Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

Pyric Herbivory and the Nexus Between Forage,Fire and Native and Introduced Large Grazing Herbivores in Australian Tropical Savannas

Earth’s tropical savannas typically support high biomass of diverse grazing herbivores that depend on a highly fluctuating resource: high-quality forage. An annual wet–dry cycle, fire and herbivory combine to influence forage quality and availability throughout the year. In the savannas of northern Australia, a depauperate suite of large native (marsupial) herbivores (wallaroos [Osphranter spp.] and the agile wallaby [Notamacropus agilis]) compete for resources with non-native large herbivores introduced in the late nineteenth century, particularly bovines (feral and managed cattle [Bos spp.] and feral water buffalo [Bubalus bubalis]) that now dominate the landscape. Anecdotal reports of recent population declines of large macropods and negative impacts of bovines highlight the need to better understand the complex relationship between forage, fire and abundance of native and introduced large herbivores. The pyric herbivory conceptual model, which posits complex feedbacks between fire and herbivory and was developed outside Australia, predicts that native and introduced large herbivores will both respond positively to post-fire forage production in Australian savannas where they co-occur. We used grazing exclosures, forage biomass and nutrient analyses and motion-sensor camera-trapping to evaluate the overall robustness of the pyric herbivory model in the Australian context, specifically whether forage quantity and quality are impacted by herbivory, season and fire activity, and which forage attributes most influence large grazing herbivore abundance. Forage quantity, as measured by live, dead and total herbaceous biomass and proportion of biomass alive, was higher inside herbivore exclosures, even at relatively low densities of herbivores. Forage quality, as measured by fibre content, was not affected by herbivory, however, crude protein content of live herbaceous biomass was greater outside herbivore exclosures. Recent fire was an important predictor of all measures of forage quantity and quality. Recent fire occurrence decreased overall quantity (biomass) but increased quality (decreased fibre content and increased crude protein content); late dry season fires resulted in forage with the highest crude protein content. The predictions of the pyric herbivory conceptual model are consistent with observations of the feeding behaviour of introduced bovines and some large macropods in northern Australian savannas, lending support to the global generality of pyric herbivory in fire-prone grassy biomes.

     

Effects of an anion channel blocker, 4, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), on human neutrophil function

We investigated the capacity of DIDS to influence degranulation and superoxide anion (O-2) generation by human neutrophils exposed to soluble, surface-active stimuli. DIDS caused a concentration-related (25-200 microM) inhibition of myeloperoxidase (MPO) release from N-formyl-methionyl-leucyl-phenylalanine (FMLP), 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) and phorbol myristate acetate (PMA)-stimulated neutrophils. In contrast, DIDS had no effect on O-2 production by FMLP and PMA activated cells, whereas it enhanced LTB4 and AGEPC-elicited O-2 generation. The respective effects of DIDS on these neutrophil activities were reversed by washing the cells prior to exposure to stimulus. Thus, DIDS represents a useful pharmacologic probe for investigating the role(s) of anions in the mechanisms of neutrophil activation with structurally and chemically dissimilar stimuli.