Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K_m for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a K_m for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.     

Ovine fetal electrocortical activity and regional cerebral blood flow

Fetuses of 12 near-term sheep were prepared for microsphere determination of cerebral blood flow. Experiments were performed 5 days postsurgery. The regional blood flows were measured in successive high (HV), low (LV) and high voltage electrocorticographic states. Comparisons were made between the observations made in the LV and averaged flanking HV cycles. Total cerebral blood flow was 95 +/- 8, 119 +/- 11 and 100 +/- 9 ml/min/100 g in HV, LV and HV, respectively. Low voltage electrocortical activity increased average cerebral blood flow by 22% (P less than 0.01). Significant changes were seen in all regions except the occipital cortex. The maximum change was observed in the thalamus in which the flows were 152 +/- 23, 243 +/- 35 and 138 +/- 20 ml/min/per 100 g tissue, respectively. The increase was 68% (P less than 0.001). The percent changes seen in the cerebrum are as follows: Frontal grey + 18%, frontal white + 22%, parietal white + 22%, temporal + 18%. A + 17% change was seen in the cord (P less than 0.03). It is concluded that in low voltage electrocortical activity all of the brain, except the occipital region, shows an increase in cerebral blood flow. This is probably secondary to a variance in cerebral activity. This preparation may be useful in localizing function in the fetal brain.     

Interaction and combination of separate A and B chains of insulin effects of D-and L-tryptophan in position A1

The S-thiomethyl derivatives of insulin A chain with A1-Gly replaced by D- or L-Trp have been prepared and their respective interaction and combination with the S-thiomethyl B chain studied. The UV difference spectra of the mixed against the separated [Trp1]A chains with the B chain at pH 10.8 are similar to those obtained for the unmodified chains except that the 295-nm-negative peak for ionized Tyr residue appears to be less marked. Fluorescence studies show very little environmental changes at the A1-Trp residues when mixed with the B chain. The intact hormone with A1-Gly replaced by D-Trp is known to be considerably more active than the analog with L-Trp replacement. However, for both derivatives the resynthesis of the whole molecules correctly joined by disulfide bridges starting from the separated reduced chains, gives similar low yields as shown by HPLC analysis and by receptor-binding assay. The replacement of A1-Gly by D-Trp appears to affect the separated A chain more than the intact hormone and replacements at A1 by both D- and L-Trp probably lead to significant conformational changes of the A chain so as to prevent its correct pairing with the B chain.     

Cleavage of territrems by alkaline hydrogen peroxide: Isolation and characterization of the aromatic moiety of territrems

The chemical reaction of cleavaging territrem B to give 3,4,5-trimethoxy benzoic acid by alkaline hydrogen peroxide was investigated. The method was applied for confirmation of the chemical structure of the aromatic moiety of territrem A, A’, B, and B’. The physicochemical properties of the aromatic cleavage product of territrem Aindicated the structure as 3,4-methylendioxy, 5-methoxy benzoic acid (or 4-methoxy, 6-carboxy, 1, 3-benzodioxole). The experiment also gave the evidences that territrem A and A’, on the other hand territrem B and B’ have the identical aromatic moieties on their structures.     

Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia

The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion.     

兜兰胚胎学的研究

云南兜兰花药壁有五层细胞,绒毡层细胞具双核,属分泌型。小孢子母细胞减数分裂为同时型。四分体中四个小孢子呈四面体或左右对称式排列。成熟花粉为二细胞型。单粒分散的花粉包裹于黄色粘性物质中。生殖细胞最初形成的壁为胼胝质的,待游离到营养细胞质中时,具一层很薄的 PAS 正反应的壁,直到花药开裂时这层壁仍存在。成熟花粉无特化的萌发孔,只具薄壁区。胚珠为薄珠心,具一层珠被,胚囊发育为葱型,成熟胚囊为6—8核,胚发育过程中,具2—4细胞胚柄。胚乳具二核。种子成熟时胚柄及胚乳核都消失。成熟种子只具单层细胞的种皮和一个未分化的球形胚。     

Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.     

A radioimmunoassay for human pro-luteinizing hormone-releasing factor [pro-LRF(14-69)OH]

A radioimmunoassay (RIA) for human pro-LRF(14-69)OH was developed with an antiserum, generated in a rabbit, to [Tyr67]pro-LRF(47-67)NH2 conjugated to BSA. This antiserum bound 28-32% of [125I]pro-LRF(14-69)OH at a final dilution of 1:2500 and the binding was inhibited by pro-LRF(14-69)OH in a dose-dependent manner. The sensitivity of the RIA was 31.2-62.5 pg and the dose that inhibited 50% of the binding to the tracer was 280-320 pg. Intra- and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. Neither LRF nor pro-LRF(14-37)OH was recognized by the antiserum. The dilution curve generated with human hypothalamic extract was parallel to that of pro-LRF(14-69)OH. In addition the extract yielded a major immunoreactive peak emerging in elution volumes concordant with [125I]pro-LRF(14-69)OH on Sephadex G-50 chromatography.     

Influence of α-interferon therapy on blood lymphoid cells

Summary The influence of natural -interferon (-IFN) therapy (3×10⁶ units i.m. daily) on blood lymphoid cells was studied in 20 patients with gynecological neoplasias (7 patients with condylomata accuminata and 13 patients with ovarian carcinoma). There was a statistically significant increase in the intracellular levels of 2'–5'oligoadenylate synthese 1 day after the first injection of IFN and with few exceptions this activity remained increased during 3 months of treatment. In most of the patients, the capacity of blood lymphoid cells to produce IgA, IgG, and IgM following stimulation with pokeweed mitogen was decreased 1 day after the first injection of IFN and with few exceptions it remained low during 6 months of IFN therapy. In most patients there was a decrease in the capacity of lymphoid cells to act as stimulator or responder cells in a mixed lymphocyte culture during IFN therapy. The -IFN therapy had no major influence on the response of lymphoid cells to mitogens. We conclude, that neither this nor our previous studies on the influence of IFN therapy on immunological functions have given support to the hypothesis that the antitumor action of IFN is mediated by the immune system.