Sulfide Production from Cysteine by Desulfovibrio desulfuricans

Two rumen nitrate-reducing isolates of Desulfovibrio desulfuricans were found to hydrolyze cysteine with the production of sulfide and pyruvate. When cultured on agar medium containing yeast extract with nitrate as the primary electron acceptor and ferrous chloride as the indicator, blackening of colonies occurred. The blackening of colonies appeared sooner and was more intense when either cysteine or sulfate was added to the culture medium with nitrate present.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose.

Fibrobacter succinogenes subsp. succinogenes S85, formerly Bacteroides succinogenes, adheres to crystalline cellulose present in the culture medium. When the cells are suspended in buffer, adhesion is enhanced by increasing the ionic strength. Heat, glutaraldehyde, trypsin, and pronase treatments markedly reduce the extent of adhesion. Treatment with dextrinase, modification of amino and carboxyl groups with Formalin or other chemical agents, and inclusion of either albumin (1%) or Tween 80 (0.5%) do not decrease the degree of adhesion. Adherence-defective mutants isolated by their inability to bind to cellulose exhibited different growth characteristics. Class 1 mutants grew on glucose, cellobiose, amorphous cellulose, and crystalline cellulose. Class 3 mutants grew on glucose and cellobiose but not on amorphous or crystalline cellulose. No substantial changes were detected in the endoglucanase, cellobiosidase, and cellobiase activities of the wild type and the mutants. These data suggest that adhesion to crystalline cellulose is specific and that it involves surface proteins.     

Treatment with different antiestrogens in the neonatal period and effects in the cervicovaginal epithelium and ovaries of adult mice: a comparison to estrogen-induced changes

Female mice of the NMRI strain were treated for the first 5 days after birth with the following compounds: diethylstilbestrol (DES), MER-25 (ethamoxytriphetol), tamoxifen, ICI 47.699 (the cis-isomer of tamoxifen, an estrogen agonist), clomiphene, nafoxidine or 17 beta-estradiol-3-benzoate (E2). Females were killed at 8 wk or 6 mo and, in the case of tamoxifen also at 12 mo. The cervicovaginal region and the ovaries were prepared for histological studies. MER-25 had no effect on either the cervicovaginal epithelium or ovarian histology. Tamoxifen, clomiphene and nafoxidine resulted in extensive regions with a heterotopic columnar epithelium (HCE) in the cervicovaginal preparations. At 8 wk these regions were more widespread than those observed after treatment with DES and E2. While earlier studies have shown a progressive development of DES-induced HCE, that induced by the antiestrogens regressed with time. All ovaries from adult females treated with DES or E2 lacked corpora lutea. For the antiestrogens there were ovaries with or without corpora lutea, and this treatment was not incompatible with fertile females. It is concluded that in the neonatal period, the cervicovaginal epithelium is more sensitive to antiestrogens than central structures (hypothalamic nuclei), but for DES the opposite is true.     

Mixing patterns in Amazon lakes

The diel mixing patterns of two small floodplain lakes, Lago Jacaretinga in the Amazon drainage, and Lago Cristalino in the Rio Negro system, were investigated during both the high-water and low-water states of the Amazon River hydrograph. Measurements included temperature, oxygen, ammonia, phosphate, and chlorophyll. In both lakes thermal stratification developed during the day and was eroded at night. During the low-water period when the lakes were shallow, nocturnal circulation extended to the lake bottom, whereas when the lakes were deeper (greater than about 5 m), circulation did not reach the bottom and an anoxic hypolimnion developed. During the low-water period, percent of oxygen concentrations were relatively high but always less than saturation. Low oxygen concentrations were observed during the high-water period. At all times nocturnal mixing supplied a significant amount of oxygen to the lake ecosystems. Nighttime upward mixing of recycled nitrogen and phosphorus also appeared to be important nutrient sources for algal productivity.     

Steroid synthesis in ovarian homogenates from immature mice treated with diethylstilboestrol in neonatal life

Female mice of the NMRI strain were treated with the synthetic oestrogen diethylstilboestrol (DES) for the first 5 days after birth. Pools of ovaries were removed from groups of 6-, 12-, 21-, 28- and 56-day-old females. An homogenate of an ovarian pool was incubated for 1 h in the presence of [3H]pregnenolone. Synthesized steroids were extracted and separated in a two-dimensional thin-layer chromatography system. Homogeneity of tentative steroids was verified with recrystallization to constant specific activity. Synthesis of [3H]progesterone and [3H]testosterone was demonstrated at 6 days, [3H]androstenedione at 12 days, [3H]17 alpha-hydroxyprogesterone at 21 days, and [3H]oestradiol-17 beta at 28 days. Up to 28 days (21 days for progesterone), the synthetic activity was lower in homogenates of DES-exposed ovaries than in control homogenates. After 28 days, values for recovered [3H]progesterone, [3H]androstenedione and [3H]oestradiol-17 beta were higher in DES homogenates than in control homogenates while the reverse was true for [3H]17 alpha-hydroxyprogesterone and [3H]testosterone. The results are compatible with an early and direct DES inhibitory effect on ovarian steroidogenesis and, later in immature life, a DES-induced disruption of the normal FSH-LH stimulation of ovarian development.     

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.