β-Glucosidase from the cellulolytic system of Alternaria alternata autolyzed cultures

Abstract A β-glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G-200, CM-Biogel A and DEAE-Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a M _r of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β-glucosidase is inhibited by gluconolactone but not by 10 mM glucose.     

Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus

The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Physiological and biochemical studies on Fusarium oxysporum f. sp. lycopersici race 2 for its biocontrol by nonpathogenic fungi

Abstract The self-degradation of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici race 2 ( F. oxysporum l. 2), which reached an autolysis degree of 72% after 60 days of incubation in stationary culture, occurred principally during the first 14 days of incubation, when considerable β-(1,3)-glucanase, pectinase, xylanase and chitinase activities were detected in the culture fluids. The levels of β-(1,3)-glucanase, pectinase, cellulase, chitinase and xylanase activities increased in the culture fluids of this fungus, when the culture medium was supplemented with different inducers. The vegetable juice (V8) that contained tomato juice, was the best inducer for most of these activities. Chitosan, glucosamine oligomers and Mucor rouxii mycelium extract were found to have an inhibitory effect on F. oxysporum l. 2 growth. When incubating cell walls from young mycelia of F. oxysporum l. 2 with enzymic precipitates obtained from autolyzed cultures of Mucor rouxii, Aspergillus nidulans, Penicillin oxalicum and Penicillium purpurogenum , degradations of 45%, 22%, 21% and 12%, respectively, were detected.     

Role of platelet-activating factor in renal function in normal rats and rats with bilateral ureteral obstruction

Platelet-activating factor (PAF) is a powerful vasodilator with important effects on kidney function. It has been suggested that the renal effects of PAF are mediated by thromboxane A2 (TxA2). We examined the effect of PAF on renal function in sham-operated rats and rats that had undergone unilateral release of bilateral ureteral obstruction (BUO) of 24-hr duration, a condition in which the synthesis of TxA2 is increased. To eliminate systemic hemodynamic changes, PAF was infused directly into the left renal artery using the lowest dose that affected renal function (2.3 x 10(-13) moles/min). Infusion of PAF significantly decreased the glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in normal rats and rats with BUO. Normal (sham-operated) rats pretreated with an inhibitor of TxA2 synthesis also had a significant decrease in GFR after administration of PAF (ERPF also decreased, but not significantly). Rats with BUO pretreated with an inhibitor of TxA2 synthesis had significantly greater basal GFR and ERPF (increases of 72 and 171%, respectively) than untreated BUO rats. Administration of PAF to the former group further increased GFR and ERPF (by 37 and 39%, respectively; P less than 0.001). The role of endogenous PAF was evaluated by administering a specific PAF receptor antagonist. Sham-operated rats pretreated with high doses of the PAF receptor antagonist had significantly higher mean arterial pressure values than normal untreated rats, and had no decrease in GFR and ERPF during PAF infusion. Rats with BUO pretreated with the PAF antagonist had a significant, dose-dependent decrease in basal GFR and ERPF. These data suggest that endogenous PAF has a vasodilatory role in obstructive nephropathy. No significant differences in eicosanoid excretion in the urine corrected per GFR were observed during infusion of PAF in any of the groups examined. In BUO rats with intact TxA2 synthesis, exogenous administration of PAF decreased renal function, presumably through further increases in the production of TxA2. However, when TxA2 production was inhibited, PAF administration increased GFR and ERPF, presumably due to its unopposed vasodilatory properties. The data suggest an important role of PAF in the hemodynamic changes seen in obstructive nephropathy.     

Rehabilitation in the Philippines

The international community has perspective and experience that will freshen our approaches to rehabilitation. Martin Grabois, MD^*, editor of this special section, has gathered articles written by experts from other countries. The intention is to stimulate thought, discussion, and action—and to broaden horizons.     

The respiratory response to heat shock in Neurospora crassa

A sharp decrease in oxygen uptake occurred in Neurospora crassa cells that were transferred from 30 degrees C to 45 degrees C, and the respiration that resumed later at 45 degrees C was cyanide-insensitive. Energization of mitochondria, measured in vivo with fluorescence microscopy and a carbocyanine dye, also declined sharply in cells at 45 degrees C. Electron microscopy showed no changes in mitochondrial complexity; however, the cytoplasm of heat-shocked cells was deficient in glycogen granules.     

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms

This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 degrees C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 mM dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls.     

Characterization of cytotoxic cells generated from in vitro cultures of murine bone marrow cells

Bone marrow cells cultured for 5-6 days generate cytotoxic activity against a number of natural killer (NK)-susceptible tumor cells. In this study, these bone marrow cytotoxic cells were compared to cells with NK activity obtained either from spleen cells activated in vitro with interferon (IFN-alpha/beta) or mitogen or from peritoneal exudate cells (PEC) obtained 4 days after bacillus Calmette-Guerin (BCG) infection. Splenic and PEC cytotoxic cells were shown to be Thy 1.2+, NK 1.1+, Asialo GM+1, Lyt 1.2-, Lyt 2.2-. In contrast, bone marrow cytotoxic cells were Thy 1.2+, NK 1.1-, Lyt 1.2-, Lyt 2.2- and expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Precursor cells for bone marrow cytotoxic activity were shown to be Thy 1.2-, NK 1.1-, Lyt 1.2-, Lyt 2.2- but also expressed low levels of Asialo GM1 antigen (Asialo GM +/- 1). Cytotoxic activity for both bone marrow and spleen cells peaked in the low-density fractions of discontinuous Percoll density gradients. The cytotoxic activity of these bone marrow cells was augmented by pretreatment with IFN (-alpha/beta, -gamma) or soluble factors (IFN free) from activated EL-4 thymoma cells. Surprisingly, the ability of bone marrow cells to generate high levels of cytotoxic activity following in vitro culture appeared to be associated primarily with mice which were of the H-2b haplotype.     

Mechanism of accessory cell requirement in inducing IL 2 responsiveness by human T4 lymphocytes that generate colonies under PHA stimulation

PHA-driven monoclonal colony formation by low concentrations of resting T4 lymphocytes in agar culture requires the presence of interleukin 2 (IL 2) and accessory cells. Using recombinant IL 2 and anti-Tac monoclonal antibody as a probe for the IL 2 receptor, we demonstrate that the requirement of accessory cells (here an irradiated B cell line) in inducing IL 2 responsiveness relies on their enhancing effect in functional IL 2 receptor expression by the T colony progenitors. Furthermore, it is shown that cell to cell interaction between accessory cells and colony progenitors results in IL 2 response, i.e., colony formation, when the IL 2 receptor density reaches a critical threshold. The asynchronism in IL 2 responsiveness expression by the T colony progenitors upon activation and the short-lived T cell-accessory cell interaction, due to accessory cell death, determine the 10% colony efficiency of the culture system. In addition, we demonstrate that the accessory function in IL 2 receptor and IL 2 responsiveness expression by the T colony progenitors can be supported by irradiated T lymphocytes as well as B cells. The absence of lineage restriction of the signal delivered by accessory cells, and the requirement of physical interaction between T colony progenitors and accessory cells, emphasize the necessity of cross-linking the activation-signal receptors in inducing IL 2 responsiveness by resting T4 cells.