Inhibitors of Trichoderma reesei β-glucosidase activity derived from autohydrolysis-exploded Eucalyptus regnans

Summary Enzymic saccharification of Eucalyptus regnans pulps pretreated by autohydrolysis-steam explosion resulted in low cellulose conversions into glucose when using trichodermal cellulase preparations. The reduced levels of glucose were attributable to the production of compounds during enzymic hydrolysis which were inhibitory to -d-glucosidase of Trichoderma reesei C-30 and in Meicelase, but not to the cellulases. Aspergillus niger -glucosidase was not inhibited, nor were -d-xylosidase(s) and 1,4--d-xylanase(s). The inhibitory compound(s) could be extracted from the enzymic hydrolyzates with ethyl acetate. The ethyl acetate extractives inhibited -glucosidase in a competitive manner, and inhibitory action was not affected by pH. Addition of the inhibitory compound(s) to trichodermal cellulase digests of cellulose resulted in reduced glucose yields compared to a control. The inhibitory effects could be overcome when cellulase digests were supplemented with A. niger -glucosidase resulting in higher cellulose-to-glucose conversions. The inhibitory compound(s) were localized mainly in the heartwood of E. regnans. An inhibitor compound of this type has not hitherto been reported. The presence of inhibitory compound(s) in the autohydrolysis liquor fraction is also reported.     

The relationship between RNA catalytic processes

Proposals that an RNA-based genetic system preceeded DNA, stem from the ability of RNA to store genetic information and to promote simple catalysis. However, to be a valid basis for the RNA world, RNA catalysis must demonstrate or be related to intrinsic chemical properties which could have existed in primordial times. We analyze this question by first classifying RNA catalysis and related processes according to their mechanism. We define: (A) thedisjunct nucleophile class which leads to 5-phosphates. These include Group I and II intron splicing, nuclear mRNA splicing and RNase P reactions. Although Group I introns and its excision mechanism is likely to have existed in primordial times, present-day examples have arisen independently in different phyla much more recently. Comparative methodology indicates that RNase P catalysis originated before the divergence of the major kingdoms. In addition, alldisjunct nucleophile reactions can be interrelated by a proposed mechanism involving a distant 2-OH nucleophile. (B) theconjunct nucleophile class leading to 3-phosphates. This class is composed of self-cleaving RNAs found in plant viruses and the newt. We propose that tRNA splicing is related to this mechanism rather than the previous one. The presence of introns in tRNA genes of eukaryotes and archaebacteria supports the idea that tRNA splicing predates the divergence of these cell types.     

Characterization of the calcium sensitivity of differentiation in SCC-13 human squamous carcinoma cells

Summary The sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of regulation at the level of mRNA accumulation or translation rather than posttranslational modification. This research was supported by Public Health Service grant AR 27130 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, and National Research Service postdoctoral fellowship ES 05336 from the National Institute of Environmental Health Sciences, Research Triangle Park, NC.     

Effect of elastin peptides on cell proliferation]

Elastin peptides were shown to act on a cell membrane receptor coupled to a G-protein, phospholipase C, and its activation increases IP3 and DAG and opens receptor-dependent Ca(++)-channels. As some growth factors also produce similar modifications in intracellular Ca++, we wanted to explore the effect of elastin peptides on cell proliferation using 3H-thymidine incorporation and cell counting. The concentration of peptides needed for the stimulation of cell proliferation varied between large limits (1 microgram/ml to 10 mg/ml) according to the origin of the cells and the nature of the peptides. The proliferation of CCL 39 chinese hamster lung fibroblasts was enhanced in a dose-dependent fashion in the concentration range of 3 to 10 mg/ml. The proliferation of human skin fibroblasts was enhanced in the concentration range of 0.5 to 3.3 mg/ml and inhibited at higher concentrations. This effect depended little on the average molecular weight (MW) of the peptide preparation, high MW peptides (average 75 kDa) and lower MW peptides (average MW 10 kDa) were both efficient approximately to the same extent. It appears probable that only a small fraction of these peptides possesses this growth promoting property; other sequences might have the opposite effect. The conformation of the peptides may also play an important role. Human sera contain circulating elastin peptides in the concentration range of 1.0 to 10 micrograms/ml, increasing in obstructive arteriopathies and in some hyperlipidemias. It appears therefore that the above findings may have physiopathological significance in the regulation of cell proliferation in normal and pathological conditions.     

Demographic responses of arctic hares Lepus arcticus placed on two predominantly forested islands in Newfoundland

During January-April 1989. we monitored survival, reproduction, and body condition of 19 radio-collared arctic hares Lepus areticus introduced to two predominantly (80%) forested islands. Merchant (66 1 ha) and Burke (82 6 ha), in Placentia Bay, Newfoundland By late April, bone-marrow fat (42 7%) and kidney fat indices were lower than found in populations on the mountain barrens of western Newfoundland However, most island hares gained weight overwinter, and litter sizes (mean. 4 2 in utero) and testis weights (mean, 8 3 g) were as large or larger than recorded from other introduced and natural populations in Newfoundland The distribution of telemetry locations, tracks and feeding sites indicated that hares frequented the scattered barrens (totalling c 30 ha) on both islands in greater proportion than available These results suggest that, in the absence of snowshoe hares and mammalian predators, forested regions interspersed with small patches of barrens can sustain arctic hares     

Rapid acquisition of three-dimensional triple-resonance experiments using pulsed field gradient techniques

Summary A rapid method for recording three-dimensional triple-resonance experiments utilising pulsed field gradient techniques is proposed, and applied to the HNCO experiment. In order to optimise the sensitivity of the method, a short phase cycle is used in conjunction with the pulsed field gradients to select the desired coherence transfer pathway. The method is demonstrated for the HU protein.     

Identification and sequence of a Na+-linked gene from the marine bacterium Alteromonas haloplanktis which functionally complements the dagA gene of Escherichia coli

A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli. We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation. The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955). Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven. Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E. coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters.     

Oestrogen receptor gene structure and function in breast cancer

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoRI, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.