Foetal and placental growth in the mouse after pre-implantation development in vitro under oxygen concentrations of 5 and 20%

Blastocysts which developed from two-cell mouse embryos in culture tubes containing an atmosphere with 20% oxygen had approximately 20% fewer blastomeres than blastocysts which developed under an oxygen concentration of 5%. When these smaller blastocysts were transferred to the uteri of pseudopregnant foster mothers, the foetuses developing were as viable as those developing from blastocysts cultured under 5% oxygen, indicating their ability to regulate for a lower blastomere number by at least day 17 of development. The transfer operation itself had no adverse effect on foetal or placental growth. However, culture of blastocysts in vitro did depress foetal though not placental growth, suggesting that the inner cell mass is more susceptible than the trophectoderm to culture in vitro. Foetal but not placental growth was lower following the transfer of blastocysts to a day-3 rather than a day-4 uterus. Four cases of placental fusion were found. In one case, the foetuses were contained within the same embryonic sac and may have been twins.     

Failure of human spermatozoa to penetrate zona free mouse and rat ova in vitro

When incubated for 8 to 26 hours with zona-free mouse or rat ova, human spermatozoa failed to attach to or penetrate any of the ova. The ova were capable of being fertilized since both intra- and inter-species penetration of spermatozoa and formation of pronuclei occurred between rat and mouse gametes. When mouse spermatozoa were incubated for three to eight hours with rat ova, a high proportion of the ova were penetrated, formation of pronuclei occurred and in 9 out of 36 ova incubated for 40 hours after insemination, regular cleavage and formation of morphologically normal 2-cell embryos occurred. Human spermatozoa retained their morphological integrity and motility only when the culture medium contained purified bovine serum albumin (3 mg/ml) or human serum (5% v/v) and not when unpurified BSA from several different commercial sources was used as a protein source. In this latter medium, the ova of both rats and mice degenerated after 8-hour incubation in the presence of human spermatozoa but not when human spermatozoa were absent or in the presence of either rat or mouse spermatozoa. Electron microscopy indicated that the human spermatozoa incubated for eight hours in medium containing purified BSA had undergone an acrosome reaction. These spermatozoa also attached to and penetrated human oocytes which had been matured in vitro.     

Behavioural thermoregulation and homing by spring chinook salmon, Oncorhynchus tshawytscha (Walbaum), in the Yakima River

Temperature-sensitive radio transmitters were employed to study the patterns of behavioural thermoregulation, habitat preference and movement of 19 adult spring chinook salmon, Oncurhynchus tshawytscha (Walbaum), in the Yakima River. During the 4 months prior to spawning, fish maintained an average internal temperature 2.5°C below ambient river temperature. This represented a 12 to 20% decrease in basal metabolic demand or a saving of 17.3 to 29.9 calkg⁻¹ h⁻¹. Fish were most commonly associated with islands, pools, and rock out-croppings along stream banks. Homing behaviour appeared to be modified to optimize temperature regimes and energy conservation. As the time of spawning approached, fish left thermal refuges and migrated to spawning grounds upstream and downstream of refuge areas. Although spring chinook salmon residing within cool-water refuges may be capable of mitigating sub-lethal temperature effects, cool-water areas need to be abundant and available to the fish. The availability of suitable thermal refuges and appropriate holding habitat within mainstem rivers may affect long-term population survival.     

Restricted Ig H chain V gene usage in the human antibody response to Haemophilus influenzae type b capsular polysaccharide

The mechanisms that govern the content of the human antibody repertoire are poorly understood. To investigate the antibody response to a clinically relevant Ag, we have produced heterohybridomas secreting human antibodies directed against the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). Immune lymphocytes were harvested 7 days after immunization with either of two vaccine formulations, a plain polysaccharide vaccine (Hib PS) or a polysaccharide-protein conjugate of Hib PS and diphtheria toxoid (Hib PS-D). H chain V region gene nucleic acid sequences were determined for five stable hybridomas. All use members of the VHIII gene family and are 83% to 98% homologous to two candidate germ-line sequences. A variety of D and JH segments are used. Thus the Ig H chain repertoire appears to be restricted to a limited group of VHIII family members. The previously reported expression of homologous sequences in the human fetal repertoire suggests that the inability of young children to respond to this Ag is not caused by deficiencies of these important elements early in development. The restricted use of VHIII gene segments suggests that this gene family plays a pivotal role in the immune response to this important childhood pathogen.     

Analysis of the monocyte chemotactic response to lysophosphatidylcholine: role of lysophospholipase C

Previously, we reported that lysophosphatidylcholine (lyso-PtdCho), a component of oxidized low-density lipoprotein, was a monocyte chemoattractant (M.T. Quinn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2805-2809). Monocyte chemotaxis was also stimulated by lyso-platelet activating factor but not by platelet activating factor itself. In the present studies, we used other analogs of lyso-PtdCho to determine structural and metabolic features required for chemotactic activity. Although both D- and L-lyso-PtdCho stimulated chemotaxis, suggesting a lack of stereospecificity, studies using propanediol and ethanediol analogs of lyso-PtdCho suggested that a free hydroxyl moiety or an ester-linked fatty acid vicinal to the phosphocholine group of the lysophospholipid was required for the expression of activity. Incubation of [3H]choline-labeled lyso-PtdCho with monocytes resulted in the formation of labeled PtdCho, glycerophosphocholine (GPC), phosphocholine, and free choline, while resident peritoneal macrophages, cells which we show do not respond chemotactically to lyso-PtdCho, metabolized the labeled substrate to generate only labeled PtdCho and GPC; no labeled phosphocholine was found, suggesting a possible role for lysophospholipase C activity in the monocyte chemotactic response. Although monoacylglycerol, the product of lysophospholipase C hydrolysis of lyso-PtdCho, was not chemotactic for monocytes, diacylglycerol demonstrated chemotactic activity, suggesting that the subsequent acylation to diacylglycerol may be involved in the monocyte chemotactic response to lyso-PtdCho. Indeed, monocytes incorporated [3H]glycerol from [3H]glycerol-labeled lyso-PtdCho into di- and triacylglycerol. Based on these results, a model is proposed whereby the monocyte chemotactic response to lyso-PtdCho involves a sequence of metabolic steps which includes hydrolysis of lyso-PtdCho to monoacylglycerol and phosphocholine by lysophospholipase C followed by acylation of monoacylglycerol to diacylglycerol. Diacylglycerol would then act as an intracellular second messenger that could activate or facilitate the chemotactic response.     

Measurement of bacterial random motility and chemotaxis coefficients: I. Stopped-flow diffusion chamber assay

Bacterial chemotaxis, the directed movement of a cell population in response to a chemical gradient, plays a critical role in the distribution and dynamic interaction of bacterial populations in nonmixed systems. Therefore, in order to make reliable predictions about the migratory behavior of bacteria within the environment, a quantitative characterization of the chemotactic response in terms of intrinsic cell properties is needed.The design of the stopped-flow diffusion chamber (SFDC) provides a well-characterized chemical gradient and reliable method for measuring bacterial migration behavior. During flow through the chamber, a step change in chemical concentration is imposed on a uniform suspension of bacteria. Once flow is stopped, diffusion causes a transient chemical gradient to develop, and bacteria respond by forming a band of high cell density which travels toward higher concentrations of the attractant. Changes in bacterial spatial distributions observed through light scattering are recorded on photomicrographs during a 10-min period. Computer-aided image analysis converts absorbance of the photographic negatives to a digital representation of bacterial density profiles. A mathematical model (part II) is used to quantitatively characterize these observations in terms of intrinsic cell parameters: a chemotactic sensitivity coefficient, mu(0), from the aggregate cell density accumulated in the band and a random motility coefficient, mu, from population dispersion in the absence of a chemical gradient.Using the SFDC assay and an individual-cell-based mathematical model, we successfully determined values for both of these population parameters for Escherichia coli K12 responding to fucose. The values obtained were mu = 1.1 +/- 0. 4 x 10(-5) cm(2)/s and chi(o) = 8 +/- 3 +/- 10(-5) cm(2)/s. We have demonstrated a method capable of determining these parameter values from the now validated mathematical model which will be useful for predicting bacterial migration in application systems.     

The grazing effect of limpets on the macroalgal community of a rocky intertidal shore

Abstract Abundance of macro-algae in the mid-littoral zone on a Victorian intertidal rocky shore varied seasonally, algae being rare in Summer and common in Winter and Spring. Field experiments demonstrated that, of two species of grazing limpets (Cellana tramoserica and Siphonana diemenensis), only Siphonaria had a major effect on the abundance of foliose algae (e.g. Scytopsiphon lomeniarid) and neither species had a great effect on the encrusting algae (Ralfsia spp.). There was no evidence of competition for food between the two species of limpets, in contrast with results found for similar species in New South Wales.     

Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis.

The kinetics of receptor-mediated cell adhesion to a ligand-coated surface play a key role in many physiological and biotechnology-related processes. We present a probabilistic model of receptor-ligand bond formation between a cell and surface to describe the probability of adhesion in a fluid shear field. Our model extends the deterministic model of Hammer and Lauffenburger (Hammer, D.A., and D.A. Lauffenburger. 1987. Biophys. J. 52:475-487) to a probabilistic framework, in which we calculate the probability that a certain number of bonds between a cell and surface exists at any given time. The probabilistic framework is used to account for deviations from ideal, deterministic behavior, inherent in chemical reactions involving relatively small numbers of reacting molecules. Two situations are investigated: first, cell attachment in the absence of fluid stress; and, second, cell detachment in the presence of fluid stress. In the attachment case, we examine the expected variance in bond formation as a function of attachment time; this also provides an initial condition for the detachment case. Focusing then on detachment, we predict transient behavior as a function of key system parameters, such as the distractive fluid force, the receptor-ligand bond affinity and rate constants, and the receptor and ligand densities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive hierarchies in marine benthic communities

Summary Patterns of competitive displacement by over-growth were examined in communities of sessile organisms in the low intertidal zone at three sites in Washington state and Alaska. Cruotose invertebrates and algae can be arranged into a hierarchy such that species of lower competitive rank rarely overgrow any higher ranking species. Erect and solitary species show a wide range of competitive abilities, but whether they fall into a strict hierarchy is unknown. Few of the solitary or erect species occupy substantial amounts of space in the communities examined.An approximate competitive hierarchy is well established in middle to high intertidal areas dominated by mussels, fleshy algae, and barncles, and has been an important concept in developing both an intuitive understanding and specific mathematical models of the dynamics of benthic marine communities. In particular, lower ranking species in such communities are thought to depend upon predation or chronic disturbance to the dominants to avoid competitive displacement. An alternative viewpoint, proposed on the basis of nonstransitive competitive relationships observed in cryptic encrusting communities on the undersides of coral plates, is that specific competitive loops or networks allow the coexistence of a number of competitors. Although the growth forms and higher taxa represented in the low intertidal bear some similarity to those in the cryptic coral reef community, there is little evidence of ecologically important competitive loops in the intertidal. A reanalysis of data from cryptic reef communities suggests that they also do not depart substantially from a competitive hierarchy, although there appear to be many more cases of local reversals in the outcome of competition. It is suggested that the ecological importance of departures from a strict hierarchy depends upon the competitive rankings of the participants, with departures involving competitively dominant species likely to contribute much more to community structure than those involving opportunistic species.     

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.