Genetic differentiation of the dengue vector, Aedes aegypti (Ho Chi Minh City, Vietnam) using microsatellite markers

Dengue haemorrhagic fever emerged in the 1950s and has become a major public health concern in most Asian countries. In Vietnam, little is known about the intraspecific variation of the vector and its consequences on vectorial capacity. Here we report the use of microsatellite markers to differentiate Aedes aegypti populations in Ho Chi Minh City, a typical, overcrowded Asian city. Six microsatellite loci, with 5-14 alleles per locus, were scored in 20 mosquito samples collected in 1998 in Ho Chi Minh City. We found substantial differentiation among Ae. aegypti populations from the outskirts, whereas populations from the centre of the city showed less differentiation. These results are consistent with the hypothesis that populations of Ae. aegypti in central Ho Chi Minh City are panmictic because there are abundant larval breeding sites and an abundance of humans for adults to feed upon. In contrast, populations on the outskirts become differentiated largely through the processes of genetic drift because larval breeding sites are not as abundant. These findings implicate human activities associated with urbanization, as factors shaping the genetic structure of Ae. aegypti populations.     

SIR2 regulates recombination between different rDNA repeats, but not recombination within individual rRNA genes in yeast

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Delta and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Delta relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.     

The calcium response of mouse sperm flagella: role of calcium ions in the regulation of dynein activity

Triton X-100-extracted mouse sperm treated with 0.1 mM ATP and 1.0 mM Ca(2+) exhibit an extremely coiled configuration that has been previously described as a curlicue. Sperm in the curlicue configuration exhibit a monotonically curved flagellum where the shear angle of the flagellum can reach a value as high as 14 radians at the flagellar tip. We utilized this strong reaction to Ca(2+) to elucidate the mechanism of the calcium response. The disintegration of the axoneme was facilitated by the use of an extraction procedure that removed the mitochondrial sheath without eliminating the calcium response. The order of emergence of the doublet microtubule outer dense fiber complexes was observed in the presence and absence of added Ca(2+). The identity of the emergent elements was confirmed by transmission electron microscopy. Ca(2+) altered the order of emergence of internal axoneme elements to favor the appearance of the elements of the 9-1-2 side of the axoneme. These elements are propelled baseward by the action of dyneins on doublets 1 and 2. It was also possible to establish that the motive force for maintaining the curlicue configuration is dynein-based. The curlicues were relaxed by inhibition with 50 μM NaVO(3) and were reestablished by disinhibiting the vanadate with 2.5 mM catechol.     

Population Structure of Wild Musk Shrews (Suncus murinus) in Asia Based on Mitochondrial DNA Variation, with Research in Cambodia and Bhutan

The musk shrew (Suncus murinus) is a small mammalian species belonging to Insectivora. It is widely distributed in Asia. To identify the genetic relationship among wild musk shrew populations and examine its migration route, we investigated the populations of Cambodia and Bhutan by using mitochondrial DNA restriction fragment length polymorphism analysis and compared them with other Asian populations previously described. Four haplotypes were detected in Cambodia and eight in Bhutan. A total of 53 haplotypes were detected in Asia and were classified largely into two groups, the Continental and Island types, based on a minimum spanning network. From the distribution of mtDNA types in wild musk shrews, three major population groups are identified in Asia: South Asia, Southeast Asia, and Malay. It is suggested that the Malay population group was a mix of South and Southeast Asian population groups and that this was a contact area of the two groups. In addition, other contact areas between the South and Southeast Asian groups exist in Myanmar, but unlike the Malay, the Myanmar area was the border of these groups.     

Antenatal antioxidant prevents adult hypertension, vascular dysfunction, and microvascular rarefaction associated with in utero exposure to a low-protein diet

Developmental programming of hypertension is associated with vascular dysfunction characterized by impaired vasodilatation to nitric oxide, exaggerated vasoconstriction to ANG II, and microvascular rarefaction appearing in the neonatal period. Hypertensive adults have indices of increased oxidative stress, and newborns that were nutrient depleted during fetal life have decreased antioxidant defenses and increased susceptibility to oxidant injury. To test the hypothesis that oxidative stress participates in early life programming of hypertension, vascular dysfunction, and microvascular rarefaction associated with maternal protein deprivation, pregnant rats were fed a normal, low protein (LP), or LP plus lazaroid (lipid peroxidation inhibitor) isocaloric diet from the day of conception until delivery. Lazaroid administered along with the LP diet prevented blood pressure elevation, enhanced vasomotor response to ANG II, impaired vasodilatation to sodium nitroprusside, and microvascular rarefaction in adult offspring. Liver total glutathione was significantly decreased in LP fetuses, and kidney eight-isoprostaglandin F2alpha (8-isoPGF(2alpha)) levels were significantly increased in adult LP offspring; these modifications were prevented by lazaroid. Renal nitrotyrosine abundance and blood levels of 1,4-dihydroxynonene and 4-hydroxynonenal-protein adducts were not modified by antenatal diet exposure. This study shows in adult offspring of LP-fed dams prevention of hypertension, vascular dysfunction, microvascular rarefaction, and of an increase in indices of oxidative stress by the administration of lazaroid during gestation. Lazaroid also prevented the decrease in antioxidant glutathione levels in fetuses, suggesting an antenatal mild oxidative stress in offspring of LP-fed dams. These studies support the concept that perinatal oxidative insult can lead to permanent alterations in the cardiovascular system development.     

Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.     

Estimation of muscle response using three-dimensional musculoskeletal models before impact situation: a simulation study

When car crash experiments are performed using cadavers or dummies, the active muscles' reaction on crash situations cannot be observed. The aim of this study is to estimate muscles' response of the major muscle groups using three-dimensional musculoskeletal model by dynamic simulations of low-speed sled-impact. The three-dimensional musculoskeletal models of eight subjects were developed, including 241 degrees of freedom and 86 muscles. The muscle parameters considering limb lengths and the force-generating properties of the muscles were redefined by optimization to fit for each subject. Kinematic data and external forces measured by motion tracking system and dynamometer were then input as boundary conditions. Through a least-squares optimization algorithm, active muscles' responses were calculated during inverse dynamic analysis tracking the motion of each subject. Electromyography for major muscles at elbow, knee, and ankle joints was measured to validate each model. For low-speed sled-impact crash, experiment and simulation with optimized and unoptimized muscle parameters were performed at 9.4 m/h and 10 m/h and muscle activities were compared among them. The muscle activities with optimized parameters were closer to experimental measurements than the results without optimization. In addition, the extensor muscle activities at knee, ankle, and elbow joint were found considerably at impact time, unlike previous studies using cadaver or dummies. This study demonstrated the need to optimize the muscle parameters to predict impact situation correctly in computational studies using musculoskeletal models. And to improve accuracy of analysis for car crash injury using humanlike dummies, muscle reflex function, major extensor muscles' response at elbow, knee, and ankle joints, should be considered.     

HLA-Cw group 1 ligands for KIR increase susceptibility to invasive cervical cancer

Inherited genetic polymorphisms within immune response genes have been shown to associate with risk of invasive cervical cancer (ICC) and its immediate precursor, cervical intraepithelial neoplasia grade 3. Here, we used the transmission/disequilibrium test to detect disease-liability alleles and investigate haplotype transmission of KIR and HLA class I polymorphisms in a large family-based population of women with cervical cancer and their biological parents (359 trios). The effect of distinct human papillomavirus types was also explored. HLA-Cw group 1 (HLA-Cw alleles with asparagine at position 80), which serves as ligand for certain killer immunoglobulin-like receptors (KIR), was significantly overtransmitted in women with ICC (P?=?0.04), and particularly in the subgroup of women infected with high risk HPV16 or 18 subtypes (P?=?0.008). These data support the involvement of the HLA-C locus in modulating the risk of cervical neoplasia perhaps through its function as ligands for KIR, but functional studies are essential to confirm this hypothesis.     

A physical map of the sorghum chloroplast genome

Summary The chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the bcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.USDA-ARS