The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress.

Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. The resulting ghost cells retain Na/K-ATPase activity. alpha-Crystallin was incorporated in the cells on resealing and the activity of Na/K-ATPase assessed by ouabain-sensitive 86Rb uptake. Incubation with fructose, hydrogen peroxide and methylglyoxal (compounds that have been implicated in diabetes and cataract formation) were used to test inactivation of the Na/K pump. Intracellular alpha-crystallin protected against the decrease in ouabain sensitive 86Rb uptake, and therefore against inactivation induced by all external modifiers, in a dose-dependent manner.     

THE TIME-COURSE OF PHOTOADAPTATION TO LOW-LIGHT IN PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE)1

The estuarine dinoflagellate, Prorocentrum mariaelebouriae (Parke & Ballantine 1957) Faust 1974 undergoes increases in pigmentation and photosynthetic efficiency within several days of downward light shifts. These changes can be described by first-order kinetics, as has been reported previously for Chlorophyll (Chl) a in several phytoplankton species. The studies described in this paper were conducted with isolates of populations of Prorocentrum from the Chesapeake Bay. We determined rates of adaptation to low-light for cultures grown at a range of photon flux densities (I₀= 2.65–26.2 E.m^?2, d^?1, shifted to 6.3–7.0% I₀) at three temperatures (10°, 15°, and 20° C), bracketing the conditions this species experiences in situ. In this paper, I report the time-course of changes in α, P_max Chl a, peridinin, and I_k and first-order rate constants, K₁ for changes in α, Chl a and peridinin. cell^?1. K₁ for changes in α cell^?1 averaged 1.58 × 10^?2 h^?1 for conditions encompassing five light treatments and three temperatures; the corresponding mean for Chl a was 1.59 × 10^?2 h^?1. Increases in peridinin measured for five light treatments at 15° C showed a mean K₁ of 1.22 × 10^?2 h^?1, Average percent changes in per cell α, Chl a, and peridinin ranged from 0.4–4.0% h^?1 (10–90% d^?1) following exposure to low-light. Photoadaptive changes are important to Prorocentrum because in nature it occupies turbid waters (K_t≥ 0.5 m^?1) where the mixing depth often exceeds the depth of the photic layer. Cells are entrained beneath a seasonally-stable density discontinuity and are exposed to very low-light (< I E.m^?2.d^?1) for days to weeks during subpycnocline transport. The ability of this species to undergo changes in pigmentation and photosynthetic physiology confers increased efficiency of light harvesting and contributes to this species’survival in the estuary where it is an important component of the dinoflagellate flora.     

PHOTOSYNTHETIC PHYSIOLOGY OF PROROCENTRUM MARIAE-LEBOURIAE (DINOPHYCEAE) DURING ITS SUBPYCNOCLINE TRANSPORT IN CHESAPEAKE BAY1

This paper presents results of field studies on the estuarine dinoflagellate Prorocentrum mariae-lebouriae (Parke & Ballantine) Faust in Chesapeake Bay. We tested the hypothesis that the photosynthetic physiology of Prorocentrum shows adaptive responses to low-light during a lengthy subpycnocline transport in estuarine circulation. Prorocentrum underwent a seasonal, northward trnasport between February and June, 1984 and 1985. Low cell densities occurred in the seaward part of the estuary during winter and early-spring, subpycnocline populations progressed up-estuary in the ensuring 2–3 months, and dense surface populations developed in the mesohaline portion of the estuary thereafter. We sampled Prorocentrum from surface and subpycnocline waters and measured photosynthesis-light (P-I) relations with in situ incubations. The photophysiology of Prorocentrum collected below the pycnoline differed from that of cells in the surface mixed layer in that photosynthetic efficiency, α-cell^?1, was higher, photosynthetic capacity, P_max-cell^?1 was ·4 times greater for subpycnocline (≦ 10m) samples than for those from the surface mixed layer (≧ 6m). Comparison of in situ photosynthetic properties to those generated in laboratory studies showed that values of α·cell^?1 for both surface and subpycnocline samples were in the range found for cultures in low-light. Concentrations of Chls a, c and peridinin·cell^?1 and molar pigment ratios peridinin: Chl a and Chl a: Chl c were not significantly different for the surface and subpycnocline samples, nor were C · cell^?1 or C : Chl a. Chloroplast and starch volume fractions and the number of thylakoids were the same for samples collected at different depths, and there was no evidence of cytoplasmic vacuolization in any field samples. These morphometric data for cells from natural populations of Prorocentrum most closely resembled data for laboratory cultures grown at or near 2.6E·m-^?2·4d^?1. A lower growth irradiance of 0.3E·m^?2·d^?1 produced indications of stress in cultures, including starch depletion and vacuolization, that were never observed in natural populations. Based on the combination of these findings, we conclude that Prorocentrum is adapted to low-light both in the surface mixed layer and beneath the pycnocline, although certain photophysiological characteristics distinguish these two groups of samples.     

Widespread protein sequence similarities: origins of Escherichia coli genes.

To learn more about the evolutionary origins of Escherichia coli genes, we surveyed systematically for extended sequence similarities among the 1,264 amino acid sequences encoded by chromosomal genes of E. coli K-12 in SwissProt release 26 by using the FASTA program and imposing the following criteria: (i) alignment of segments at least 100 amino acids long and (ii) at least 20% amino acid identity. Altogether, 624 extended alignments meeting the two criteria were identified, corresponding to 577 protein sequences (45.6% of the 1,264 E. coli protein sequences) that had an extended alignment with at least one other E. coli protein sequence. To exclude alignments of questionable biological significance, we imposed a high threshold on the number of gaps allowed in each of the 624 extended alignments, giving us a subset of 464 proteins. The population of 464 alignments has the following characteristics expressed as median values of the group: 254 amino acids in the alignment, representing 86% of the length of the protein, 33% of the amino acids in the alignment being identical, and 1.1 gaps introduced per 100 amino acids of alignment. Where functions are known, nearly all pairs consist of functionally related proteins. This implies that the sequence similarity we detected has biological meaning and did not arise by chance. That a major fraction of E. coli proteins form extended alignments strongly suggests the predominance of duplication and divergence of ancestral genes in the evolution of E. coli genes. The range of degrees of similarity shows that some genes originated more recently than others. There is no evidence of genome doubling in the past, since map distances between genes of sequence-related proteins show no coherent pattern of favored separations.     

High diversity of alpha-globin haplotypes in a Senegalese population, including many previously unreported variants.

RFLP haplotypes at the alpha-globin gene complex have been examined in 190 individuals from the Niokolo Mandenka population of Senegal: haplotypes were assigned unambiguously for 210 chromosomes. The Mandenka share with other African populations a sample size-independent haplotype diversity that is much greater than that in any non-African population: the number of haplotypes observed in the Mandenka is typically twice that seen in the non-African populations sampled to date. Of these haplotypes, 17.3% had not been observed in any previous surveys, and a further 19.1% have previously been reported only in African populations. The haplotype distribution shows clear differences between African and non-African peoples, but this is on the basis of population-specific haplotypes combined with haplotypes common to all. The relationship of the newly reported haplotypes to those previously recorded suggests that several mutation processes, particularly recombination as homologous exchange or gene conversion, have been involved in their production. A computer program based on the expectation-maximization (EM) algorithm was used to obtain maximum-likelihood estimates of haplotype frequencies for the entire data set: good concordance between the unambiguous and EM-derived sets was seen for the overall haplotype frequencies. Some of the low-frequency haplotypes reported by the estimation algorithm differ greatly, in structure, from those haplotypes known to be present in human populations, and they may not represent haplotypes actually present in the sample.     

Conformational changes induced in bovine lens alpha-crystallin by carbamylation. Relevance to cataract.

Carbamylation of lens proteins may contribute to cataractogenesis in certain medical conditions where blood urea is elevated for prolonged periods. This paper reports on the effects of carbamylation on the physicochemical properties of one of the major lens structural proteins, alpha-crystallin. In particular it is shown that carbamylation alters the tertiary and secondary structure of the protein, leading to an increased reactivity of protein thiols, resulting in interchain disulphide bonding.     

Reduction of chronic hypoxic pulmonary hypertension in the rat by beta-aminopropionitrile

We administered antifibrotic agent beta-aminopropionitrile (BAPN) to rats exposed to 10% O2-90% N2 for 3 wk to prevent excess vascular collagen accumulation. Groups of Sprague-Dawley rats studied were air breathing, hypoxic, and hypoxic treated with BAPN, 150 mg/kg twice daily intraperitoneally. After the 3-wk period, we measured mean right ventricular pressure (RVP), the ratio of weight of right ventricle to left ventricle plus septum (RV/LV + S), and hydroxyproline content of the main pulmonary artery (PA) trunk. Hypoxia increased RVP from 14 to 29 mmHg; RVP was 21 mmHg in hypoxic BAPN-treated animals. Hypoxia increased the RV/LV + S ratio from 0.28 to 0.41; the ratio was 0.32 in hypoxic BAPN-treated animals. Hypoxia increased PA hydroxyproline from 20 to 239 micrograms/artery; hydroxyproline was 179 micrograms/artery in hypoxic BAPN-treated animals. Thus BAPN prevented pulmonary hypertension, right ventricular hypertrophy, and excess vascular collagen produced by hypoxia. We conclude that vascular collagen contributes to the maintenance of chronic hypoxic pulmonary hypertension.