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P Fernando

Publications and source records attributed to P Fernando.

12 recordsLinked to original sources
PubMed2005

Population differentiation within and among Asian elephant (Elephas maximus) populations in southern India.

Southern India, one of the last strongholds of the endangered Asian elephant (Elephas maximus), harbours about one-fifth of the global population. We present here the first population genetic study of free-ranging Asian elephants, examining within- and among-population differentiation by analysing mitochondrial DNA (mtDNA) and nuclear microsatellite DNA differentiation across the Nilgiris-Eastern Ghats, Anamalai, and Periyar elephant reserves of southern India. Low mtDNA diversity and 'normal' microsatellite diversity were observed. Surprisingly, the Nilgiri population, which is the world's single largest Asian elephant population, had only one mtDNA haplotype and lower microsatellite diversity than the two other smaller populations examined. There was almost no mtDNA or microsatellite differentiation among localities within the Nilgiris, an area of about 15,000 km2. This suggests extensive gene flow in the past, which is compatible with the home ranges of several hundred square kilometres of elephants in southern India. Conversely, the Nilgiri population is genetically distinct at both mitochondrial and microsatellite markers from the two more southerly populations, Anamalai and Periyar, which in turn are not genetically differentiated from each other. The more southerly populations are separated from the Nilgiris by only a 40-km-wide stretch across a gap in the Western Ghats mountain range. These results variably indicate the importance of population bottlenecks, social organization, and biogeographic barriers in shaping the distribution of genetic variation among Asian elephant populations in southern India.

Animals↗
PubMed2002

Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30.

Small heat shock proteins (shsps) act as molecular chaperones by preventing heat-induced aggregation and unfolding of cellular proteins by a mechanism that is still unclear. Previously we found that the C-terminal end of Xenopus shsp, hsp30C (30C), was essential for optimal chaperone activity. Examination of the C-terminal tail of 30C revealed that it had a net negative charge. Involvement of this negative charge in chaperone activity was assessed by the creation of two mutants, D209G (Asp converted to the more neutrally charged and less polar Gly at position 209) and D209/213G (Asp to Gly at position 209 and 213). Compared to 30C and D209G, D209/213G was impaired in inhibiting heat-induced citrate synthase aggregation. In rabbit reticulocyte lysate and Xenopus oocyte microinjection refolding assays the mutants were not as efficient as 30C in maintaining heat-treated luciferase in a folding competent state. Circular dichroism analysis revealed that D209G was similar in secondary structure to 30C whereas D209/213G displayed a loss of alpha-helical-like and beta-sheet structure. Also, C-terminal truncation of 30C or 30D (an hsp30 isoform) resulted in a loss of secondary structure and function. This study clearly shows that mutation of aspartic acid residues in the C-terminal end of hsp30 or its truncation disrupts secondary structure and impairs its chaperone activity.

Amino Acid Sequence↗
PubMed2000

Mitochondrial DNA variation, phylogeography and population structure of the Asian elephant.

We report the first genetic analysis of free-ranging Asian elephants (Elephas maximus). We sampled 118 elephants from Sri Lanka, Bhutan/North India, and Laos/Vietnam by extracting DNA from dung, PCR amplifying and sequencing 630 nucleotides of mitochondrial DNA, including part of the variable left domain of the control region. Comparison with African elephant (Loxodonta africana) sequences indicated a relatively slow molecular clock in the Proboscidea with a sequence divergence of approximately 1%/Myr. Genetic diversity within Asian elephants was low, suggesting a small long-term effective population size. Seventeen haplotypes were identified within Asian elephants, which clustered into two well-differentiated assemblages with an estimated Pliocene divergence of 2.5-3.5 million years ago. The two assemblages showed incomplete geographical partitioning, suggesting allopatric divergence and secondary admixture. On the mainland, little genetic differentiation was observed between elephant populations of Bhutan and India or Laos and Vietnam. A significant difference in haplotype frequencies but relatively weak subdivision was observed between the regions Bhutan-India and Laos-Vietnam. Significant genetic differentiation was observed between the mainland and Sri Lanka, and between northern, mid-latitude and southern regions in Sri Lanka.

Animals↗
PubMed2000

Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity.

Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.

Animals↗
PubMed1999

Spatial pattern of constitutive and heat shock-induced expression of the small heat shock protein gene family, Hsp30, in Xenopus laevis tailbud embryos.

We employed whole-mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock-induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33 degrees C heatshock. The lowest temperature capable of inducing this pattern was 30 degrees C. Placement of embryos at 22 degrees C following a 1-h 33 degrees C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues.

Animals↗
PubMed1998

Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells.

In this study, we report the detection of a new group of five stress-inducible basic small heat shock proteins (BShsps) in Xenopus laevis kidney epithelial A6 cells by means of two-dimensional non-equilibrium pH gradient gel electrophoresis. These basic 30-kDa small hsps are distinct from the previously described X. laevis acidic hsp30 family on the basis of their charge and lack of cross-reactivity with an hsp30 antibody. Furthermore, at least two of the five BShsps were present constitutively, an observation that has not been made with the acidic hsp30 family. The heat inducibility of the BShsps was regulated at the level of transcription as indicated by their inhibited synthesis in the presence of the transcriptional inhibitor actinomycin D. Furthermore, the optimal temperature of BShsp induction, temporal pattern of synthesis, and induction of BShsps by other stressors such as herbimycin A and sodium arsenite were similar to those reported for the acidic hsp30 family. This study suggests that X. laevis contains at least two unique groups of small heat shock proteins that are coordinately expressed.

Animals↗
PubMed1997

Preferential activation of HSF-binding activity and hsp70 gene expression in Xenopus heart after mild hyperthermia.

We have examined the effect of mild hyperthermia on the pattern of heat shock transcription factor (HSF) binding activity, heat shock protein 70 (hsp70) and hsp30 gene expression and protein denaturation in selected tissues of adult Xenopus namely, heart, hind limb muscle, eye, liver and spleen. In these studies it was found that heart tissue was the most thermally sensitive of all of the tissues examined since maintenance of adult frogs at 26 degrees C resulted in a preferential activation of HSF binding. Thus, heart has a lowered set point temperature for HSF activation compared to the other tissues examined. At 30 degrees C HSF activation was observed in all of the tissues examined. Heart HSF activation at 26 degrees C was correlated with an increase in hsp70 mRNA and Hsp70 protein accumulation. At 28 degrees C the largest amount of hsp70 and hsp30 mRNA accumulation was detected in heart and skeletal muscle compared to other tissues while hsp70 mRNA accumulation was relatively low in spleen and hsp30 mRNA accumulation was not detectable in eyes, liver and spleen. Incubation of adult frogs at 30 degrees C resulted in enhanced hsp70 and hsp30 mRNA accumulation in all of the tissues. Finally, we have used differential scanning calorimetry (DSC) to compare the temperatures at which protein denaturation occurs in heart and liver tissue. The onset of protein denaturation (T0) occurred approximately 8.5 degrees C lower in heart compared to liver. Also the midpoint of the DSC profile (T1/2) was approximately 10.4 degrees C lower in heart than in liver. Thus, heart proteins are generally more thermolabile than proteins in liver tissue. Taken together these data suggest that heart is more sensitive than the other tissues examined with respect to moderate increases in environmental temperature.

Animals↗
PubMedDate not supplied

Reliable noninvasive genotyping: fantasy or reality?

Noninvasive genotyping has not gained wide application, due to the notion that it is unreliable, and also because remedial measures are time consuming and expensive. Of the wide variety of noninvasive DNA sources, dung is the most universal and most widely used in studies. We have developed collection, extraction, and amplification protocols that are inexpensive and provide a high level of success in amplifying both mitochondrial and nuclear DNA from dung. Here we demonstrate the reliability of genotyping from elephant dung using these protocols by comparing results from dung-extracted DNA to results from blood-extracted DNA. The level of error from dung extractions was only slightly higher than from blood extractions, and conducting two extractions from each sample and a single amplification from each extraction was sufficient to eliminate error. Di-, tri-, and tetranucleotide loci were equally reliable, and low DNA quantity and quality and PCR inhibitors were not a major problem in genotyping from dung. We discuss the possible causes of error in genotyping with particular reference to noninvasive samples and suggest methods of reducing such error.

Animals↗
PubMedDate not supplied

Predicting submaximal oxygen consumption during treadmill running in mice.

Treadmill exercise is the most common form of exercise used in studies involving laboratory rodents. However, oxygen consumption (VO2) is not usually monitored since the measurement of this parameter is particularly difficult in small, exercising animals. The purpose of this study was to develop a regression equation for predicting submaximal VO2 in mice during treadmill running. Twenty female mice of three strains were tested, in an Oxyscan metabolic treadmill, on different days at running speeds of 10, 13, 15, 17, and 20 m/min at a 0% grade. Each mouse was placed in the treadmill chamber for a 10-min period before running commenced. Running speed was increased in 2-min intervals by 5 m/min until the assigned speed was reached. Each mouse then ran for 10 min to reach steady state, followed by another 20 min at the steady-state plateau. During this period a 30-s expired gas sample was collected every minute. Gas samples were analyzed for O2 and CO2, using an Oxyscan gas analyzer. Trials were conducted during the nocturnal phase of the animals' photoperiod, and running speeds were assigned randomly to minimize order effects. This approach provided highly reliable measures of oxygen consumption (r = 0.96). By using the running speeds (10-20 m/min) and the animal's body weight it was possible to predict the submaximal oxygen consumption (mL/min) during running with a high degree of accuracy (i.e., R2 = 0.85). In an independent group of animals (n = 14) we compared the predicted VO2 with the measured VO2. This yielded a good correlation coefficient (r = 0.79) and a low prediction error (+2.4 +/- 2.9%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗