Functional Restoration of a Midforearm Level Amputation using the Second Toe Transfer.

Unilateral midforearm level amputation is a severely debilitating situation. The present solution for this problem is either restoration of function using a prosthetic hand, or a vascularized composite allotransplant (VCA) in some very selected cases. In cases of distal forearm level amputations, even vascularized second toe transfer could be done on the radial side of the hand to achieve good functional restoration. We present a case of midforearm level amputation in which the second toe transfer has been done above the level of insertion of pronator teres muscle to achieve meaningful function. This report highlights the fact that a single toe transfer could be helpful in achieving meaningful prehension, even at this proximal.

Preparation and characterization of (125)i labeled bovine serum albumin.

Bovine serum albumin is a model protein, which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology and medicine. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer ((125)I-BSA) with required specific activity without impairing the immune reactivity and biological activity. Optimized radioiodination procedure involving 10 µg of chloramine-T along with 20 µg of sodium metabisulphite with 60 seconds incubation at 2° yielded (125)I-BSA with high integrity.

Clitoria ternatea root extract enhances acetylcholine content in rat hippocampus.

Treatment with 100 mg/kg of Clitoria ternatea aqueous root extract (CTR), for 30 days in neonatal and young adult age groups of rat, significantly increased acetylcholine (ACh) content in their hippocampi as compared to age matched controls. Increase in ACh content in their hippocampus may be the neurochemical basis for their improved learning and memory.

Clitoria ternatea (Linn) root extract treatment during growth spurt period enhances learning and memory in rats.

Neonatal rat pups (7 days old) were intubated with either 50 mg/kg body weight or 100 mg/kg body weight of aqueous root extract of Clitoria ternatea (CTR) for 30 days. These rats were then subjected to open field, two compartment passive avoidance and spatial learning (T-Maze) tests (i) immediately after the treatment and (ii) 30 days after the treatment, along with age matched normal and saline control rats. Results showed no change in open field behaviour, but showed improved retention and spatial learning performance at both time points of behavioural tests, indicating the memory enhancing property of CTR which implicates a permanent change in the brain of CTR treated rats.

Mosquito genomes: structure, organization, and evolution.

A great deal of information has been accumulated on chromosome numbers and heterochromatin distribution as well as on genome size and organization in the mosquito family Culicidae. A number of trends in genome evolution emerge when these data are reviewed in light of recent cladistic phylogenies of Culicidae and its sister families. Anophelinae have heteromorphic sex chromosomes and a small genome size, and repetitive elements are distributed in a long-period interspersion pattern. In contrast, Culicinae have homomorphic sex chromosomes, and repetitive DNA is organized in a short-period interspersion pattern. There has been a general increase in genome size during the evolution of culicine tribes. The organization of the ancestral culicid genome remains uncertain awaiting studies on genome organization in Chaoboridae-Corethrellidae taxa. The most parsimonious hypothesis for the evolution of sex chromosomes and genome organization in Culicidae would be that homomorphic sex chromosomes and a long-period interspersion pattern was ancestral in lineages leading to Toxorhynchitinae and Culcinae. Larger genomes developed in subsequent culicine lineages through accumulation of short-period interspersed repetitive elements. Heteromorphic sex chromosomes evolved early in the evolution of Anophelinae, and a long-period interspersion pattern was retained. The alternative scenario proposed by Rao and Rai (1987a) is that Culicidae arose from a chaoborid Mochlonyx-like ancestor with heteromorphic sex chromosomes and possibly short-period interspersion. This scenario would require the loss of heteromorphic sex chromosomes in the lineage leading to Toxorhynchitinae and Culicinae and the "shedding" of repetitive elements in the lineage leading to Anophelinae. Several interesting patterns have emerged from studies of C-banding, and the distribution of heterochromatin in Culicidae and phylogenies derived from these studies are supported by the modern cladistic analyses. Recent intensive multipoint linkage map studies suggest that recombination frequencies per genome have remained relatively constant over the course of culicid evolution such that Anophelinae, with a relatively small genome size, has a linkage map of similar size to Aedini. As a consequence, taxa in Anophelinae have higher amounts of recombination per haploid genome size than Culicinae. Although several key questions have yet to be addressed, the Culicidae remain one of the best-studied systems of genome evolution in animals.

An estimate of phylogenetic relationships among culicine mosquitoes using a restriction map of the rDNA cistron.

Restriction maps of the rDNA cistron of twelve species of mosquitoes in six genera of the subfamily Culicinae were constructed using eight 6 bp recognition restriction enzymes. Anopheles albimanus was used as an outgroup. The size of the rDNA cistron ranged from 8.5 kb in Aedes katherinensis to 12.9 kb in Ae. polynesiensis. A total of twenty-six sites were scored; eighteen were polymorphic among ingroup taxa. The proportion of polymorphic nucleotide sites (Pnuc) was 0.059 and the heterozygosity per nucleotide site (Hnuc) was 0.028. Wagner and Fitch Parsimony, Dollo Parsimony and Nei-Li distance/neighbour-joining methods were used to construct phylogenetic trees. The rDNA RFLP dataset did not provide a well-supported phylogeny among culicine taxa. The RFLP phylogenies are incongruent with the morphology character based and molecular phylogenies and derived relationships did not correspond with current taxonomic classifications. The lack of resolution was due to homoplasy arising from frequent independent loss or gain of restriction sites among unrelated taxa.

Molecular organization and evolution of mosquito genomes.

Given the importance of mosquitoes as disease vectors, relatively little is known about the molecular organization and evolution of mosquito genomes as compared to other insects such as fruit flies. The advances in recombinant DNA technology and the possibility that mosquito populations could be controlled and genetically manipulated using such technology has stimulated considerable research during the last few years in the areas of genome organization and evolution, genome mapping, endogenous transposable elements, and mapping and characterization of genes conferring susceptibility to different parasites and pathogens. This review summarizes research currently underway in our laboratory and elsewhere in these areas.

Midgut basal lamina thickness and dengue-1 virus dissemination rates in laboratory strains of Aedes albopictus (Diptera: Culicidae).

Strain differences in midgut basal lamina thickness, assessed by measurement in transmission electron micrographs, and disseminated infection rates of dengue-1 virus were compared among three laboratory strains of Aedes albopictus (Skuse). Mean basal lamina thickness for the New Orleans and Houston strains were significantly greater than those for the Oahu strain, which exhibits a higher disseminated infection rate than the former two. Although basal lamina thickness among the F1 progeny of reciprocal crosses of the Oahu and Houston strains were intermediate between the parental strains, they were too variable to be useful as markers in genetic studies. Measurements of basal laminae among individuals of the New Orleans strain, with disseminated or nondisseminated infections, failed to demonstrate a role for basal lamina thickness as a modulator of dengue-1 virus dissemination across the midgut epithelium of Ae. albopictus.

Random amplified polymorphic DNA of mosquito species and populations (Diptera: Culicidae): techniques, statistical analysis, and applications.

A polymerase chain reaction (PCR)-based method is described for the identification and differentiation of mosquito species and populations. The method, described first by Williams et al. (1990), employs single 10 base-long primers of arbitrary DNA sequence and results in the amplification of random segments of DNA known as random amplified polymorphic DNA (RAPD). We wished to determine if RAPD of mosquito DNA could be used for the differentiation of species and populations, identification of unknown specimens, and the reconstruction of phylogeny. RAPD of mosquito DNA results in the amplification of a series of DNA fragments of varying length. Most amplified fragments are unique to an individual; however, our data indicated that in each of the five species of Aedes examined, some fragments are species-specific and are present in all individuals of that species. This enabled us to derive a diagnostic profile for each of the five species. A nearest-neighbor analysis of all the amplified DNA fragments discriminated among species on a multivariate basis. Several individuals of Aedes albopictus (Skuse), included in the analysis as "unknowns," were correctly identified as belonging to Ae. albopictus. UPGMA clustering of presence-absence data enabled the separation of different Aedes species as well as different populations of Ae. albopictus. The entomological applications of RAPD include the construction of diagnostic profiles for species identification and differentiation among conspecific populations.

Variation in the efficiency of vertical transmission of dengue-1 virus by strains of Aedes albopictus (Diptera: Culicidae).

Five geographical strains of Aedes albopictus (Skuse) were compared for their ability to transmit vertically a dengue-1 isolate from Jamaica. The OAHU strain of Ae. albopictus and a strain of Aedes aegypti (L.) from the United States were included as controls. The offspring of orally infected females were assayed individually for vertical infection. Vertical transmission rates among strains ranged from 11 to 41%, and filial infection rates of strains ranged from 0.5 to 2.9%. Filial infection rates of individual positive families within strains ranged from 1.4 to 17.4%. These rates were higher than those previously recorded for Ae. albopictus. The observed differences in rates of vertical transmission among the strains were not statistically significant, because 95% of the measured variation was attributed to families within strains. The most significant source of variation in vertical transmission of dengue-1 by Ae. albopictus was at the individual level.

Frequencies of mitochondrial DNA haplotypes in laboratory cage populations of the mosquito, Aedes albopictus.

A laboratory cage experiment was undertaken to study changes over time in the frequencies of two mitochondrial DNA (mtDNA) haplotypes in the mosquito, Aedes albopictus, under two conditions: bidirectionally compatible matings and unidirectionally incompatible matings. Frequencies were monitored for 10 generations in three replicate cages for each of the two conditions above. In cages with bidirectionally compatible strains, changes in haplotype frequencies were nondirectional and neither haplotype increased in frequency. Statistical analysis of relative proportions of the two haplotypes in each generation indicated that the magnitude of the observed fluctuations could be expected under an assumption of random genetic drift alone. In cages with unidirectionally incompatible matings, mtDNA of females that lay inviable eggs upon mating with males of another strain, decreased significantly in the F1 generation and was completely replaced in the F2 generation.

Conservation of a highly repeated DNA family of Aedes albopictus among mosquito genomes (Diptera: Culicidae).

The genomic organization and chromosomal localization of a cloned 0.79-kb highly repeated DNA fragment, H-115, isolated from Aedes albopictus has been examined. The cloned fragment is a part of a larger unit of 1.86 kb that is tandemly repeated in the Ae. albopictus genome. The H-115 family of sequences are located at the intercalary position on chromosome 1 in Ae. albopictus. Similar patterns of in situ and Southern blot hybridization results are obtained in Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. Alcasidi, and Ae. katherinensis. The H-115 sequences are widely conserved in Culicidae and are found in Haemagogus equinus, Tripteroides bambusa, and Anopheles quadrimaculatus by hybridization under high stringency conditions. The H-115 sequences are also tandemly repeated in Hg. equinus with a monomer unit of 1.86 kb and in Tp. bambusa with a slightly diverged monomer unit of 1.90kb. In Anopheles quadrimaculatus, the H-115 sequences are dispersed throughout the genome. Partial sequence analysis shows that the H-115 insert is 62% AT and contains two perfect inverted repeats and numerous perfect direct repeats. The occurrence of inverted repeats with potential to form intrastrand palindromic structure suggests that the H-115 family of sequences may be involved in chromatin condensation.

Organization of a cloned repetitive DNA fragment in mosquito genomes (Diptera: Culicidae).

The structure and genomic organization of a cloned 5.2-kb repetitive DNA fragment, H-85, isolated from the Aedes albopictus genome have been examined. In situ hybridization of the 3H-labeled H-85 DNA to the meiotic and mitotic chromosome preparations of Ae. albopictus shows that the sequences homologous to H-85 DNA are dispersed throughout the length of all three pairs of chromosomes. A similar pattern of in situ hybridization appears in Aedes seatoi, Aedes flavopictus, and Aedes aegypti. The study shows that the arrangement of sequences in the cloned 5.2-kb fragment is rare in the Ae. albopictus genome. Dot-blot hybridization reveals that the sequences homologous to H-85 DNA are present in 12 species of mosquitoes examined, belonging to six genera in subfamilies Culicinae ad Anophelinae. The H-85 sequences are also present in the genome of Mochlonyx velutinus of the nematocerous family Chaoboridae, earlier proposed as the ancestor of the mosquito family Culicidae. Although the sequences homologous to H-85 DNA are present in different species of mosquitoes, they have diverged in their structure and organization. The cloned 5.2-kb fragment is composed of elements of different and independently evolving repetitive DNA families.

Geographic origin of the US and Brazilian Aedes albopictus inferred from allozyme analysis.

A survey of genetic variation using allozymes was conducted on 57 populations of Aedes albopictus, an Asian mosquito that was introduced into the US and Brazil in the mid-1980s. Our objective was to quantify the patterns of genetic variation among its populations and to use that information to trace genetically the geographic origin of the US and Brazilian populations. Populations from the various regions were genetically distinct from one another. Populations from within a region were genetically similar to one another, which resulted in a significant positive correlation between geographic and genetic distances. A discriminant analysis of allele frequencies separated populations from the various countries into nine non-overlapping clusters; the US, Japanese, Chinese and Brazilian populations formed closely placed, but distinct, clusters. The probability of assigning a population to the correct country was 98 per cent. The US and the Brazilian populations were closest in terms of genetic distance from the Japanese populations. Based on discriminant and genetic distance analyses, we conclude that the US and the Brazilian Aedes albopictus originated in Japan.

Temporal variation in the ribosomal DNA nontranscribed spacer of Aedes albopictus (Diptera: Culicidae).

Length variation in ribosomal DNA (rDNA) nontranscribed spacer within and among populations of the mosquito Aedes albopictus was studied over a 3-year period in eight different populations from Texas. Statistically significant variation was detected among populations at each sampling date. Significant, but nondirectional, changes were observed within populations over time. An average of 78% of the total variance in the frequencies of spacer variants was attributable to the within-population component, suggesting local differentiation. The results corroborate those from a previous study on spacer length diversity in populations of A. albopictus from around the world and are similar to those from a study on temporal variation in allozyme frequencies in some of the same populations and sampling dates. We propose that a high rate of production of length variants and localized breeding structure could explain the observed high levels of within- and among-population variation in A. albopictus.

Mitochondrial DNA variation within and among populations of the mosquito Aedes albopictus.

A survey of restriction fragment polymorphism in mitochondrial DNA (mtDNA) of 17 populations of the mosquito Aedes albopictus was undertaken. The mtDNA size was estimated to be about 17.5 kbp. The level of polymorphism was low, with over 99% of the fragments being shared in common among the 17 populations. Three populations, Mauritius, Singapore, and Hong Kong, contained individuals with both the ancestral and novel mtDNA haplotypes. We conclude that the low level of mtDNA polymorphism in A. albopictus is a result of recent range expansion and that the mixture of haplotypes is a likely result of human-aided gene flow among populations.

Temporal variation in genetic structure of a colonising species: Aedes albopictus in the United States.

The medically important mosquito, Aedes albopictus, has recently become established in several locations within the United States. A three-year survey of temporal variation in allele frequencies at ten enzymatic loci in 17 populations was undertaken to determine the extent and direction of change since the colonization event. Significant, but non-directional, changes were detected in allele frequencies at most of the loci in all populations. Some alleles detected at low frequencies in earlier samples were not found in subsequent samples. Variance of allele frequencies remained unchanged. In at least two locations, there was an increase in mean heterozygosity, perhaps because of gene flow. There was no significant change in the levels of genetic variation within and among populations. The results from this study indicate that the breeding structure of Ae. albopictus in the U.S. did not differ substantially from that in a native habitat, either soon after colonisation or after several generations in the new habitat. We suggest that a large founder population, gene flow and a rapid population expansion could explain the present breeding structure of Ae. albopictus.

Genome evolution in the mosquitoes and other closely related members of superfamily Culicoidea.

Karyotypes and nuclear DNA amounts of 36 species belonging to twelve genera of mosquitoes and other closely related taxa in the superfamily Culicoidea (Dixidae, Chaoboridae, and Culicidae) were studied. The results showed an eight-fold range in haploid DNA amounts and a 4.5-fold range in total chromosomal lengths. It is concluded that the evolutionary trend in this superfamily has been an overall increase in total haploid DNA amounts from the primitive Dixidae to the highly evolved Culcidae, with a concomitant increase in overall chromosome sizes.