Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7–8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g · 1^-1 · h^-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, -glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5--lactone, a specific inhibitor of -glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.     

Palaeolimnological aspects of a Late-Glacial shallow lake in Sandy Flanders,Belgium

A summary account is given of the development of a small Late-Glacial lake at Snellegem-St. Andries, Belgium. Sedimentation, hydrology, water quality and biotic succession clearly depended on climatic conditions and catchment processes (soil stability and leaching, vegetation). Special attention is drawn to a period of low water level near the end of the Allerød and the abundance of Fragilaria in certain periods.     

KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas.

DNAs from human pancreatic adenocarcinomas were analyzed for the presence of mutations in codons 12, 13 and 61 of the NRAS, KRAS and HRAS gene. Formalin-fixed and paraffin-embedded tissue was used directly in an in vitro amplification reaction to expand the relevant RAS sequences. The mutations were detected by selective hybridization using mutation-specific synthetic oligonucleotides. In 28 of the 30 patients we found a mutation in codon 12 of the KRAS gene. This result confirms the findings of Almoguera et al. [Cell 53 (1988) 549-554] that KRAS mutations occur frequently in adenocarcinomas of the exocrine pancreas. The mutations are predominantly G-T transversions, in contrast to the KRAS mutations in colon tumors which are mainly G-A transitions. Furthermore, in a portion of the tumors the mutation appears to be homozygous.     

Natural cytotoxicity of rat hepatic natural killer cells and macrophages against a syngeneic colon adenocarcinoma

Summary The in vitro cytotoxic activity of two types of hepatic sinusoidal cells, i.e., natural killer (NK) cells and macrophages (Kupffer cells), was tested against a syngeneic rat colon adenocarcinoma cell line (DHD-K12). Purified hepatic NK cells (85% cells with large granular lymphocyte morphology) were spontaneously cytolytic, whereas Kupffer cells (90% pure) were not able to kill the DHD-K12 cells. This carcinoma cell line was found to be resistant to the action of mouse recombinant tumor necrosis factor which is considered as the major cytolytic molecule secreted by macrophages. However, colon carcinoma cells were readily lysed by soluble factors present in the culture supernatant of NK cells. It is postulated that hepatic NK cells, which are strategically located within the lumen of the sinusoids, may form a first line of defense to metastasizing colon carcinoma cells.Senior research assistant of the National Fund for Scientific Research (N.F.W.O., Belgium)Supported by an A.S.L.K. cancer grant     

The involvement of an enantioselective transaminase in the metabolism of D-3- and D-4-hydroxyphenylglycine inPseudomonas putida LW-4

Summary Pseudomonas putida LW-4, isolated on D-phenylglycine as sole carbon and energy source, was also able to grow on D-3-and D-4-hydroxyphenylglycine. Both D-3-and D-4-hydroxyphenylglycine were initially converted to the corresponding hydroxyphenylglyoxylates by means of an enantioselective transaminase. Subsequently, the hydroxyphenylglyoxylates were decarboxylated and then oxidized to 3-and 4-hydroxybenzoate, respectively. These latter compounds in turn were oxidized by NADPH-dependent hydroxylases to protocatechuate, which was further oxidized via an intradiol cleavage. Preliminary experiments with cell extracts in which the 4-hydroxyphenylglyoxylate decarboxylase was partially removed by an ammonium sulfate fractionation showed that D-4-hydroxyphenylglyoxycine could be formed from 4-hydroxyphenylglyoxylate by the enantioselective transaminase.     

Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane

Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein.     

Two new methods for recovery and genetic analysis of hybrids after fusion of yeast protoplasts

A method for regeneration of yeast protoplasts and fusants in a gelatin-agar mixture, followed by total recovery of the regenerated cells from the gelatin-agar mixture and isolation of the fusants, is described. A one-step method for obtaining intergeneric fusants in which the greater part of the genome is derived fromSaccharomyces cerevisiae, and in which the fusant can be sporulated directly and tetrad analysis carried out without construction of further hybrids, is also described.     

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acute Effect of Protein or Carbohydrate Breakfasts on Human Cerebrospinal Fluid Monoamine Precursor and Metabolite Levels

Patients with normal pressure hydrocephalus who had three lumbar punctures during 1 week ingested either water, a protein breakfast, or a carbohydrate breakfast 2.5 h before each of the lumbar punctures. The CSF was analyzed for biogenic amine precursors and metabolites. The protein meal raised CSF tyrosine levels, a finding consistent with animal data, but did not alter those of tryptophan or any of the biogenic amine metabolites. The carbohydrate meal increased CSF 3-methoxy-4-hydroxyphenylethylene glycol, an unexplained finding. The carbohydrate meal did not affect CSF tryptophan, tyrosine, 5-hydroxyindoleacetic acid, or homovanillic acid. Our results support the idea that in humans protein or carbohydrate meals do not alter plasma amino acid levels sufficiently to cause appreciable changes in CNS tryptophan levels or 5-hydroxytryptamine synthesis.