Effects of acyl groups and ethanol ratios on lipase-catalyzed regioselective deacylation in peracylated methyl glycopyranosides

Summary Regioselective ethanolysis of peracylated methyl , -D-glucopyranoside and methyl -D-mannopyranoside in anhydrous organic solvent (n-hexane/EtOH = 99/1) could afford 6-OH derivatives exclusively by Candida rugosa lipase (CRL). No 4 6 acyl migration was observed in such an anhydrous solvent system. Substrates with propanoyl groups were more reactive than with acetyl groups on CRL-catalyzed reactions.     

Efficient excision of the upstream large intron from P4-generated pre-mRNA of the parvovirus minute virus of mice requires at least one donor and the 3' splice site of the small downstream intron.

We have previously shown that efficient excision of the upstream large intron from P4-generated pre-mRNA of the autonomous parvovirus minute virus of mice depends upon at least the initial presence of sequences within the downstream small intron (Q. Zhao, R. V. Schoborg, and D. J. Pintel, J. Virol. 68:2849-2859, 1994). In this report, we show that the requirement of downstream small intron sequences is complex and that efficient excision of the upstream intron requires at least one small intron donor and the 3' splice site. In the absence of both small intron donors, a new spliced product is produced in which the intervening exon is skipped and the large intron donor at nucleotide 514 is joined to a small intron acceptor. Exon skipping caused by the loss of the two small intron donors can be overcome, and the excision of the large intron can be regained by mutations that improve the large intron polypyrimidine tract. These results are consistent with a model in which the binding of multiple splicing factors that assemble at both a downstream donor and acceptor facilitates the binding of splicing factors to the weak polypyrimidine tract of the upstream large intron, thereby defining the intervening exon and promoting excision of the upstream intron.     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.     

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSO_A type, as well as with the conversion signal of the Escherichia coli phage X174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

大鼠不同脑区突触体钙水平的年龄差异

本实验使用荧光指示剂Ｆｕｒａ－２与Ｔｂ~（３＋）,检测了不同年龄组大鼠的不同脑区（海马、皮层、间脑、小脑）突触体内游离钙与膜结合钙水平。结果显示,与青年对照组相比,老年大鼠大部分脑区（海马、皮层、间脑）突触体内游离钙水平显著增高,尤其是海马突触体内游离钙增高极为显著;其突触体膜结合钙水平表现为：海马、小脑两脑区明显升高,而皮层、间脑两脑区明显下降,呈现一种全脑范围内的钙水平失衡。提示动物的衰老与其脑内钙自体平衡失调有关。     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.     

Fed-batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor

Fed-batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed-batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. (c) 1995 John Wiley & Sons, Inc.