A small heat-shock protein, p26, from the crustacean Artemia protects mammalian cells (Cos-1) against oxidative damage

A small heat-shock protein (p26) purified from stress-resistant embryos of the crustacean, Artemia franciscana, was introduced into cultured cells of green monkey kidney (Cos-1) using the BioPORTER delivery system. Cells containing p26 exhibited impressive resistance to hydrogen peroxide compared to controls. Introduction of the disaccharide trehalose did not provide protection against oxidative damage, but enhanced substantially the protective performance of p26 when both were present. These studies extend previous research on the protective role played by p26 in cells exposed to various forms of stress, presumably through its ability to function as a molecular chaperone.     

The 'island rule' in birds: medium body size and its ecological explanation

Do birds show a different pattern of insular evolution from mammals? Mammals follow the ''island rule'', with large-bodied species getting smaller on islands and small-bodied species getting bigger. By contrast, the traditional view on birds is that they follow no general island rule for body size, but that there is an insular trend for large bills. Insular shifts in feeding ecology are, therefore, widely assumed to be the primary cause of divergence in island birds. We use a comparative approach to test these ideas. Contrary to the traditional view, we find no evidence for increased bill size in insular populations. Instead, changes in both bill size and body size obey the ''island rule''. The differences between our results and the traditional view arise because previous analyses were based largely on passerines. We also investigate some ecological factors that are thought to influence island evolution. As predicted by the traditional view, shifts in bill size are associated with feeding ecology. By contrast, shifts in body size are associated with the potential for intraspecific competition and thermal ecology. All these results remain qualitatively unchanged when we use different methods to score the ecological factors and restrict our analyses to taxa showing pronounced morphological divergence. Because of strong covariation between ecological factors, however, we cannot estimate the relative importance of each ecological factor. Overall, our results show that the island rule is valid for both body size and bill length in birds and that, in addition to feeding ecology, insular shifts in the level of intraspecific competition and the abiotic environment also have a role.     

Phylogeographical approaches to assessing demographic connectivity between breeding and overwintering regions in a Nearctic-Neotropical warbler (Wilsonia pusilla)

We characterized the pattern and magnitude of phylogeographical variation among breeding populations of a long-distance migratory bird, the Wilson's warbler (Wilsonia pusilla), and used this information to assess the utility of mtDNA markers for assaying demographic connectivity between breeding and overwintering regions. We found a complex pattern of population differentiation in mitochondrial DNA (mtDNA) variation among populations across the breeding range. Individuals from eastern North America were differentiated from western individuals and the eastern haplotypes formed a distinct, well-supported cluster. The more diverse western group contained haplotype clusters with significant geographical structuring, but there was also broad mixing of haplotype groups such that no haplotype groups were population specific and the predominance of rare haplotypes limited the utility of frequency-based assignment techniques. Nonetheless, the existence of geographically diagnosable eastern vs. western haplotypes enabled us to characterize the distribution of these two groups across 14 overwintering locations. Western haplotypes were present at much higher frequencies than eastern haplotypes at most overwintering sites. Application of this mtDNA-based method of linking breeding and overwintering populations on a finer geographical scale was precluded by the absence of population-specific markers and by insufficient haplotype sorting among western breeding populations. Our results suggest that because migratory species such as the Wilson's warbler likely experienced extensive gene flow among regional breeding populations, molecular markers will have the greatest utility for characterizing breeding-overwintering connectivity at a broad geographical scale.     

Differential binding to and biofilm formation on,HEp-2 cells by Salmonella enterica serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster

Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.     

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.     

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.     

Multiple glucose 6-phosphate dehydrogenase-deficient variants correlate with malaria endemicity in the Vanuatu archipelago (southwestern Pacific).

In studying the relationship between genetic abnormalities of red blood cells and malaria endemicity in the Vanuatu archipelago in the southwestern Pacific, we have found that of 1,442 males tested, 98 (6.8%) were G6PD deficient. The prevalence of GdPD deficiency varied widely (0%-39%), both from one island to another and in different parts of the same island, and generally correlated positively with the degree of malaria transmission. The properties of G6PD from GdPD-deficient subjects were analyzed in a subset of 53 samples. In all cases the residual red-blood-cell activity was < 10%. There were three phenotypic patterns. PCR amplification and sequencing of the entire coding region of the G6PD gene showed that the first of these patterns corresponded to G6PD Union (nucleotide 1360C-->T; amino acid 454Arg-->Cys), previously encountered elsewhere. Analysis of samples exhibiting the second pattern revealed two new mutants: G6PD Vanua Lava (nucleotide 383T-->C; amino acid 128Leu-->Pro) and G6PD Namoru (nucleotide 208T-->C; amino acid 70Tyr-->His); in three samples, the underlying mutation has not yet been identified. Analysis of the sample exhibiting the third pattern revealed another new mutant: G6PD Naone (nucleotide 497G-->A; amino acid 166Arg-->His). Of the four mutations, G6PD Union and G6PD Vanua Lava have a polymorphic frequency in more than one island; and G6PD Vanua Lava has also been detected in a sample from Papua New Guinea. G6PD deficiency is of clinical importance in Vanuatu because it is a cause of neonatal jaundice and is responsible for numerous episodes of drug-induced acute hemolytic anemia.     

Molecular Evolution of the Ac/Ds Transposable-Element Family in Pearl Millet and Other Grasses

We report an Ac-like sequence from pearl millet (Pennisetum glaucum) and deletion derivative Ac-like sequences from pearl millet and another grass species, Bambusa multiplex. Sequence relationships between the pearl millet and maize Ac elements suggest the Ac/Ds transposable-element family is ancient. Further, the sequence identity between the Bambusa Ac-like sequence and maize Ac implies that the Ac/Ds transposable-element family has been in the grass family since its inception. The Ac-like sequences reported from pearl millet and maize Ac are statistically heterogeneous in pair-wise distance comparisons to each other. Yet, we are unable to discriminate between differential selection or ectopic exchange (recombination and conversion) between nonidentical transposable element homologues, as the cause of the heterogeneity. However, the more extreme heterogeneity exhibited between the previously described pearl millet element and maize Ac seems likely to derive from ectopic exchange between elements with different levels of divergence.     

Thermal resistance, developmental rate and heat shock proteins in Artemia franciscana, from San Francisco Bay and southern Vietnam

Cysts (encysted gastrula embryos) of Artemia franciscana collected from salterns in San Francisco Bay, California, USA (SF) were inoculated into much warmer growth ponds in the Mekong Delta region of Vietnam (V) in 1996. V adults arising directly from these cysts during 17 April to 15 May produced their own cysts, which were collected, processed and stored until shipped to the USA for study. Adults grown in the laboratory from SF cysts (those used for the inoculation) were less resistant to high temperature than adults cultured from V cysts. V cysts produced heat-resistant adults, even though cultured under the same laboratory conditions as SF animals, at much lower temperatures than they ever experienced in Vietnam. Differences in thermal performance between SF and V adults were retained in the second generation, cultured from cysts produced in the laboratory by first generation adults, suggesting a genetic basis for the better heat resistance of V adults. We propose that the operation of natural selection in the Vietnam growth ponds produced adults with improved thermal tolerance, and that the basis for this tolerance was incorporated into the developmental program of their cysts. Surprisingly, differences in heat resistance of laboratory reared animals were not reflected in constitutive levels of the hsp70 family which were similar in first generation SF and V adults. A conditioning heat shock (HS, 37 degrees C, 30 min) led to the same level of induced thermotolerance in SF and V first generation adults when evaluated 24 h post-HS. Levels of hsp70 were also up-regulated at that time, but to about the same extent in SF and V adults. Developmental rates of SF cysts used for the inoculation were faster than those of cysts produced in Vietnam when both were incubated at 21+/-1 degrees C, suggesting that V cysts have become adapted to develop at higher temperatures.