Isolation of Aulacomya paramyosin

Tropomyosin A or paramyosin has been isolated from the adductor muscle of Aulacomya magellanica. It has in common with other tropomyosins A the method used for extracting it from adductor muscle, its solubility, facility of crystallization, ammonium sulphate range of precipitation, amino acid composition and behaviour when digested with trypsin. As a particular feature it exhibits an unusual low viscosity for this type of tropomyosin. Its molecular weight, determined by the Archibald approach-to-sedimentation-equilibrium method, is 258000+/-16000.     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Measures of food quality as demographic predictors in freshwater copepods

This study examines the ability of a number of short-term measuresof algal food quality to predict longer term demographic parametersfor two species of freshwater calanoid copepods, Diaptomus minuiusand Epischura lacustris. Food quality of two species of algaethat are usually considered highly edible (Cryptomonas erosavar. reflexa and Chlamydomonas reinhardtii) are compared withrespect to: (i) the biochemical constituents of the algae (totalN, total C and protein); (ii) short-term foraging responsesby D.minutus to either algal species (e.g. clearance, ingestionand assimilation rates); (iii) longer term demographic responses(e.g. survivorship and reproduction) by both D.minutus and E.lacustrisfed a diet of either algal species. Demographic responses ofthe two copepod species indicate that C.erosa is a higher qualityfood. In fact, survival and reproduction of both copepod speciesfed C.reinhardtii were not different from starved treatments.Cryptomonas erosa treatments also had greater C, N and protein.However, D.minutus ingested five times more C.reinhardtii thanC.erosa, indicative of ‘compensatory feeding’ inthe presence of poor-quality food. Based upon these higher ingestionrates, individuals fed C.reinhardai actually ingested greateramounts of C, N and protein. Hence, ingestion rates taken aloneor coupled with biochemical parameters are not reliable predictorsof consumer demographic response. Assimilation rate, which waspositive for C.erosa and zero for C.reinhardtii, was the singlebest short-term predictor of food quality.     

Contraception across Cultures: Technologies, Choices, Constraints.

Contraception across Cultures: Technologies, Choices, Constraints. Andrew Russell. Elisa J. Sobo. and Mary S. Thompson. Oxford: Berg Publishers, 2000. 224 pp.     

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide

Summary The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 s at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.     

Two-Dimensional Crystallization of Brush Border Myosin I

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22₁2₁or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 Å and show that the asymmetric unit is the same in both crystal types.     

A carbon monoxide irreducible form of cytochrome c oxidase and other unusual properties of the “monomeric” shark enzyme

Contrary to previous reports, the functional and spectral properties of “monomeric” shark cytochrome c oxidases are not entirely similar to those of the “dimeric” beef enzyme. Most significantly, unlike the behavior of beef oxidase, the fully oxidized shark enzyme is not reducible by carbon monoxide. Also, preparations of the shark enzyme, isolated at pH 7.8-8.0, lead to more than 60% of the sample always being obtained in a resting form, whereas similarly prepared beef oxidase is very often obtained, both by ourselves and others, exclusively in the pulsed form. Although the electronic absorption, magnetic circular dichroism and electron paramagnetic resonance (EPR) spectra of cytochrome c oxidase obtained from several shark species are similar to those of the beef enzyme, there are some significant differences. In particular, the Soret maximum is at 422 nm in the case of the fully oxidized resting shark oxidases at physiological pH and not 418 nm as commonly found for the beef enzyme. Moreover, the resting shark oxidases do not necessarily exhibit a “g = 12” signal in their EPR spectra. The turnover numbers of recent preparations of the shark enzyme are higher than previously reported and, interestingly, do not differ within experimental uncertainty from those documented for several beef isoenzymes assayed under comparable conditions.     

Localization of serotonin within tanycytes of the rat median eminence

Summary Formaldehyde and glyoxylic acid histochemical methods were employed to examine monoamine fluorescence of the rat median eminence. Tanycytes of the median eminence contained a yellow histofluorescence which was verified with microspectrofluorometry as due to the presence of serotonin. Catecholamine-containing varicosities, arranged in linear profiles throughout the depth of the median eminence, were observed. These linear profiles appeared to follow the contours of serotonin-containing tanycytes. Organculture experiments supported the hypothesis that the serotonin associated with tanycytes is localized within the tanycytes and does not arise from an extrahypothalamic source of nerve terminals. These data provide evidence that a tanycytic catecholamine-indoleamine morphological juxtaposition occurs in a manner reminiscent of that of another circumventricular organ, the pineal.Supported by USPHS Grants NS11642 and AM-19761USPHS Career Development Awardee NS-00259     

Microcommunities of algae on a Sphagnum mat

The algal epiphytes of three species of Sphagnum from the mat edge of an acid bog were quantitatively sampled over three months. On the basis of differences in total algal abundance, algal diversity, and the growth of certain algal species and species groups, two significantly different algal communities were recognized. While these communities were only centimeters apart, they differed in physical, biological and nutrient parameters, with lower, moister plots having more algae, higher algal diversity, and lower concentrations of phosphate, ammonium, and silicate ions.