Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant

Summary The secretion of multiple forms of cellulolytic enzymes by a Trichoderma reesei QM 9414 selectant exhibiting high protease activity (T. reesei QM 9414/A 30) was investigated using monoclonal, domain-specific antibodies against cellobiohydrolase (CBH) I, CBH II and -glucosidase, and a polyclonal antibody against endoglucanase I. The pattern of appearance of these proteins was followed during growth of the fungus on Avicel cellulose, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting/immunostaining. Evidence was obtained that, at late cultivation stages, CBH I and II became partially modified to lower molecular weight components, whereas -glucosidase and endoglucanase I appeared to remain largely intact. Modification of CBH I appeared to commence from the carboxy-terminal AB region, whereas CBH II appeared to become modified both from the amino- (ABB') and the carboxy-terminal. Evidence for a protease activity that modifies the already truncated cellobiohydrolases in the culture filtrate was obtained. These results show that proteolysis at late culture stages may contribute to the multiplicity of cellulases found in T. reesei culture fluids. Initial proteolytic cleavage of CBH I and II may, however, involve an unusual protease not detectable by the azocasein method.Offprint requests to: C. P. Kubicek     

Fragile×expression and×inactivation

Summary The inactive fragile×chromosomes of a 47,fra(X),fra(X),Y male with a typical fragile×phenotype were successfully separated from the active homologues by means of somatic cell hybridization. It was shown by FUdR-induction and caffein-posttreatment that the separated inactive×chromosomes expressed their fragile sites and that the presence of an active mutated \sxchromosome was not a prerequisite for fragile X expression. The fragility seems to be an intrinsic property of the individual fragile site. This result is in favour of the classical concept that the fragile site at Xq27.3 has a primary pathogenetic function in this syndrome, although the fragility itself could represent a secondary phenomenon related to an unknown alteration of the DNA in this chromosome region. It is also concluded that inactivation of the fragile\sxchromosome in females is not responsible for either false negative fragile\sxfindings or the observation of fragile\sxnegative colonies isolated from fragile\sxpositive fibroblasts in heterozygotes.     

Monoclonal antibodies to interferon-gamma inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors

In this study we tested the effect of monoclonal antibodies (moAb) AN-18 to murine IFN-gamma on the generation of cytolytic T cells (CTL) from a homogeneous population of precursor cells (CTL-P). As responder cells, highly purified Lyt-2+ C57BL/6 lymph node T cells were used that had been positively selected by flow cytofluorometry on a cell sorter. Lyt-2+ cells were set up in bulk culture or in limiting dilution (LD) either with Con A or with P815 tumor cells as antigen and recombinant human interleukin 2 (rec.hIL 2) in the presence or absence of moAb AN-18 and tested for growth and development of CTL. The results show that moAb AN-18 but not the unrelated moAb AN-37 diminished or abrogated proliferative and cytolytic responses of Lyt-2+ lymphocytes to lectin and rec.hIL 2 in a dose-dependent manner. The inhibitory activity of the antibodies could be abolished by neutralizing moAb AN-18 with recombinant murine IFN-gamma (rec.mIFN-gamma) before their addition to culture. Kinetic analysis shows that the inhibitory effect of moAb AN-18 is only optimal when added at the beginning of culture or up to 48 hr after initiation. The frequencies of CTL-P responding either to Con A or to P815 tumor cells and rec.hIL 2 were reduced up to 10-fold in the presence of moAb AN-18. The inhibitory capacity of moAb AN-18 was also operative in cultures containing on the average one antigen-specific CTL-P. Together with the finding that activated CTL-P secrete IFN-gamma in response to rec.hIL 2 in a dose-dependent manner, the data suggest that endogenous IFN-gamma collaborates with exogenous IL 2 in the induction of CTL-P. The generation of CTL may therefore represent a case of autocrine growth regulation of normal lymphocytes, in which the same cell synthesizes and responds to its own factor.