Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.     

Fatty Acid Patterns in <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. as a Marker for Nutritional Regimes and Temperature under Extremely Acidic Conditions

Fatty acid profiles were used to characterize nutritional pathways in Chlamydomonas sp. isolated from an acidic mining lake (pH 2.7). Surprisingly, profiles of Chlamydomonas sp. grown in the lab under photoautotrophic, mixotrophic, and heterotrophic conditions at in situ deep strata lake water temperatures (8°C) were very similar, polyunsaturated fatty acids including -linolenic acid (18:33) and 16:43 along with palmitic acid (16:0) being most abundant. Therefore, heterotrophic growth of Chlamydomonas sp. at low temperatures can result in high concentrations of polyunsaturated fatty acids, as previously only described for some psychrophilic bacteria. By contrast, the cultivation of isolated Chlamydomonas sp. at 20°C, reflecting surface water temperatures, provided fatty acid patterns characteristic of the nutrition strategy applied: the concentration of polyunsaturated fatty acids decreased when the growth pathway changed from photoautotrophic via mixotrophic to heterotrophic. Total fatty acid concentration also diminished in this order. Principal component analysis confirmed the significance of FA profiling to mirror nutritional pathways. Lake-water analysis revealed low concentrations of dissolved organic carbon, mainly consisting of polymeric fulvic acids that are unable to support heterotrophic growth of Chlamydomonas sp. Polymeric fulvic acids present in the deeper strata of the lake turned out to be formed in situ on the basis of organic monomers including reduced sulfur-containing ones, as revealed by thermochemolysis and pyrolysis. Growth of Chlamydomonas sp. in the deep chlorophyll maximum is therefore assumed to mainly result from photosynthesis, despite very low photon densities. Phytol-including metabolites proved to be significant biomarkers to indicate the nutritional pathway of Chlamydomonas sp. , -Dicarboxylic acids—light-induced degradation products of unsaturated fatty acids—appeared to be good indicators of photooxidative alterations to the algal species under study.     

Antibodies to Brucella spp. among blue wildebeest and African buffalo in Kenya

A serologic survey of blue wildebeest (Connochaetes taurinus Burchell) and African buffalo (Syncerus caffer Sparrman) in the Masai Mara area was conducted. Antibodies to Brucella spp. were found in 18% of the blue wildebeest and 30% of the African buffalo examined. There were titers in all age groups and in both sexes. Hygromata were seen in both species. The increase in numbers of blue wildebeest and African buffalo which share grazing and watering areas with cattle of the Masai people, makes the presence of infections by Brucella spp. in wildlife an important consideration in any program for control of brucellosis.     

Antibodies to Brucella spp. in Pacific bottlenose dolphins from the Solomon Islands

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.     

Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus

Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.     

Resistance to azolic compounds in clinical Trichophyton spp. strains

The increase of dermathophytosis in patients with poor therapeutic response leads us to study the antifungal susceptibility of 36 clinical isolates to itraconazole, ketoconazole and fluconazole by the E-test method. According to established parameters by the Clinical Laboratory Standards Institute, the resistance to one or more antifungal drugs was demonstrated in seven isolates (19.4%) as follows: three Trichophyton rubrum, three T. mentagrophytes and one T. tonsurans. A T. rubrum isolate was resistant to the three azolic drugs; the other six only to fluconazole. It is important to establish the antifungal susceptibility as part of the study procedures in patients with dermatophytosis and a poor antifungal response.     

Resistance to freshwater exposure in White Sea Littorina spp. II: Acid-base regulation

Parameters of acid-base and energy status were studied by in vivo ³¹P-nuclear magnetic resonance spectroscopy in three White Sea Littorina spp. (L.littorea, L. saxatilis and L. obtusata) during prolonged anaerobiosis in freshwater. Intracellular pH decreased significantly, especially during the early period of anaerobiosis, but later the decrease in intracellular pH slowed down considerably, suggesting a capacity for intracellular pH regulation in all three species. There was a trend for intracellular pH to fall most rapidly in the least freshwater-resistant species, L. obtusata, as compared to the most resistant, L. littorea. Non-bicarbonate, non-phosphate buffer values estimated by the homogenate technique were similar in the three studied species (28–37 mmol pH⁻¹ kg⁻¹ wet weight) and did not change during freshwater exposure. The CaCO₃ buffer value of the foot tissues was considerably higher (171–218 mmol pH⁻¹ kg⁻¹ wet weight) and decreased significantly during freshwater exposure. The contribution of the multiple tissue buffering systems to intracellular pH regulation in Littorina spp. shifts between different stages of freshwater exposure. Initially, the non-bicarbonate, non-phosphate tissue buffering system seems to be of major importance for metabolic proton buffering at intracellular pH between 7.5 and 7.0. During later stages of anaerobiosis and at lower intracellular pH, the CaCO₃ buffer is involved in proton buffering. Decrease in the CaCO₃ buffer value during freshwater exposure was in quantitative agreement with the amount of metabolic protons buffered, thus suggesting that CaCO₃ tissue stores may serve as a major buffering system during prolonged anaerobiosis in Littorina spp. Accepted: 23 December 1999     

Contribution of Brain Size to IQ and Educational Underperformance in Extremely Preterm Adolescents

Objectives

Extremely preterm (EP) survivors have smaller brains, lower IQ, and worse educational achievement than their term-born peers. The contribution of smaller brain size to the IQ and educational disadvantages of EP is unknown. This study aimed (i) to compare brain volumes from multiple brain tissues and structures between EP-born (<28weeks) and term-born (≥37weeks) control adolescents, (ii) to explore the relationships of brain tissue volumes with IQ and basic educational skills and whether this differed by group, and (iii) to explore how much total brain tissue volume explains the underperformance of EP adolescents compared with controls.

Methods

Longitudinal cohort study of 148 EP and 132 term controls born in Victoria, Australia in 1991-92. At age 18, magnetic resonance imaging-determined brain volumes of multiple tissues and structures were calculated. IQ and educational skills were measured using the Wechsler Abbreviated Scale of Intelligence (WASI) and the Wide Range Achievement Test(WRAT-4), respectively.

Results

Brain volumes were smaller in EP adolescents compared with controls (mean difference [95% confidence interval] of -5.9% [-8.0, -3.7%] for total brain tissue volume). The largest relative differences were noted in the thalamus and hippocampus. The EP group had lower IQs(-11.9 [-15.4, -8.5]), spelling(-8.0 [-11.5, -4.6]), math computation(-10.3 [-13.7, -6.9]) and word reading(-5.6 [-8.8, -2.4]) scores than controls; all p-values<0.001. Volumes of total brain tissue and other brain tissues and structures correlated positively with IQ and educational skills, a relationship that was similar for both the EP and controls. Total brain tissue volume explained between 20-40% of the IQ and educational outcome differences between EP and controls.

Conclusions

EP adolescents had smaller brain volumes, lower IQs and poorer educational performance than controls. Brain volumes of multiple tissues and structures are related to IQ and educational outcomes. Smaller total brain tissue volume is an important contributor to the cognitive and educational underperformance of adolescents born EP.     

Resistance to freshwater exposure in White Sea Littorina spp. I: Anaerobic metabolism and energetics

 Anaerobic metabolism and changes in the osmotic concentration of extravisceral fluid were studied in the White Sea periwinkles (Littorina littorea, Littorina saxatilis and Littorina obtusata) during freshwater exposure. Resistance to hypoosmotic stress increased in the order: L. obtusata < L. saxatilis < L. littorea. Our data suggest that osmotic shock is not a primary reason for mortality of the periwinkles under these conditions. During environmental anaerobiosis, considerable succinate accumulation (up to 10–19 μmol g⁻¹ wet weight), and depletion of phosphagen and ATP pools were found in the studied species. Other metabolic end products (alanopine, strombine, lactate, acetate or propionate) were not detected. Succinate accumulation and net ATP breakdown were the fastest in the least resistant species, L. obtusata, and slowest in the most resistant, L. littorea. Rate of ATP turnover decreased during freshwater exposure in L. littorea and L. saxatilis, but not in L. obtusata. Our data suggest that differential resistance of three studied Littorina spp. to extreme hypoosmotic stress may be related to their different abilities to reduce metabolic rate and ATP turnover during sustained anoxia. Species-specific variations in anaerobic capacity of Littorina spp. are discussed in relation to their vertical distribution, size and ecology. Accepted: 4 October 1999     

Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep

Outer membrane proteins (OMPs) of Rev-1 strain of Brucella melitensis were used in a Western blotting assay for the serological diagnosis of brucellosis in ovine sera. Fifty-four sheep sera were tested and divided into the following groups: Group A) n. 9 samples from one sheep that had been experimentally infected with Y. enterocolitica O:9; Group B) n. 10 samples collected from sheep infected with Brucella melitensis and 1 sample from a sheep vaccinated with the Rev 1 strain; Group C) n. 10 samples collected in "officially brucellosis-free" herds; Group D) n. 12 samples classified as "suspicious"; Group E) n. 12 samples classified as "positive". Antibodies were detected by routine tests performed for the diagnosis of brucellosis in serum samples of the sheep infected with Y. enterocolitica O:9 after the 2nd week post infection. In the WB assay, sera of group B recognised a 17 kDa protein, whereas sera of groups A, and D and 9 out of 12 of group E exhibited no reactivity to this protein. The results obtained encourage the use of the WB assay as a confirmatory test for the diagnosis of brucellosis.     

Mesophyll Resistance and Carboxylase Activity: A Comparison under Water Stress Conditions

The response of several leaf gas exchange parameters were monitored with decreasing leaf water potential in Phaseolus vulgaris L. leaflets. These included photosynthesis, transpiration, CO₂ compensation point, ribulose 1,5-diphosphate carboxylase activity, boundary layer plus stomatal, and mesophyll resistance to diffusion of CO₂. Mesophyll resistance was calculated under two assumptions: (a) the CO₂ concentration at the chloroplast was zero, and (b) it was equal to the CO₂ compensation point.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

Distribution of γ-Aminobutyric Acid and Glutamate Decarboxylase in the Layers of Rat Oviduct

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to yield a high sensitivity and a low blank. The 20-microns thick freeze-dried sections (0.2-1.5 micrograms dry weight) were prepared from the oviduct and ovary of rat. The analysis of these microsamples by the improved method showed that, contrary to the previous observations, the rat ovary is devoid of GAD activity and contains a trace amount of GABA. Both are present abundantly in the oviduct. In the oviduct mucosa, significant GAD activity was found in the estrous phase, whereas the activity was nearly null during other phases of the estrous cycle. GABA concentration in the oviduct mucosa was 10-fold higher than in the cerebral cortex; its variation during the estrous cycle was not remarkable. In the muscle layer of oviduct, GAD activity had a low peak in the estrous phase and GABA concentration was almost constant during the estrous cycle. The denervation experiment showed that GAD is present in the nerve terminals innervating the oviduct.     

The Alcohol Dehydrogenase Polymorphism in Drosophila Melanogaster: Fitness Measurements and Predictions under Conditions with No Alcohol Stress

The relative fitnesses of the different Adh genotypes under normal laboratory conditions and in the absence of alcohol stress were estimated in Drosophila melanogaster according to Prout's method. The larval component (viability) did not reveal fitness differences between the genotypes but for the adult component significant differences were observed. The female adult component (fecundity) showed an overdominant pattern: both homozygous genotypes showed a relative fitness significantly lower than the heterozygous genotype. For the male adult component (virility) also differences were observed. The homozygous SS genotype showed a lower relative fitness than the other two genotypes. Predictions for gene frequency changes based on the estimated fitness values do show a reasonably good correspondence with frequency changes actually observed in a number of experimental cage populations and indicate a globally stable equilibrium around a frequency of the F allele of 0.40-0.70. The relevance of these fitness estimates, obtained under conditions with no alcohol stress, for the explanation of the Adh polymorphisms observed in nature is discussed.     

极端干旱条件下杠柳的落叶相对休眠-复水复苏型生存策略

以3种梯度恒定干旱胁迫(土壤水分分别控制在最大田间持水量的80%、55%和35%)为对照,人工模拟持续自然干旱(干旱15~54 d后复水),研究黄土丘陵区广泛分布的乡土灌木杠柳(Periploca sepium)的形态、耗水、生长、生理在持续干旱、极端干旱以及旱后复水时的变化,探讨长期恒定胁迫及极端干旱下植物的适应性策略。结果表明:(1)在恒定干旱胁迫下,杠柳可调整生长和生理状况使其达到适应有限水分供应的新稳定状态;植株冠层减小,叶片可溶性糖积累以及过氧化物酶活性升高是杠柳适应长期恒定干旱胁迫的重要机制。(2)在长期自然干旱胁迫下,杠柳的形态与生理状况均持续变化。在叶片死亡脱落前,叶片中大量积累的脯氨酸、游离氨基酸和可溶性糖提高了杠柳对干旱胁迫的耐受性;随着干旱胁迫的加剧,杠柳叶片加速衰老死亡、以休眠芽形式进入相对休眠状态。(3)复水后根系和茎复苏长出新芽并补偿生长,新芽中高含量的脯氨酸和游离氨基酸以及高活性状态的SOD和POD表明新芽活跃的生理状态是其补偿生长的重要机制,即杠柳在极端干旱条件下具有落叶相对休眠-复水后复苏型生存策略。     

Importance of Aerodynamic Resistance to Water Use Efficiency in Three Conifers under Field Conditions

The quantitative importance of aerodynamic resistance to H₂O vapor and CO₂ exchange was determined for shoots from saplings of three conifers (Abies lasiocarpa [Hook] Nutt., Pinus contorta Dougl., Juniperus communis L.) under natural conditions in the field. A combination of relatively low stomatal resistances (<300 seconds per centimeter) and low wind speeds (<30 centimeters per second) led to substantial contributions of the aerodynamic resistance (R_wv^a) to water use efficiency (WUE = photosynthesis/transpiration) for all three species. For A. lasiocarpa, transpiration was calculated to be 44% less and photosynthesis 17% less due to the presence of R_wv^a, which led to a predicted increase in WUE of 57% compared to the calculated WUE when R_wv^a was assumed negligible. Similar increases in WUE were computed for P. contorta (48%) with somewhat smaller values for J. communis (34%). These results are discussed in terms of the estimated importance of R_wv^a on water and photosynthetic relations of plants that have relatively low stomatal resistances and grow in microhabitats with low winds.     

Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ-Aminobutyric Acid Release

Using cerebellar, neuron-enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D-[3H]aspartate release, whereas N-methyl-D-aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20-100 microM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 microM. Kainate and dihydrokainate (20-100 microM) inhibited the initial rate of D-[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N-methyl-DL-aspartate were ineffective. D-[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D-[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50-200 microM) and, to a lesser extent, 2,3-cis-piperidine dicarboxylic acid (100-200 microM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20-100 microM) also stimulated gamma-[3H]-aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D-[3H]aspartate neuronal uptake by interfering with the acidic amino acid high-affinity transport system.     

Contribution of Vesicular Glutamate Transporters to Stress Response and Related Psychopathologies: Studies in VGluT3 Knockout Mice

Maintenance of the homeostasis in a constantly changing environment is a fundamental process of life. Disturbances of the homeostatic balance is defined as stress response and is induced by wide variety of challenges called stressors. Being the main excitatory neurotransmitter of the central nervous system glutamate is important in the adaptation process of stress regulating both the catecholaminergic system and the hypothalamic-pituitary-adrenocortical axis. Data are accumulating about the role of different glutamatergic receptors at all levels of these axes, but little is known about the contribution of different vesicular glutamate transporters (VGluT1-3) characterizing the glutamatergic neurons. Here we summarize basic knowledge about VGluTs, their role in physiological regulation of stress adaptation, as well as their contribution to stress-related psychopathology. Most of our knowledge comes from the VGluT3 knockout mice, as VGluT1 and 2 knockouts are not viable. VGluT3 was discovered later than, and is not as widespread as the VGluT1 and 2. It may co-localize with other transmitters, and participate in retrograde signaling; as such its role might be unique. Previous reports using VGluT3 knockout mice showed enhanced anxiety and innate fear compared to wild type. Moreover, these knockout animals had enhanced resting corticotropin-releasing hormone mRNA levels in the hypothalamus and disturbed glucocorticoid stress responses. In conclusion, VGluT3 participates in stress adaptation regulation. The neuroendocrine changes observed in VGluT3 knockout mice may contribute to their anxious, fearful phenotype.