The Rex proteins of human T-cell leukemia virus type II differ by serine phosphorylation.

The Rex proteins of human T-cell leukemia virus types I and II (HTLV-I and HTLV-II) induce cytoplasmic expression of unspliced gag-pol mRNA and singly spliced env mRNA and are critical for virus replication. Two rex gene products, p27rex and p21rex of HTLV-I and p26rex and p24rex of HTLV-II, have been detected in HTLV-infected cells; however, the structural and biological relationship of the proteins has not been clearly elucidated. Endoproteinase digestion and phosphoamino acid analysis of HTLV-II Rex indicated that p24rex has the same amino acid backbone as p26rex and that the larger apparent molecular size of p26rex is attributable to serine phosphorylation.     

Splenocyte subsets in normal and protein malnourished mice after long-term exposure to cocaine or morphine

An experimental model which resembles human drug addiction was developed to study the effect of chronic drug (cocaine or morphine) administration on the immune system. As malnutrition has been associated with drug use, a low protein diet has been evaluated for its contribution to the impairment of the immune system during cocaine/morphine addiction. Female C57BL/6 mice that received a 20% or 4% casein diet were studied. Both drugs were administered intraperitoneally daily for 11 weeks and drugs were administered in increasing daily doses, beginning after 3 weeks of diet consumption. Doses of cocaine began with 5 mg/kg body weight and reached the maximum dose of 40 mg/kg/day at the fourth week. Doses of morphine gradually increased from 10 mg/kg to 75 mg/kg body weight with the maximum dose reached after 5 weeks of treatment. Cocaine administration reduced body weight, particularly in the low protein diet group, and spleen weight in protein malnourished mice. Cocaine as well as saline injected mice showed a decrease in the percentage of CD4+ CD8+ and Mac-1+ cells and an increase in B cells in the spleens of well nourished mice. Morphine-treated mice showed similar results to those observed in cocaine or saline treated mice. These results suggest that cocaine, morphine or saline injection can alter the percentage of cells that express a defined phenotype independently of the nutritional status of the subject. Moreover, the effect appears dependent on a stress mediated process.     

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.     

Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.     

Simultaneous bilateral forearm revascularization

A successful simultaneous bilateral forearm revascularization was performed on a 17-year-old boy. Functional recovery of both forearms was evaluated 42 months after injury. The patient can use both hands for the activities of daily living. So far, he has been employed and has no significant psychological problems. Temporary intraluminal silicone shunts are extremely helpful for reducing ischemic damage to the injured limb. The sufficient skeletal shortening of the upper limb replantation is crucially important. The wounds must be managed by aggressive and repeated debridement. Accurate primary nerve repair is essential, and the early postoperative rehabilitation is also important to achieve a satisfactory functional return. The functional replanted or revascularized upper extremity is superior to an amputation or prosthesis, especially in the cases of bilateral upper extremity amputation or devascularization.     

Photoperiod control of poplar bark storage protein accumulation

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.     

3,4-Dihydroxyxanthone dioxygenase from Arthrobacter sp. strain GFB100.

Bacterial extradiol ring-fission dioxygenases play a critical role in the transformation of multiring aromatic compounds to more readily biodegradable aromatic or aliphatic intermediates. Arthrobacter sp. strain GFB100 utilizes an extradiol meta-fission dioxygenase, 3,4-dihydroxyxanthone dioxygenase (DHXD), in the catabolism of the three-ring oxygen heterocyclic compound xanthone. In this paper, we show that DHXD is a cytosolic enzyme, induced by growth on xanthone and maximally expressed during the stationary phase of growth. In addition, we characterize the DHXD activity in terms of its basic enzymological properties. 1,10-Phenanthroline and H2O2 treatments eliminated DHXD activity, indicating that the enzyme required Fe2+ ions for activity. Other divalent cations were either inhibitory or had no effect on activity. DHXD had a temperature optimum of 30 degrees C and a pH optimum of 7.0. DHXD followed typical saturation kinetics and had an apparent Km of 10 microM for 3,4-dihydroxyxanthone. The dye celestine blue served as a noncompetitive DHXD inhibitor (Ki, 5 microM). Several other structural analogs served neither as substrates nor inhibitors. DHXD was thermally labile at temperatures above 40 degrees C. The half-life for thermal DHXD inactivation was 5 min at 40 degrees C. DHXD activity was completely stable through one freeze-thaw cycle, and about 80% of the DHXD activity remained after 2 days of incubation at 0 degree C. The apparent tight binding of the Fe2+ cofactor to DHXD may be a factor contributing to the stability of this extradiol dioxygenase when it is stored.     

Kinetic resolution of N-protected amino acid esters in organic solvents catalyzed by a stable industrial alkaline protease

Summary An industrial alkaline protease Alcalase has been found to be very stable in organic solvents and usable as a catalyst for resolution of N-protected amino acids, in both aqueous solution and organic solvent with high yield and optical purity. Only the L-amino acid ester has been hydrolysed.Abbreviation Cbz- carbobenzyloxy- - OMe methyl ester - Hop homophenylalanine - Nol norleucine - Aba -amino butyric acid - Nov norvaline - Fug furylglycine     

Irreversible inactivation of diacylglycerol kinase-II requires a mediator.

Cytosolic diacylglycerol kinase was irreversibly inactivated by 5'-AMP since the enzyme remained less active after the removal of 5'-AMP by P-10 gel chromatography. The inactivation was time-dependent, suggesting the involvement of a covalent bond modification. A reconstitution experiment detected a rat brain cytosolic mediator for the effect of 5'-AMP. A protein kinase rich fraction prepared from rat liver was also capable of restoring the sensitivity of diacylglycerol kinase-II to 5'-AMP. We propose that 5'-AMP-activated protein kinase is the mediator which inactivates diacylglycerol kinase-II, possibly by phosphorylation.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis