Ammonium assimilation by Candida albicans and other yeasts: a 13N isotope study.

Nuclear magnetic resonance spectra of cultures of Candida albicans incubated in the presence of 15N-labelled ammonium demonstrated that glutamine and glutamate were the only initial products of ammonium assimilation. The nature of the route of assimilation in the yeasts Candida albicans, Saccharomyces cerevisiae, and Candida tropicalis was further examined by the use of the short-lived isotope 13N. [13N]ammonium was generated in the reaction 16O(p,alpha)13N, induced by proton bombardment of water in tandem accelerator. High-pressure liquid chromatography was used to separate and identify the products of assimilation, and radioactivity was detected and corrected for decay, using a computer-linked NaI scintillation detector. In the three yeasts studied, the labelled ammonium was assimilated into the acid-extractable fraction of cell suspensions within 1 min, and over 75% was converted to glutamine and glutamate. Subsequent to exhaustion of the labelled ammonium, the stoichiometry of the distribution of radiolabel was consistent with a net transfer of radiolabel from glutamine to glutamate, confirming the operation of glutamate synthase (EC 1.4.1.14) in these yeasts. Initial assimilation of label was mostly into glutamine (at a maximal rate within 10 s in C. albicans), whereas accumulation in glutamate did not occur at maximal rate until more than 70% of the labelled ammonium had been assimilated (between 30 and 60 s in C. albicans). We conclude that the glutamine synthetase-glutamate synthase pathway is the major route of ammonium assimilation in C. albicans and also in nitrogen-starved cultures of S. cerevisiae and C. tropicalis.     

Diabetes decreases sensitivity of adipocyte lipolysis to inhibition by G-linked receptor agonists

(1) Streptozotocin-diabetes decreased the responsiveness of noradrenaline- or forskolin-stimulated lipolysis to inhibition by phenylisopropyladenosine (PIA), prostaglandin (E₁ (PGE₁) and nicotinate in rat adipocytes. (2) Diabetes had no effect on high affinity binding of [³H]PIA to adipocyte plasma membranes. (3) Plasma membranes from diabetic animals had increased abundance of -subunits of G_i1 and G_i2. The effect of pertussis toxin in overcoming inhibition of lipolysis by PIA was delayed in adipocytes from diabetic rats. (4) Diabetes decreased the GTP-dependent right-wards shift in the dose-curve for displacement of the antagonist [³H]DPCPX by PIA in adipocyte plasma membranes. (5) It is concluded that, despite increased abundance of G_i in diabetic adipocytes, less of this functional. This may contribute to reduced sensitivity to PIA, PGE₁ and nicotinate and explains some of the loss of control of lipolysis in insulin-dependent diabetes.     

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium.

When olfactory receptor neurons are exposed to sustained application of odours, the elicited ionic current is transient. This adaptation-like effect appears to require the influx of Ca2+ through the odour-sensitive conductance; in the absence of extracellular Ca2+ the current remains sustained. Odour transduction proceeds through a G-protein-based second messenger system, resulting finally in the direct activation of an ion channel by cyclic AMP. This channel is one possible site for a negative feedback loop using Ca2+ as a messenger. In recordings of single cyclic AMP gated channels from olfactory receptor neurons, the open probability of the channel in saturating cAMP concentrations was dependent on the concentration of intracellular Ca2+. It could be reduced from 0.6 in 100 nm Ca2+ to 0.09 in 3 microM Ca2+. However, as neither the single channel conductance nor the mean open time were affected by Ca+ concentration, this does not appear to be a mechanism of simple channel block. Rather, these results suggest that intracellular Ca2+ acts allosterically to stabilize a closed state of the channel.     

Glucose tolerance factor potentiation of insulin action in adipocytes from rats raised on a torula yeast diet cannot be attributed to a deficiency of chromium or glucose tolerance factor activity in the diet

The nature of the dietary component responsible for adipocytes having the ability to respond to Glucose Tolerance Factor (GTF) was investigated. Rats were raised on either a control diet or one of three diets differing only in the protein source (torula yeast, brewer's yeast, or casein). Only in adipocytes from rats fed the torula yeast diet did a GTF fraction prepared from brewer's yeast potentiate the action of suboptimal concentrations of insulin in the incorporation of label fromd-[1-¹⁴C]-glucose andd-[U-¹⁴C]-glucose into CO₂ and fatty acids. It was concluded that this potentiation was not the result of a deficiency of GTF activity in torula yeast, because a GTF fraction prepared from torula yeast had similar insulin potentiating activity. Differences in response among diets were not owing to differences in levels of amino acids or owing to concentrations of 22 (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mo, Na, Ni, P, Pb S, Se, Si, Sn, Sr, Zn) of the 23 trace elements investigated. The level of Mn, was low in all diets, but particularly low in the torula yeast diet. Mn deficiencies have previously been implicated in perturbations of glucose metabolism, so that it is possible that this deficiency may be responsible for the effects attributed to the torula yeast diet.     

Preparation and characterization of a beta-lactamase-Fab' conjugate for the site-specific activation of oncolytic agents.

Antibody-directed catalysis (ADC) is a two-step method for the targeted delivery of chemotherapeutic agents in which enzyme-antibody conjugates, prelocalized to antigen-bearing cells, activate prodrugs designed to be substrates for the enzyme. An enzyme-Fab' conjugate exhibiting both native beta-lactamase activity and immunoreactivity toward carcinoembryonic antigen (CEA) was constructed. Treatment of CEA-expressing LS174T cells with this conjugate imparted beta-lactamase activity to the cells; beta-lactamase activity was not imparted by treatment with unconjugated beta-lactamase and not to CEA negative cells treated with conjugate. Cephalosporin-based prodrugs, and other substrates synthesized as model compounds, were found to have wide variations in their kinetic parameters toward the conjugate, with kcat values ranging from 16 to 3300 s-1 and KM values ranging from 5 to 160 microM. The prodrug derived from desacetylvinblastine-3-carboxylic acid hydrazide (DAVLBHYD) was studied in vitro and found to be 5-fold less cytotoxic to LS174T cells than the parent DAVLBHYD. For antigen-positive cells preincubated with conjugate, however, the prodrug showed the same potency as the parent drug. Thus, the combination of conjugate and prodrug appears to provide antigen-dependent toxicity to tumor cells.     

Morphological and radiochemical evidence for the metabolism of exogenous proteins by the preimplantation sheep blastocyst.

The ability of the trophoblast of the ovine preimplantation blastocyst to take up and metabolise proteins has been investigated using two experimental approaches, microscopical and radiochemical. The ultrastructure of the expanded blastocyst obtained from 14 and 17 day pregnant ewes was examined. The morphology of tissues maintained in culture for 24 h has been compared with that of fresh tissues. After culture, the cellular morphology of the explants was well preserved. Fresh and 24 h cultured tissues were incubated with horse-radish peroxidase and ferritin and these proteins subsequently were found to be localized in coated pits, caveolae and secondary lysosomes of the trophoblast. Comparison of the uptake of [3H]dextran and of 125I-labelled bovine serum albumin indicated that proteins could be taken up by cultured tissue by mechanisms in addition to simple fluid phase endocytosis. During culture of explants of blastocyst with 125I-labelled bovine serum albumin, a large fraction of the radioactivity taken up by the tissue appeared in the TCA-soluble fraction of the culture medium indicating that cultured trophoblast hydrolysed proteins. That amino acids released from captured protein could be used for protein synthesis by the trophoblast was indicated by the labelling of tissue and medium proteins after culturing explants with beta-lactamase labelled with [14C]leucine. A major product (Mr approximately 17 x 10(3) present in the medium was likely to have been ovine trophoblast protein-1. It is concluded that, during the expansion of the ovine blastocyst, the trophoblast has the ability to take up proteins, transport them to lysosomes and degrade them to amino acids which are used for protein synthesis. Thus proteins, as well as free amino acids, present in the histotrophe may be an important source of nitrogen for the sheep conceptus in the critical period just prior to implantation.     

Two-step one-dimensional SDS-PAGE analysis of LMW subunits of glutelin

Summary A collection of 222 hexaploid wheat cultivars (including the 207 cultivars studied by Gupta and Shepherd in 1988) from 32 countries was analyzed for variation in the banding patterns of LMW subunits of glutenin using a modified two-step 1-D SDS-PAGE. Seventy percent ethanol at high temperature (50 °C) was used to selectively dissolve the native glutenins containing A, B, and C subunits and not the albumins and globulins (non-prolamins). This procedure allowed the glutenin subunits A, B and C to be separated in a background free of albumins and globulins, which normally overlap the B and C subunits (LMW subunits of glutenin). Although 40 different B and C subunits were detected, except where the cultivars carried a 1BL-1RS translocation or 1B/1R substitution, each cultivar exhibited from 7 to 16 subunits. These subunits could be divided into 20 band patterns which fell into three groups on the basis of their mutual exclusiveness, with 6, 9, and 5 patterns. Analysis of substitution lines revealed that the different patterns in these groups are controlled by genes on chromosomes 1A, 1B, and 1D, respectively. The least number of subunits was controlled by chromosome 1A and approximately 40% of the cultivars did not contain any band controlled by this chromosome. Thirteen of the cultivars were found to consist of two biotypes with respect to LMW subunits of glutenin. The genetic, evolutionary, and technological implications of these findings are discussed.The term glutelin refers to the polymeric prolamins of cereals and, in the case of wheat, it is called glutenin     

Multi-Donor Longitudinal Antibody Repertoire Sequencing Reveals the Existence of Public Antibody Clonotypes in HIV-1 Infection

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