The synergistic lethal interaction of cis-diamminedichloroplatinum and natural nucleosides is related to increased DNA cross-links

Human tumor cells were treated in vitro with combinations of cis- or trans-dichlodiammineplatinum (DDP) and natural nucleosides (thymidine, uridine, cytidine and adenosine). Effects were measured by inhibition of colony-formation (cell survival) and DNA alkaline elution (DNA cross-links). No increments in cell lethality or DNA cross-links were elicited by any combination of trans-DDP and nucleosides. In contrast, every combination of cis-DDP and nucleoside was eminently synergistic with 5- and 10-fold increases in cell lethality over the predicted sum of each agent alone. These increments in cell kill correlated linearly with increases in DNA crosslinks suggesting that the nucleosides interact with cis-DDP to enhance its cytotoxic crosslinking mode of action.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons.

Apoptosis is a prominent mechanism of programmed cell death in lymphocytes and in cancer cells not previously found in neurons. We have identified apoptosis and internucleosomal DNA degradation in cultures of cerebellar granule neurons. 1-methyl-4-phenylpyridinium, a selective neurotoxin that destroys the dopaminergic nigrostriatal pathway and results in a parkinsonian syndrome, increases the rate of apoptosis and kills cerebellar granule cells in culture via induction of programmed cell death. Inhibition of gene expression in granule cells with cycloheximide prevents the MPP(+)-induced apoptosis and the DNA fragmentation. Our findings demonstrate a new pathway of neuron death and suggest the possibility that neurodegenerative diseases may result from the inappropriate activation of programmed cell death by apoptosis.     

Multiple synthesis by the multipin method as a methodological tool

The multipin method of peptide synthesis is demonstrated as a potent methodological tool, where large numbers of comparative studies can be performed concurrently. Two studies are presented. In each study, the test peptides were simultaneously synthesized, and the products examined by high throughput ion spray mass spectrometry and reverse-phase HPLC. In the first study, comprising 24 experiments, peptides 1 (AELFSTHYLAFKEDYSQ-NH₂) and 2 (LKDFRVYFREGRDQLWKGPG-NH₂) were prepared using Fmoc-Axx/BOP/HOBt/NMM (100: 100: 100: 150 mM ) and Fmoc-Axx/HATU/HOAt/NMM (100: 100: 100: 150 mM ) with 60.90 and 120 min coupling times. The two reagent combinations were found to give comparable results. The second study compared the N-terminal coupling of Fmoc-Asn-OH, Fmoc-Asn(Mbh)-OH, Fmoc-Asn(Mtt)-OH, Fmoc-Asn(Tmob)-OH and Fmoc-Asn(Trt)-OH in the synthesis of seven test peptides: 3 , NVQAAIDYIG-cyclo(Kp); 4 , NTVQAAIDYIG-cyclo(KF); 5 , NRVYVHPFNL; 6 , NRVYVHPFHL: 7 , NEAYVHDAPVRSLN: 8 , NQLVVPSEGLYLIYSQVLFK. 9 , NPNANPNANPNA. A total of 33 experiments were performed. Peptides 3 and 4 were selected to highlight the effect of steric bulk of each Asn derivative on coupling efficiency. Reagent efficiency, as measured by target peptide purity, was as follows: Fmoc-Asn(Tmob)-OH > Fmoc-Asn-OH > Fmoc-Asn(Mtt)-OH = Fmoc-Asn(Trt)-OH > Fmoc-Asn(Mbh)-OH.     

Locomotory activity of epithelial cells in culture.

The movement of epithelial cells in vitro has been studied with time lapse cinemicrography, micromanipulation, marking of the cell surface, and electron microscopy. The cells, in contrast to fibroblasts, spread as contiguous sheets. Locomotion results primarily from the activity of the marginal cells, as determined by the extent and location of cell adhesions to the plane substratum. The locomotory activity of epithelial cells as members of a sheet is similar to that of chick heart fibroblasts, consisting of a fluctuation of the flattened free edge, a backward movement of particles adhering to the upper surface of the lamellipodium, ruffling, blebbing, and microspike activity. Of these, only the first two are invariably associated with movement. These phenomena are discussed in relation to the mechanism of epithelial cell movement. The basic differences between epithelial cells and fibroblasts, as far as locomotory and adhesive properties are concerned, are the tendency of isolated epithelial cells to bleb more vigorously than fibroblasts and the more extensive and apparently stronger lateral adhesion of epithelial cells.     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

<Emphasis Type="Italic">Salinivibrio sharmensis</Emphasis> sp. nov., a novel haloalkaliphilic bacterium from a saline lake in Ras Mohammed Park (Egypt)

A novel haloalkaliphilic, facultative anaerobic and Gram-negative Salinivibrio-like microorganism (designated strain BAG^T) was recovered from a saline lake in Ras Mohammed Park (Egypt). Cells were motile, curved rods, not spore-forming and occurred singly. Strain BAG^T grew optimally at 35°C (temperature growth range 25–40°C) with 10.0% (w/v) NaCl [NaCl growth range 6.0–16.0% (w/v)] and at pH 9.0 (pH growth range 6.0–10.0). Strain BAG^T had phosphatidylethanolamine (PEA) and phosphatidylglycerol (PG) as the main polar lipids, C16:0 (54.0%) and C16:1 (26.0%) as the predominant cellular fatty acids and Q-8 as the major respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BAG^T was a member of Salinivibrio genus, with the highest sequence similarities of 99.1, 98.4 and 98.1% to Salinivibrio siamensis JCM 14472^T, Salinivibrio proteolyticus DSM 19052^T and Salinivibrio costicola subsp. alcaliphilus DSM 16359^T, respectively. DNA–DNA hybridization values of strain BAG^T with members of Salinivibrio genus were lower than 55.0%. DNA G + C content was 51.0 mol%. On the basis of the polyphasic taxonomic results revealed in this study, strain BAG^T should be classified as a novel species of Salinivibrio genus, for which the name Salinivibrio sharmensis sp. nov. is proposed, with the type strain BAG^T (=ATCC BAA-1319^T = DSM 18182^T).