Mutation and Evolutionary Rates in Adélie Penguins from the Antarctic

Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent.     

脂肪酶活力测定方法及其在筛选产脂肪酶微生物中的应用

比较了罗丹明平板法、橄榄油乳化法、对硝基苯酚法测脂肪酶水解酶活和对硝基苯酚法测脂肪酶酯合成酶活4种常用的脂肪酶活力测定方法。结果表明,罗丹明平板法只适合脂肪酶活力的定性和初步定量判断,后3种方法适合于脂肪酶活力的定量检测,但只有对硝基苯酚法有较好的重现性。而且,脂肪酶水解酶活力和其合成活力无对应关系,所以在筛选产脂肪酶微生物时要选择合适的脂肪酶活力测定方法。也即,筛选产水解酶活的菌株应选择水解酶活测定方法,否则,选择酯合成酶活力测定方法。基于这一原则筛选到了预期产脂肪酶微生物。     

A Sensitive Radiometric Assay for Ornithine Aminotransferase: Regional and Subcellular Distributions in Rat Brain

Abstract: A radiometric assay for ornithine aminotransferase was developed using [1-¹⁴C]α-ketoglutarate as the labeled substrate and glutamate decarboxylation as a linking step. This assay gives near total measurement of ornithine aminotransferase activities that are, respectively, about 1.5 and 10 times larger than those obtained by the spectrophotometric assay and the radiometric assay using [1-¹⁴C]ornithine. It is also the most sensitive of the three assay procedures.
Consistent with previous reports, brain ornithine aminotransferase was found to be present predominantly in synaptosomes. Regional distribution of the enzyme correlated with that of the high-affinity uptake of glutamate, but not with the distribution of glutamate decarboxylase. Ornithine aminotransferase may be responsible for the synthesis of glutamate in glutamatergic neurons but it is clearly not localized exclusively in such neurons.     

Platelet Aggregation Inhibitors from Philippine Marine Invertebrate Samples Screened in a New Microplate Assay

A new microplate assay for Ca²⁺-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 µg/ml, and epinephrine-induced aggregation by 78% at 20 µg/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 µg/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity.     

Screening of Marine Bacteria for the Production of Microbial Repellents Using a Spectrophotometric Chemotaxis Assay

A method for screening marine bacteria for the production of microbial repellents has been developed. The spectrophotometer provided quantitative information on bacterial chemotaxis in response to extracts from other strains of marine bacteria. Aqueous extracts were incorporated into an agar plug at the base of a cuvette, which was overlaid with a suspension of a motile strain. Negative chemotaxis of the motile strain in response to diffusion of repellent compounds from the agar could be measured by a fall in the optical density, allowing the direct screening of supernatants for repellent activity. Three strains producing metabolites with a repellent effect on a motile marine bacterium were identified. Antibiotic activity and the repellent effect of the supernatants were compared, with no significant correlation being found. The screening method will therefore allow the identification of bioactive metabolites that would be overlooked using traditional antibiotic screening strategies. Received March 4, 1998; accepted November 11, 1998.     

Highly Sensitive,Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.     

Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.     

A Mathematical Model of the Controlled Plant of the Réspiratory System

Ability to predict the dynamic response of oxygen, carbon dioxide tensions, and pH in blood and tissues to abrupt changes in ventilation is important in the mathematical modeling of the respiratory system. In this study, the controlled plant (the amount and distribution of O₂ and CO₂) of the respiratory system is modeled. Although the body tissues are divided into a finite number of “compartments” (three tissue groups), in contrast to earlier models, the blood and tissue gas tensions within each compartment are considered to be continuously distributed in time and in one spatial coordinate. The mass conservation equations for oxygen and carbon dioxide involved in the blood-tissue gas exchange are described by a set of partial differential equations which take into account convection of O₂ and CO₂ caused by the flow of blood as well as diffusion due to local tension gradients. Nonlinear algebraic equations for the dissociation curves, which take into account the Haldane and Bohr effects in blood, are used to obtain the relationships between concentrations and partial pressures. Time-variable delays caused by the arterial and venous transport of the respiratory gases are also included. The model so constructed successfully reproduced actual O₂ and CO₂ tensions in arterial blood, and in muscle venous and mixed venous blood when ventilation was abruptly changed.     

Temporal Variations of Microbial Activity and Diversity in Marine Tropical Sediments (New Caledonia Lagoon)

Temporal variations of oxygen consumption, sensitivity to metal spiking, and microbial diversity were investigated during a one-year survey at the sediment–water interface in the tropical lagoon of New Caledonia. Sediment oxygen consumption (SOC) exhibited strong variations with time with maximum rates during February (Austral summer) and minimum values during July (cold period). SOC was strongly positively correlated with temperature, with an apparent activation energy (E _a) of 41 kJ mol⁻¹, corresponding to an apparent Q ₁₀(20–30 °C) of 1.75. Strong short-term variations of SOC were also observed with ratios between two consecutive samplings reaching up to twofold of magnitude within one week, whereas the maximum/minimum ratio over the whole year was equal to 2.73. In most cases, metal spiking led to a strong decrease of SOC; however, in a third of sampling dates, spiking did not significantly decrease activity. These periods of apparent metal tolerance were not characterized by a particular bacterial community structure. Bacterial community structure estimated from terminal restriction fragment length polymorphism (T-RFLP) analysis exhibited strong variations over the one-year survey, and no seasonality was observed for bacterial richness. However, on average, the Whittaker similarity index between two consecutive T-RFLP profiles was above 60% suggesting a relative stability of the bacterial community structure on the short timescale with prominent T-RFs representing on average more than 67% of relative abundance occurring over most of the year, whereas other T-RFs only occurred during some periods.     

A Novel Bacteroidetes Symbiont Is Localized in Scaphoideus titanus, the Insect Vector of Flavescence Dorée in Vitis vinifera

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.     

绵羊肺腺瘤病特异性PCR及套式PCR诊断方法的建立

绵羊肺腺瘤是由外源性反转录病毒JSRV引起的接触性传染的肺肿瘤性疾病。感染羊没有针对JSRV的循环抗体,但在感染羊的肺肿瘤组织、淋巴网状系统及外周血单核细胞中可检测到外源性前病毒exJSRV及JSRV转录产物。健康羊基因组中存在15~20拷贝的内源性前病毒enJSRV,内外源性前病毒的结构基因高度相似,而在U3区存在差别,因而,设计了针对exJSRVU3区的特异性引物并在国内首次建立了一步法特异性PCR检测法及套式PCR检测法。以700ng健康羊基因组DNA为背景,梯度稀释阳性质粒pJSRV-LTR作为模板,比较两种方法的敏感性,结果表明套式法的敏感性是一步法的10倍以上,套式法也是目前可用于检测感染羊血液样品的唯一方法,可望在绵羊肺腺瘤病的流行病学调查及防控方面起重要作用。     

Formation of Siderite in Microbial Microcosms Derived from a Marine Sediment

Abstract

Exceptionally well-preserved fossils are frequently encased by carbonate concretions. The initial steps of their formation in marine and freshwater sediments are induced by microbial activity. The role of the involved microbial communities, however, is not well understood. In this study, siderite (FeCO₃) formation in microbial microcosms is observed, with various fatty acyl compounds (lipids, surfactants) as substrates and Wadden Sea sediment samples as inocula. In actively growing microcosms, sulfate-reducing bacteria (the genus Desulfofrigus in particular) dominate the microbial community and submicroscopic siderite precipitates on bacterial cell surfaces were identified. We suggest that these biologically induced mineralization processes may, in the natural environment, initiate the formation of large concretions under suboxic conditions in coastal sediments.     

Development and Preliminary Evaluation of a Loop-Mediated Isothermal Amplification Procedure for Sensitive Detection of Cryptosporidium Oocysts in Fecal and Water Samples

A loop-mediated isothermal amplification (LAMP) procedure for the detection of Cryptosporidium in environmental and fecal samples was developed and evaluated. This is the first demonstration of LAMP applied to detection of Cryptosporidium. Due to its specificity and simplicity, the method could become a useful diagnostic tool for epidemiologic studies of Cryptosporidium presence.     

HrpA, a new ribosome-associated protein which appears in heat-stressed Mycobacterium bovis bacillus Calmette-Guérin.

A novel 18-kDa heat shock protein, HrpA, has been identified from Mycobacterium bovis BCG. HrpA was rapidly synthesized in membrane and ribosome fractions but not in the cytoplasmic fraction under heat shock stress. HrpA bound tightly to 70S ribosomes, mainly in 30S subunits. HrpA might be involved in the initiation step of translation at high temperature.     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.     

Cross-trait familial resemblance for body fat and blood pressure: familial correlations in the Québec Family Study.

Cross-trait resemblance between body fat and blood pressure (BP) was examined among families in the Québec Family Study by using a bivariate familial correlation model assessing both intraindividual (e.g., comparison of father's body fat with his own BP) and interindividual (e.g., comparison of father's body fat with son's BP) cross-trait correlations. Each of six body-fat measures-(i) percent body fat, (ii) body-mass index, (iii) the sum of six skinfolds, (iv) the ratio of the sum of six skinfolds to total fat mass, (v) the ratio of the trunk skinfold sum to the extremity skinfold sum, and (vi) the regression of the trunk-extremity skinfold ratio on the sum of six skinfolds--was analyzed separately with systolic BP and with diastolic BP. Results showed that (1) upper-body fat was the strongest interindividual correlate of BP (especially the correlation of trunk-extremity ratio with diastolic BP), suggesting shared pleiotropic genetic and/or common familial environmental effects; (2) summary body-fat measures either were inconsistent (in the case of both percent body fat and sum of six skinfolds) or gave no evidence of interindividual cross-trait resemblance with BP (in the case of body-mass index); and (3) intraindividual resemblance between the sum of six skinfolds and BP largely vanished once the skinfold sum was adjusted for fat mass, suggesting that the intraindividual association may be mediated largely by the absolute amount of subcutaneous fat rather than by the subcutaneous proportion. Finally, the magnitude of the spouse resemblance for the trunk-extremity ratio with diastolic BP suggests that a significant proportion of the resemblance may be due to environmental influences. In summary, our investigation confirms a heritable link between BP and truncal-abdominal fat as predicted by the metabolic-syndrome hypothesis. That this result is obtained in primarily normotensive, nonobese families, suggests the connection involves normal metabolic paths.     

Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay

Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.Mycobacteriosis is a common disease affecting a large variety of wild and aquacultured fishes worldwide (9). Chronic disease is most commonly observed and is characterized by granulomatous inflammation that may affect all host tissues. External clinical signs include scale loss, dermal ulceration, spinal defects, emaciation, and ascites (5, 6, 16, 25, 31).Mycobacteriosis in Chesapeake Bay striped bass (Morone saxatilis) was first observed in 1997 from histologic findings of acid-fast bacilli in granulomatous lesions (W. Vogelbein, unpublished data). Since the initial finding, surveys have demonstrated a very high prevalence of this disease in Chesapeake Bay striped bass, exceeding 50% in many samples (8, 17). Concomitantly with detection of high prevalence, tag recapture analysis has indicated that natural, nonfishing mortality of Chesapeake Bay striped bass has increased since 1999 (13), and modeling of apparent prevalence data has indicated that some mortality is associated with disease (8). Because the striped bass is an ecologically and economically important finfish along the U.S. Atlantic coast, the high prevalence of this disease creates considerable concern about the continuing health of the resource.Mycobacteriosis of fishes has traditionally been considered to be caused by Mycobacterium marinum, M. fortuitum, or M. chelonae; however, the recognized diversity of Mycobacterium spp. infecting fishes has increased markedly in recent years (9). To date, neither M. fortuitum nor M. chelonae have been isolated from internal tissues of striped bass in the Chesapeake Bay, and M. marinum has been cultured from only a small fraction (3%) of fish (20). Instead, a variety of slow-growing mycobacteria have been isolated, dominated by the recently described species M. pseudoshottsii and M. shottsii (9, 20-22). The 16S rRNA gene sequences of M. pseudoshottsii, M. shottsii, M. marinum, and M. ulcerans are highly similar (≥99.4%), and like M. ulcerans, M. pseudoshottsii possesses the insertion sequences IS2404 and IS2606 and produces mycolactone toxin (19). M. shottsii has been reported to be positive for IS2404 under specific PCR conditions by some authors (22), but not by others (10), and this species is not known to produce mycolactone. IS2606 has been reported to amplify weakly or not at all in M. shottsii (22). M. pseudoshottsii and M. shottsii differ in pigment production, with the former being a photochromogen and the latter being nonpigmented (22).In this study, we performed a quantitative real-time PCR-based survey of the presence and density of M. pseudoshottsii and M. shottsii in water and sediments of Chesapeake Bay, as well as in two dominant prey of striped bass, the Atlantic menhaden (Brevoortia tyrannus) and the bay anchovy (Anchoa mitchilli) (12, 30). Mycobacterium pseudoshottsii was detected by amplification of IS2404 in a manner similar to that used in previous studies (7, 24). We also amplified and sequenced mycobacterial interspersed repetitive unit (MIRU) loci from menhaden, water, and sediment samples in order to confirm that IS2404 amplification in these samples was likely to represent the presence of M. pseudoshottsii and not another IS2404-positive bacterium.No unique insertion sequences have yet been described for M. shottsii, and the high degree of similarity between M. pseudoshottsii and M. shottsii in genes for which sequences are available (e.g., hsp60, erp, 16S rRNA, 23S rRNA, internal transcribed spacer [ITS]) makes development of M. shottsii-specific assays problematic. We therefore performed genomic subtractive hybridization in a manner similar to that originally described by Akopyants et al. (3) to characterize sequences specific to M. shottsii relative to M. pseudoshottsii. An M. shottsii-specific quantitative PCR (qPCR) assay was developed to target sequences identified in this manner.     

Some Digenetic Trematodes of Marine Fishes from the Barrier Reef and Reef Lagoon of Belize

A total of 200 marine fishes (36 species) from the Caribbean Sea off Belize were examined for digenetic trematodes and 163 (81.5%) harbored at least one species. Five species are described as new: Lasiotocus asymmetricus (Monorchiidae) from Haemulon flavolineatum; Stephanostomum belizense (Acanthocolpidae) from Caranx bartholomaei; Neolepidapedon belizense (Lepocreadiidae) from Sphyraena barracuda; Opecoeloides belizensis (Opecoelidae) from Priacanthus arenatus; Saturnius belizensis (Hemiuridae) from Mugil curema. 72 previously known species were identified, and all represent new geographical distribution records while many are recorded from new hosts. Their zoogeographical affinities are very strongly with the tropical western Atlantic, although some also occur in the eastern Atlantic, Mediterranean, and Indo-Pacific regions.