The kinetics of haemolysis of spherocytic erythrocytes

Spherocytosis is a hereditary disease. It results from mutations in genes that encode proteins participating in the attachment of the membrane skeleton to the plasma membrane bilayer of the erythrocyte. In affected cells, interaction between the spectrin-actin meshwork and integral membrane proteins is altered. This results in the weakening of plasma membrane mechanical resistance and diminishing its elasticity. Since defective cells are prone to mechanical destruction and phagocytosis in the spleen, the fraction of morphologically-altered erythrocytes is rather small; this in turn means such an examination is prone to errors. In this paper, we describe a simple method which could be useful in the identification of red blood cells with altered osmotic properties. The method is based on the measurement of the amount of light scattered by a suspension of the red blood cells, during which cells are exposed to osmotic stress in the stopped-flow regime. The obtained plots are fitted to a mathematical formula, the parameters of which can be used as quantitative indicators of the changes in red blood cells' osmotic features. Two types of spherocytotic samples were examined: those with a proven deficiency in ankyrin and those with a decrease in the band 3 anion transporting protein. The presented data show that this method gives a reliable indication of altered osmotic properties of the spherocytic cells.     

Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil

A highly sensitive and specific PCR-based method of monitoring 16S rRNA genes of Pseudomonas stutzeri was developed for searching P. stutzeri DNA in environmental samples. This monitoring was combined with a reliable and sensitive method for isolating P. stutzeri colony formers from soil and sediment, depending on their utilization of ethylene glycol, starch, and maltose. With these techniques, P. stutzeri populations (n = 2 to 170) were obtained from five of six sites giving positive PCR signals (including three marine sediment and two soil samples). The phylogenetic positions of isolates from the five sites, based on their 16S ribosomal DNA sequences, indicated that the environmental isolates were affiliated with different genomovars of P. stutzeri. Using the broad-host-range plasmid pNS1 with kanamycin and gentamicin resistance determinants as the transforming DNA, naturally transformable strains were identified among the isolates from all sites. For one population from soil, the genetic relationship of the 120 members was determined by randomly amplified polymorphic DNA-PCR with three PCR primers. Among the population members which are taxonomically closely related as determined by 16S sequence comparisons of group representatives, a rather high genetic diversity and a characteristic clustering into subgroups were found. Remarkably, within the population, nontransformability and different levels of transformability (a frequency between about 10(-9) and 10(-4) per cell) were often associated with distinct genetic subgroups. It is concluded that transformability is widespread among environmental P. stutzeri strains and that its specific level is a heritable trait that may vary strongly within a local population.     

Computer simulations of de novo designed helical proteins.

In the context of reduced protein models, Monte Carlo simulations of three de novo designed helical proteins (four-member helical bundle) were performed. At low temperatures, for all proteins under consideration, protein-like folds having different topologies were obtained from random starting conformations. These simulations are consistent with experimental evidence indicating that these de novo designed proteins have the features of a molten globule state. The results of Monte Carlo simulations suggest that these molecules adopt four-helix bundle topologies. They also give insight into the possible mechanism of folding and association, which occurs in these simulations by on-site assembly of the helices. The low-temperature conformations of all three sequences have the features of a molten globule state.     

Isolation and purification of proteins from the symbiosome membrane of yellow lupine root nodules

A unique feature of the symbiotic association between legume plants and rhizobia is the plant-derived membrane which separates the symbionts within root nodule; this membrane is termed the peribacteroid membrane (PBM). Although this membrane plays a vital role in facilitating transport and other processes in nodules, little is known about the proteins that are associated with and are an integral part of it. The objective of this work was to apply modern methods of protein purification to the characterisation of proteins of peribacteroid membrane from nodules of yellow lupine (Lupines luteus). The 17-kDa protein was isolated from purified peribacteroid membrane using size exclusion and ion exchange chromatography (FPLC). The N-terminal amino acid sequence of this protein was determined; the sequence does not match any of the previously reported lupine and other legume sequences. Following detergent solubilisation of purified peribacteroid membrane, integral proteins of 15 to 20 kDa were purified by size exclusion chromatography.     

Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli.

Recombinant DNA techniques were used to isolate the Rickettsia prowazekii citrate synthase gene on the plasmid vector pBR322 by functional complementation of a gltA mutation of Escherichia coli K-12. Analysis of citrate synthase activity in crude extracts revealed that the enzyme expressed in E. coli retains the regulatory control mechanisms characteristic of the rickettsial enzyme.     

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

NAD⁺ levels in resting human lymphocytes obtained from 20 donors were found to be 69.9 ± 21.7 pmols/10⁶ cells. After 3 days of phytohemagglutinin (PHA) stimulation the NAD⁺ levels rose to 452 ± 198 pmols/10⁶ cells. NADH, NADP⁺ and NADPH also increased in mitogen-stimulated lymphocytes, but the major portion of the increase in total pyridine nucleotide pools was accounted for by the increase in NAD⁺. When PHA-stimulated lymphocytes were incubated in nicotinamide-deficient growth medium, there was no significant increase in their total pyridine nucleotide pools; however, the ratios of oxidized to reduced pyridine nucleotides changed in a similar fashion to cells grown in medium containing nicotinamide. When lymphocytes in nicotinamide-deficient medium were stimulated with PHA they increased their levels of DNA synthesis and cell replication in a similar fashion to cells growing in nicotinamide-supplemented media. Human lymphocytes were able to synthesize pyridine nucleotides from nicotinamide or nicotinic acid; however, in the absence of a preformed pyridine ring they did not efficiently use tryptophan for the synthesis of NAD. Uptake of [carbonyl-¹⁴C]nicotinamide and conversion to NAD was markedly increased in PHA-stimulated lymphocytes; these cells also showed a marked increase in activity of the enzyme adenosine-triphosphate-nicotinamide mononucleotide (ATP-NMN) adenylyl transferase.     

Discovery of novel heteroaryl-substituted chalcones as inhibitors of TNF-alpha-induced VCAM-1 expression

Novel chalcone derivatives have been discovered as potent inhibitors of TNF-alpha-induced VCAM-1 expression. Thienyl or benzothienyl substitution at the meta-position of ring B helps boost potency while large substitution at the para-position on ring B is detrimental. Various substitutions are tolerated on ring A. A lipophilicity-potency relationship has been observed in several sub-series of compounds.     

Crystal structure of methyl 3-amino-2,3-dideoxy-beta-D-arabino-hexopyranoside. Stabilization of the crystal lattice by a double network of N-H...O, O-H...N and O-H...O interactions

The structure, conformation and configuration of methyl 3-amino-2,3-dideoxy-beta-D-arabino-hexopyranoside were investigated by (1)H NMR, (13)C NMR and IR spectroscopy, as well as by optical rotation. The crystal structure was confirmed by single-crystal X-ray crystallographic analysis at 293 K and R = 0.0434 based on 910 independent reflections. The crystal belongs to the monoclinic system, space group of P2(1) with cell dimensions a = 6.050(1) Angstroms, b = 7.284(1) Angstroms, c = 10.289(2) Angstroms, beta = 104.69(3) degrees, D(c) = 1.341 Mg cm(-3) and V = 438.9(1) Angstroms(3) for Z = 2. Furthermore, the molecule has a typical (4)C(1) chair conformation. Hydrogen bonds between sugar molecules are responsible for stabilizing the crystal lattice.