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)     

Tritiated borohydride reduction of carbohydrates: reduction of individual monosaccharides, alone or in groups, results in highly divergent specific activities.

Reduction of carbohydrates by tritiated borohydride resulted in the production of alditols or glycosides with characteristically divergent specific radioactivities. Simultaneous reduction of individual sugars in the presence of a reference standard, talose, permitted the assignment of a unique specific radioactivity with respect to talitol as 100%. A variety of structures was examined, including neutral hexoses, free and acetylated aminosugars, ketohexoses and glycosides containing a fixed pyranose ring adjacent to a carbonyl group. In the latter case, the resulting steric hindrance severely restricted the incorporation of tritium. In both of the ketohexoses tested, the minor product of the two epimeric alditols exhibited the higher specific radioactivity. In all cases, reduction produced a characteristic and reproducible specific activity in which the values varied from 51 to 182% of that found for talose. These results are interpreted on the basis of generalizations concerning mechanism and predictive value.     

Preformed PAF-acether and lyso PAF-acether are bound to blood lipoproteins

PAF-acether (PAF) is a newly formed mediator not normally present in circulating blood. A compound exhibiting all of its biological characteristics but coeluting with phosphatidylcholine (PC) in high-pressure liquid chromatography (HPLC) was unveiled ('peak X') in normal human plasma. A second HPLC run of peak X HPLC fractions revealed the presence of PAF itself with concomitant disappearance of peak X. Beside PAF, immunoreactive apolipoproteins A-I and E were found in peak X. Also lipoproteins (Ls) purified using either ultracentrifugation or immunoaffinity chromatography yielded peak X and, in a second HPLC run, authentic PAF. L-free plasma was devoid of peak X. Finally, after preincubation with plasma, labeled PAF was found associated with Ls. Thus in human blood preformed PAF is bound in high amounts to Ls, a result of interest given the role of Ls and platelets in vascular diseases and the present knowledge on PAF biosynthesis.     

Host-Symbiont Specificity Expressed during Early Adsorption of Rhizobium meliloti to the Root Surface of Alfalfa

Early (4 h) adsorption of Rhizobium meliloti L5-30 in low numbers to alfalfa roots in mineral solution was examined for competition with other bacterial strains. All tested competitor strains decreased the adsorption of L5-30 by extents which depended on the strain and its concentration. The decrease of adsorption by R. meliloti competitors (all of them infective in alfalfa) was nearly complete at saturation (97 to 99% decrease). All other heterologous rhizobia and Agrobacterium tumefaciens at saturating concentrations (10⁶ to 10⁷ per ml) decreased adsorption of L5-30 only partially, less than 60%. The differential effects of homologous and heterologous competitors indicate that initial adsorption of R. meliloti to the root surface of its host occurs in symbiont-specific as well as nonspecific modes and suggest the existence of binding sites on roots which are highly selective for the specific microsymbiont in the presence of other heterologous bacteria even in very unfavorable (less than 10⁻⁴) symbiont-competitor concentration ratios.     

The nifHDK genes are contiguous with a nifA-like regulatory gene in Rhodobacter capsulatus

Summary A 15.2 kb DNA fragment was isolated from Rhodobacter capsulatus (ex. Rhodopseudomonas capsulata), which was able to complement mutations both in a nifA-like regulatory gene and in the nifH gene. Physical mapping of this fragment revealed that the nifA-like gene was adjacent to, and downstream from, the nifHDK operon. Hybridization experiments were carried out using a cloned Klebsiella pneumoniae DNA fragment containing nifA and the flanking portions of nifB and nifL. This fragment failed to hybridize with a 2.15 kb HindIII fragment of R. capsulatus DNA containing the nifA-like gene, but hybridized instead with a 2.6 kb EcoRI fragment adjacent to the nifA-like gene. The homologous region was found to be located within the K. pneumoniae nifB gene. The adjacent 2.6 kb and 2.15 kb fragments also hybridized with each other, indicating the presence of repeated sequences in this region.     

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.     

Gastric mucosal binding studies with enprostil: A potent anti-ulcer prostaglandin

The potent antiulcer prostaglandin enprostil binds with high affinity to porcine gastic mucosal tissues. This binding is saturable, dissociable and displaceable by compounds with similar structures. Various characteristics of binding such as pH optimum and displacement potencies suggest that enprostil binds to mucosal PGE₂ sites. Structure-activity and gastric mucosal binding relationships were also examined.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

A simplified method for the preparation of rat thyroxine-binding prealbumin. Factors influencing its circulating level

Rat thyroxine-binding prealbumin (TBPA) was isolated in three simple steps by means of a serum precipitation by a 5% phenol solution and two consecutive semi-preparative polyacrylamide gel electrophoreses. The overall yield was 15% and the TBPA preparation contained less than 1% impurities. In addition a monospecific antiserum was raised in the rabbit. In polyacrylamide gel, rat TBPA, as with its human counterpart, migrated anodally to albumin while in agarose gel, its electrophoretic mobility was similar to that of albumin. Serum TBPA measured in adult male Wistar rats did not exhibit a circadian rhythm. However, a significant 13% decrease was observed between 9 and 15 h, followed by the restoration of the initial value by 21 h. TBPA concentration was measured in 1-, 15- and 28-day-old male and female pups as well as in adult rats. The level of this protein increased from 1 to 28 days of age and did not display any sexual difference. Yet, while TBPA concentrations in adult males were similar to those recorded in the 28-day-old pups, for adult females, they returned to the levels measured in the 1-day-old pups.     

Tracer diffusion coefficients of proteins by means of holographic relaxation spectroscopy: application to bovine serum albumin

Holographic relaxation spectroscopy has been used to measure tracer diffusion coefficients for photochromically labeled bovine serum albumin in solutions having total bovine serum albumin concentrations in the range 3.25 to 257 g/liter. In the limit of zero concentration, the diffusion coefficient (20 degrees C, 0.1 M NaCl, 0.05 M Tris, pH 8.0) was found to be (5.9 +/- 0.1) X 10(-7) cm2/s and the initial slope was zero. The concentration dependence of the diffusion coefficient was not significantly affected by the fraction of protein molecules which were labeled. Holographic relaxation spectroscopy permits rapid, accurate determination of tracer diffusion coefficients for proteins in mixtures.