Binding activities of thyroxine binding globulin versus thyroxine binding prealbumin in rat sera: differential modulation by thyroid hormone ligands, oleic acid and pharmacological drugs

We use gel equilibration and electrophoretic techniques to compare the binding properties of thyroxine binding globulin and thyroxine binding prealbumin in rat sera. The evidence indicates that TBG bears the serum lowest capacity highest affinity sites for thyroxine (T4) and triiodothyronine (T3) (Ka1 greater than or equal to 10(9) M-1) as well as weaker saturable T3 sites (Ka2 approximately 10(8) M-1). TBPA bears for T4 only Ka2 approximately 10(8) M-1 sites and for T3 only Ka approximately 10(6) M-1 sites. Consistent with these parameters are the specific responses of TBG and TBPA binding activities to varying serum concentrations of T4, T3, oleic acid, the drugs diphenylhydantoin or salicylate. The primary attack of these compounds is aimed at TBG. Small T4, oleate or DPH doses chase the TBG-bound T4 to TBPA, high doses of T4 or oleate but not of DPH inhibiting the T4 binding to both proteins. In the T3-serum interactions, all tested compounds displace the TBG-bound hormone without chasing it to TBPA. The high reactivity of TBG sites designates the protein as crucially involved in modulating the free vs bound serum levels of T4 and T3 against physiological or pathological variations of binding competitors.     

Size variation in Brachionus plicatilis resting eggs

The effect of temperature and salinity on resting egg size of two Brachionus plicatilis (Rotifers) clones was investigated. Clones were selected according to their different behaviour in laying resting eggs: one clone ejects them, whereas they remain inside the females body in the other clone. The difference in resting eggs size between the two clones is noticeable, although the difference is not as great as that between female body size. An important temperature-salinity interaction on resting egg size has been observed. The general inverse relationship between size and temperature is only true at lower temperatures. At high temperatures size varies around the mean although could be greater than at intermediate temperatures. This is more evident at the intermediate salinity tested which is considered to be the closest to the optimum in our experiments. This pattern of variation suggests that mean size is bigger than expected, in relation to temperature and salinity, when these factors have values close to the extremes of their range, normally found in nature, and to which adaptative mechanisms can evolve. Size is bigger at the salinity — temperature low - low and high - high combinations which are the most commonly found in the temperate environments.     

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH: In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of disassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles:Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups: The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride: Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides: With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride: Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride:     

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone γ-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.     

Addendum: Recovery of cellulases from spent broth of sugar beet pulp fermentation byTrichoderma reesei

Summary The exo- and endo-glucanases ofTrichoderma reesei were recovered after growth on sugar beet pulp by vacuum concentration followed by (NH₄)₂SO₄ or acetone precipitation. Final respective recoveries were 53% and 57% for the exoglucanase and 41% and 42% for the endoglucanase. The resuspended acetone precipitates of both enzymes showed no loss of activity after 30 d at 4°C, pH 4.8 and in the presence of 0.5% sorbic acid. (NH₄)₂SO₄-precipitated exoglucanase lost activity in the same period.

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Resumen Las exo y endoglucanasas deTrichoderma reesei se recuperaron después de su crecimiento en pulpa de remolacha azucarera mediante concentración al vacío seguida de precipitación con (NH₄)₂SO₄ o acetona. La recuperación final fue de 53% y 57% para la exoglucanasa y de 41% y 42% para la endoglucanasa. La resuspensión de los precipitados cetónicos de ambos enzimas no mostró perdidas de actividad después de 30 d a 4°C y pH 4.8 en presencia de ácido sórbico. La exoglucanasa precipitada con (NH₄)₂SO₄ perdió actividad durante este mismo periodo.

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Résumé Les exo- et endoglucanases deTrichoderma reesei ont été récupérées après croissance sur pulpe de betterave sucrière, par concentration sous vide après précipitation par le sulfate ammonique ou l'acétone. Les récupérations ont été respectivement de 53% et 57% pour l'exoglucanase et de 41% et 42% pour l'endoglucanase. Les resuspensions des deux enzymes après précipitation à l'acétone n'ont révélé aucune perte d'activité après 30 jours à 4°C, pH 4.8 et en présence de 0.5% d'acide sorbique. L'exoglucanase précipitée au sulfate ammonique avait perdu toute activité dans le même laps de temps.

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(Based on a paper presented at the First Latin American Congress on Biotechnology, Tucumán, Argentina, October 4–8, 1987)     

Isolation and characterization of a recombination defective-dependent bacteriophage ofRhodobacter sphaeroides

A new virulent bacteriophage, designated RZ1, was isolated from a local pond on the facultative phototrophic bacteriumRhodobacter sphaeroides ZZ101. Electron microscopic studies revealed that, in general morphology, phage RZ1 resembles the bacteriophage ofEscherichia coli. The host range of phage RZ1 is limited to some strains ofR. sphaeroides. The phage genome consists of double-stranded DNA of about 44 kb lacking cohesive ends and seems to present terminal redundancy and cyclic permutation. RZ1 phage may carry out a lytic cycle only in recombination-defective mutants ofR. sphaeroides. Nevertheless, a derivative of the RZ1 phage, termed RW1, able to grow in recombination-proficient strains ofR. sphaeroides, has also been obtained. In vitro restriction analysis of both RZ1 and RW1 phages shows the presence of a rearrangement in their DNA. Generalized transduction of Str^r and Rif^r chromosomal markers has not been detected with either RZ1 or RW1 phages.     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.     

Expression of Microtubule-Associated Proteins During the Early Stages of Neurite Extension by Brain Neurons Cultured in a Defined Medium

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.