Kinetic properties and contribution to cellulose saccharification of a cloned Pseudomonas beta-glucosidase

The plasmid pND71, which encodes beta-glucosidase (cellobiase) activity, cloned from the cellulolytic Pseudomonad, PS2-2, was mobilized by conjugation into 10 Pseudomonas strains. The highest specific activity was produced by 17498 (pND71) and the properties of the enzyme produced from this transconjugant were studied. The enzyme was shown to be cell associated, to have a temperature optimum of 37 degrees C, a pH optimum of 7.0 and Km values of 1.33 and 2.94 mM for pNPG and cellobiose respectively. It was competitively inhibited by glucose, with a Ki of 30 mM. Evidence was obtained which suggested that the enzyme was produced constitutively and that synthesis was not repressed by glucose. When culture preparations were used in combination with Trichoderma reesei QM9414 and C30 enzyme preparations to saccharify cellulose, 17498 (pND71) was more effective than preparations of PS2-2 in acting synergistically with T. reesei to solubilize more carbohydrate and produce more glucose.     

The autocrine production of transforming growth factor-beta 1 during lymphocyte activation. A study with a monoclonal antibody-based ELISA

We describe the production and characterization of three mAb to transforming growth factor-beta (TGF-beta) and the use of two of them for the development of a TGF-beta 1-specific ELISA and for the study of the regulation of immune function in vitro. All three mAb bound recombinant human TGF-beta 1 (rHuTGF-beta 1) with high affinity and recognized the dimer form of this molecule in immunoblots. mAb 2G7 immunoprecipitated rHuTGF-beta 1, TGF-beta 2, and rHuTGF-beta 3 and neutralized the growth inhibitory activity of all three molecules in vitro on mink lung epithelial-like cells, Mv1Lu, indicating a shared neutralization epitope. mAb 4A11 neutralized and immunoprecipitated only rHuTGF-beta 1, and mAb 12H5 immunoprecipitated rHuTGF-beta 1 but had no effect on the bioactivity of either rHuTGF-beta 1, TGF-beta 2, or rHuTGF-beta 3. These results suggest that a second neutralization epitope may be unique to TGF-beta 1. The ELISA was developed with mAb 4A11 and 12H5, with a range of 0.63 to 40 ng/ml, i.e., a sensitivity of 0.63 ng/ml or 63 pg/sample. The assay is accurate, precise, and specific for the active but not the inactive or latent TGF-beta 1 complex and fails to react with TGF-beta 2, rHuTGF-beta 3, inhibin A, and activin A. Supernatants obtained from serum-free cultures of human PBMC from multiple donors contained significant quantities of TGF-beta 1 (3 to 15 ng/ml), which was detected in the ELISA only after pH 2 treatment to convert latent TGF-beta to the active form. Treatment of the PBMC with either recombinant human IL-2 (rHuIL-2) or PHA-P/PMA enhanced the production of latent TGF-beta 1. mAb 4A11 and 2G7, but not mAb 12H5 enhanced both the proliferative response of PBMC to rHuIL-2/rHuTNF-alpha and PHA-P and the development of the rHuIL-2/rHuTNF-alpha treated PBMC into LAK cells with cytotoxic activity against COLO target cells. These findings suggest that although PBMC secrete latent TGF-beta 1, mechanisms that convert the latent TGF-beta complex into an active form exist in vitro and that the endogenously produced TGF-beta can regulate immune functions in an autocrine fashion.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Catabolite repression of the synthesis of inducible polygalacturonase and pectinesterase byAspergillus niger sp.

Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.     

Re-evalution of “Typothorax”meadei,a Late Triassic aetosaur from the United States

Aetosaur specimens from Howard County, Texas, USA, namedTypothorax meadei by Sawin (1947) represent a new genus here namedLongosuchus (type and only species -L. meadei). Longosu-chus is characterised by the possession of seven dentary teeth, a dentary excluded from the ventral margin of the lateral mandibular fenestra and by the angular, spinose lateral scutes throughout the dorsal region with lateral horns which have faceted sides.Longosuchus is found in mid-late Carnian strata in Texas, New Mexico and North Carolina, USA. Aetosaurs can be used to distinguish three successive biochrons in Late Triassic strata of the American Southwest, which are, in ascending order of age, theLongosuchus biochron, theStagonolepis biochron and theTypothorax biochron.     

Alternative seed-handling strategies in primates: seed-spitting by long-tailed macaques (Macaca fascicularis)

Summary The seeds in fruits consumed by primates may be chewed and digested, swallowed and defecated intact, or separated from the flesh and spat out. We show by a combination of close field observations and experiments with caged animals, that long-tailed macaques (Macaca fascicularis) have a remarkably low threshold of 3–4 mm for swallowing seeds and also that wild macaques rarely break them. The seeds of 69% of the ripe fruit species eaten are spat out intact or cleaned outside the mouth and dropped. Seed-spitting significantly reduces the swallowed food bulk and may lessen the risk of releasing seed toxins during mastication. However, it requires that even small fruits are processed in the mouth one or a few at a time. We suggest that fruit storage in the cheek pouches of cercopithecine monkeys allows them to spit seeds individually without excessively slowing fruit intake while feeding on patchily distributed fruit. In contrast, Apes and New World monkeys apparently swallow and defecate most ripe seeds in their diet and colobine monkeys break and digest them, detoxifying seed defenses by bacterial fermentation.     

Application of Asymmetric Alternating Voltage Pulse Series for Investigation of the Action Potential in Chara

An alternating (asymmetric) bipolar staircase voltage clamppulse series was used to investigate the action potential inChara corallina. Using this protocol, we found that the actionpotential was present in the hyperpolarized region of the current-voltagerelationship of the whole cell membrane. Effects of strong hyperpolarizingvoltage steps, during the excitation process, can thus be studied. (Received August 22, 1989; Accepted October 14, 1989)