Prostate androgen receptor: immunohistological localization and mRNA characterization

Four androgen receptor (AR) specific monoclonal antibodies were used for the immunohistochemical localization of AR in the human prostate tissue. The prostate tissue consisted of alveoli embedded in fibromuscular stroma and lined with a single layer of columnar secretory epithelial cells. The immunoreactive ARs were found predominantly in the nuclei of epithelial cell, suggesting ARs, like estrogen receptors and progesterone receptors, are mainly nuclear proteins. Northern blot hybridization showed that AR mRNA is about 9 kilobases (kb) and relative abundant in the androgen-sensitive organs, such as ventral prostate, dorsolateral prostate and seminal vesicle.     

Identification and expression of a human cytomegalovirus early glycoprotein.

A human cytomegalovirus early gene which possesses three temporally regulated promoters is located in the large unique component of the viral genome between 0.054 and 0.064 map units (C.-P. Chang, C.L. Malone, and M.F. Stinski, J. Virol. 63:281-290, 1989). This gene contains a major open reading frame (ORF) located 233 bases downstream of the cap site of an early unspliced RNA. The major ORF predicts a polypeptide of 17 kilodaltons (kDa) which contains a glycoproteinlike signal and anchor domains as well as potential N-glycosylation sites. Antisera were prepared against synthetic peptides derived from amino acid sequences within the major ORF. The antisera detected a viral glycoprotein of 48 kDa in infected cells and recognized the in vitro-translated 17-kDa protein early-gene product. The viral glycoprotein, designated gp48, was modified by N-linked glycans and possibly O-linked glycans. The synthesis of gp48 occurred in the absence of viral DNA replication but accumulated to the highest levels at late times after infection. Since gp48 was found in the virion, it is considered an early structural glycoprotein.     

Cloning of a creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate.

A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proteus mirabilis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was observed in recombinant E. coli by minicell analysis.     

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.     

Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.

We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.     

Assay for L-pipecolate oxidase activity in human liver: detection of enzyme deficiency in hyperpipecolic acidaemia.

A direct assay method is described for L-pipecolate oxidase. The assay uses NaHSO3 to trap the L-alpha-amino [3H]adipate delta-semialdehyde (AAS) formed as a direct reaction product of L-pipecolate oxidase from L-[3H]pipecolic acid. The adduct so formed was separated from the substrate on Dowex 50 (H+) column. The product was identified as [3H]AAS by amino acid analysis after breaking down the adduct by boiling under acidic conditions. The assay is simpler and more specific than fluorometric methods; it is also more sensitive, requiring at most 16 micrograms of liver peroxisome-enriched protein per assay. We have used this assay procedure to detect L-pipecolate oxidase in skin fibroblasts obtained from a control subject and from patients of hyperpipecolic acidaemia and Zellweger syndrome and found that this enzyme activity is present in the control, but absent or decreased in the patients with the peroxisomal disorders.     

Increased shikonin production inLithospermum erythrorhizon suspension cultures within situ extraction and fungal cell treatment (elicitor)

Summary The effect ofin situ extraction and elicitor treatment on shikonin production was studied with the suspension cultures ofLithospermum erythrorhizon. Shikonin concentration of 60 mg/L was achieved by the use of both techniques which was 24 times higher than that of control culture, and 65 times higher in terms of shikonin productivity. The host-pathogen effect of elicitor treatment andin situ extraction for product removal were effective for shikonin production.     

Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryote Dictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication in D. discoideum, provided that the Rep gene, which acts in trans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp "TGTCATGACA" palindromes separated by a poly(T.A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Oxidation and cleavage of 3-aminopyridine adenine dinucleotide phosphate with periodate

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate in the neutral pH resulted in oxidation of the ribose linked to 3-aminopyridine and cleavage of the dinucleotide into adenosine- and 3-aminopyridine-containing moieties. Separation of these moieties was afforded by thin-layer chromatography, high-performance liquid chromatography, and fast protein liquid chromatography. From fast atom bombardment mass spectra and nuclear magnetic resonance spectra, the adenosine-containing moiety was identified as 2'-phosphoadenosine 5'-phosphate while the aminopyridine moiety was present in a mixture of the hydrated 3-aminopyridine mononucleotide/nucleoside dialdehyde. Separation of the completely oxidized product by Pharmacia fast protein liquid chromatography gave three major peaks corresponding to 2'-phosphoadenosine 5'-phosphate, 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside, with minor amount of oxidized 3-aminopyridine mononucleotide. Thus the oxidized 3-aminopyridine adenine dinucleotide phosphate was shown to cleave by two pathways: it may either undergo beta-elimination to give 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside; or the phosphodiester linkage may be hydrolyzed to give 2'-phosphoadenosine 5'-phosphate and oxidized 3-aminopyridine mononucleotide. The latter compound may further undergo beta-elimination and eventually give oxidized 3-aminopyridine nucleoside. Hydrolysis could be prevented by storing the sample as lyophilized powder, while beta-elimination was diminished by lowering the storage temperature. We found that the lyophilized powder of oxidized 3-aminopyridine adenine dinucleotide phosphate can be stored at -50 degrees C for several months with minimum decomposition.     

植物冷锻炼对于胁强敏感度的影响(英文)

前文由柑桔枝条在不同低温下、不同冷冻时间的电解质外渗测定,提出胁强(stress)、作用时间与胁变(strain)之间关系的数学模型。在这个模型中共有3个参数:屈服点温度(yield point temperature),胁强敏感度(stress sensitivity)和作用时间敏感度(sensitivity to duration),用以描述植物的抗性。抗性强的植物应表现为屈服点温度较低,胁强敏感度或者时间敏感度较低。为验证此数学模型,本工作以经冷锻炼与未经冷锻炼的盆栽柑桔枝条为材料,作不同温度与时间处理的电解质外渗率的测定,研究了冷锻炼对于上述3个参数的影响。发现胁强敏感度和屈服点温度受冷锻炼影响而下降,时间敏感度未表现明显变化。对于田间柑桔、油桐与毛竹的定期测定,在固定冷冻时间下,得到了类似于盆栽柑桔的结果。入冬时,植物抗冻性提高,3种植物都表现出下列两种变化:1.胁强敏感度的明显下降;2.屈服点温度和/或时间敏感度亦下降。开春时的变化则相反。胁强敏感度的变化与后一种变化有各自的规律,且因植物种类而不同。拐点胁强(stress at inflection point)具有与半致死温度(50％killing point temperature)不同的意义,它的变化是上述两种变化的综合结果。本试验结果表明,冷锻炼对于植物胁强敏感度有明显影响,用本数学模型的3个抗性指标描述