responsive ducts) but larger bi-ionic potentials (–94±6 mV, n=35, nonresponsive vs. –65±5 mV, n=17, responsive ducts) and dilution diffusion potentials (–40±5 mV, n=11, nonresponsive vs. –29±3 mV, n=7, responsive ducts). These results are consistent with an inherently (prestimulus) maximal activation of G_Cl in nonresponsive ducts and submaximal activation of G_Cl in responsive ducts. We conclude that cAMP activates CF-G _Cl which is expressed and abnormal in both apical and basal membranes of this absorptive epithelium in CF.Abbreviations CF cystic fibrosis - G _t transepithelial conductance - V _b electrical potential across the basolateral membrane - V _a electrical potential across the apical membrane - V _t transepithelial potential - V _b transepithelial currentinduced voltage deflections across the basolateral membrane - V _a transepithelial current-induced voltage deflections across the apical membrane - V _t transepithelial current-induced voltage deflection across the epithelium - VDR voltage divider ratio - G_Cl transepithelial Cl^– conductance - CF-G_Cl cystic fibrosis-affected Cl^– conductance - EMF electromotive force - IPR isoproterenol - IBMX 3-isobutyl-1-methylxanthine - CPT-cAMP chlorophenylthio-adenosine 3-5 cyclic monophosphate - PGE₂ prostaglandin E₂     

Drought acclimation among tropical forest shrubs (Psychotria,Rubiaceae)

Summary Mechanisms of dry-season drought resistance were evaluated for five evergreen shrubs (Psychotria, Rubiaceae) which occur syntopically in tropical moist forest in central Panama. Rooting depths, leaf conductance, tissue osmotic potentials and elasticity, and the timing of leaf production were evaluated. From wet to dry season, tissue osmotic potentials declined and moduli of elasticity increased in four and five species, respectively. Irrigation only affected osmotic adjustment by P. furcata. The other seasonal changes in leaf tissue properties represented ontogenetic change. Nevertheless, they made an important contribution to dry-season turgor maintenance. Small between-year differences in dry season rainfall had large effects on plant water status. In 1986, 51 mm of rain fell between 1 January and 31 March, and pre-dawn turgor potentials averaged <0.1 MPa for all five Psychotria species in March (Wright 1991). In 1989, 111 mm of rain fell in the same period, pre-dawn turgor potentials averaged from 0.75 to 1.0 MPa for three of the species in April, and only P. chagrensis lost turgor. The relation between leaf production and drought differed among species. P. limonensis was buffered against drought by the lowest dry-season conductances and the deepest roots (averaging 244% deeper than its congeners) and was the only species to produce large numbers of leaves in the dry season. P. chagrensis was most susceptible to drought, and leaf production ceased as turgor loss developed. For the other species, water stress during severe dry seasons may select against dry-season leaf production.     

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.     

Cooling rate influences cryoprotectant distribution and organ dehydration in freezing wood frogs.

Ice formation in the freeze-tolerant wood frog (Rana sylvatica) induces the production and distribution of the cryoprotectant, glucose. Concomitantly, organs undergo a beneficial dehydration which likely inhibits mechanical injury during freezing. Together, these physiological responses promote freezing survival when frogs are frozen under slow cooling regimes. Rapid cooling, however, is lethal. We tested the hypothesis that the injurious effects of rapid cooling stem from an inadequate distribution of glucose to tissues and an insufficient removal of water from tissues during freezing. Accordingly, we compared glucose and water contents of five organs (liver, heart, skeletal muscle, eye, brain) from wood frogs cooled slowly or rapidly during freezing to -2.5 degrees C. Glucose concentrations in organs from slowly cooled frogs were significantly elevated over unfrozen controls, but no significant increases occurred in rapidly cooled frogs. Organs from slowly cooled frogs contained significantly less water than did those from controls, whereas water contents from rapidly cooled frogs generally were unchanged. Rapid cooling therefore inhibited the production and distribution of cryoprotectant and organ dehydration during freezing. This inhibition may result from an accelerated, premature failure of the cardiovascular system.     

Crystal structure of a wheat germ agglutinin/glycophorin-sialoglycopeptide receptor complex. Structural basis for cooperative lectin-cell binding.

The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at 2.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 dimers, composed of two identical four-domain (A-D) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 dimers. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha 2,6-NeuNAc and alpha 2,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C2 (monomer 2) on opposing dimers, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha 2,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A2, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha 2,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.     

Ovarian development, spawning frequency and batch fecundity in Encrasicholina heteroloba (Ruppell, 1858)

A histological and gravimetric analysis of oocyte development in Encrasicholina heteroloba (Ruppell, 1858) indicated that this species spawns serially and has a group-synchronous mode of ovarian development. A six stage maturity scale, based on both external morphology and oocyte composition, was proposed to classify ovarian development in E. heteroloba . The incidence of females with hydrated oocytes and post-ovulatory follicles in samples from two regions; the south Java Sea and Roviana lagoon, in the Solomon Islands were used to estimate spawning frequency. Estimates of mean inter-spawning intervals ranged from around 2 days in fish from Roviana lagoon to up to 16.7 days in fish from the south Java Sea. Batch fecundity was determined from the number of oocytes in the largest oocyte size class in ripe stage ovaries. Batch fecundity was related to size and was significantly greater for a given size in the Roviana lagoon population ( F = 0.081 × L ^4.89, Roviana population; F = 1.682 × L ^2.83, Jepara population).     

Peripheral 5-carboxamidotryptamine induces hindlimb scratching by stimulating 5-HT1A receptors in rats.

Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.     

Superovulation of cattle with equine pituitary extract and porcine ESH

Superovulation has been practiced in cattle for more than 50 years but the results have been highly variable. Scientists at six locations compared a horse pituitary extract (HAP) with a single batch of porcine FSH (pFSH) to determine the efficacy of these hormones to induce superovulation and to test for variability in the superovulatory response. Acetone-dried equine pituitaries were suspended in 40% ethanol containing 6% ammonium acetate, and the supernatant was mixed with 2.5 volumes of cold ethanol. The resulting precipitate was washed with cold ether and dried. Total doses of 18 mg of HAP and 36 mg of pFSH were injected intramuscularly (i.m.) over 4 days, two injections per day, and prostaglandin (PGF(2)alpha; 25 mg, i.m.) was administered on Day 3. Injections were begun on Days 6 to 13 of the estrous cycle. The overall ovulation rates (mean +/- SEM) for HAP and FSH were 8.8 +/- 0.7 and 15.1 +/- 1.0, respectively (n=231; P<0.01). Location interacted (P<0.01) with the type of gonadotropin for the ovulation rate. When expressed as a proportion of the number of corpora lutea, the total number of embryos recovered was greater (P=0.03) for pFSH than for HAP, but there was no difference in the number of Quality 1 and 2 embryos. The results show that HAP can induce a satisfactory superovulatory response, but there was no evidence of reduced variability of response to HAP compared with pFSH.