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A population study of heteroplasmy in the hypervariable region 1 (HV1) portion of the human mtDNA control region was performed. Blood samples from 253 randomly chosen individuals were examined using a sensitive denaturing gradient-gel electrophoresis (DGGE) system. This method is capable of detecting heteroplasmic proportions as low as 1% and virtually all heteroplasmy where the minor component is > or = 5%. Heteroplasmy was observed in 35 individuals (13.8%; 95% confidence interval [CI] 9.6-18.0). Of these individuals, 33 were heteroplasmic at one nucleotide position, whereas 2 were heteroplasmic at two different positions (a condition known as "triplasmy"). Although heteroplasmy occurred at a total of 16 different positions throughout HV1, it was most frequently observed at positions 16093 (n=13) and 16129 (n=6). In addition, the majority of heteroplasmic variants occurred at low proportions and could not be detected by direct sequencing of PCR products. This study indicates that low-level heteroplasmy in HV1 is relatively common and that it occurs at a broad spectrum of sites. Our results corroborate those of other recent reports indicating that heteroplasmy in the control region is more common than was previously believed-a finding that is of potential importance to evolutionary studies and forensic applications that are based on mtDNA variation.

The complete mitochondrial cytochrome oxidase II gene was sequenced from 17 black flies, representing 13 putative species, and used to infer phylogenetic relationships. A midge (Paratanytarsus sp.) and three mosquitoes (Aedes aegypti, Anopheles quadrimaculatus, and Culex quinquefasciatus) were used as outgroup taxa. All outgroup taxa were highly divergent from black flies. Phylogenetic trees based on weighted parsimony (a priori and a posteriori), maximum likelihood, and neighbor-joining (log-determinant distances) differed topologically, with deeper nodes being the least well-supported. All analyses supported current classification into species groups but relationships among those groups were poorly resolved. The majority of phylogenetic signal came from closely related sister taxa. The CO-II gene may be useful for exploring relationships at or below the subgeneric level, but is of questionable value at higher taxonomic levels. The weighting method employed gave phylogenetic results similar to those reported by other authors for other insect CO-II data sets. A best estimate of phylogenetic relationships based on the CO-II gene is presented and discussed in relation to current black fly classification., (Copyright 2000 Academic Press.)

Objective: To identify factors in childhood which might influence the development of obesity in adulthood., Background: The prevalence of obesity is increasing in the UK and other developed countries, in adults and children. The adverse health consequences of adult obesity are well documented, but are less certain for childhood obesity. An association between fatness in adolescence and undesirable socio-economic consequences, such as lower educational attainment and income, has been observed, particularly for women. Childhood factors implicated in the development of adult obesity therefore have far-reaching implications for costs to the health-services and economy., Search Strategy: In order to identify relevant studies, electronic databases--Medline, Embase, CAB abstracts, Psyclit and Sport Discus-were searched from the start date of the database to Spring 1998. The general search structure for electronic databases was (childhood or synonyms) AND (fatness or synonyms) AND (longitudinal or synonyms). Further studies were identified by citations in retrieved papers and by consultation with experts., Inclusion Criteria: Longitudinal observational studies of healthy children which included measurement of a risk factor in childhood (<18 y), and outcome measure at least 1 y later. Any measure of fatness, leanness or change in fatness or leanness was accepted. Measures of fat distribution were not included. Only studies with participants from an industrialized country were considered, and those concerning minority or special groups, e.g. Pima Indians or children born preterm, were excluded., Findings: Risk factors for obesity included parental fatness, social factors, birth weight, timing or rate of maturation, physical activity, dietary factors and other behavioural or psychological factors. Offspring of obese parent(s) were consistently seen to be at increased risk of fatness, although few studies have looked at this relationship over longer periods of childhood and into adulthood. The relative contributions of genes and inherited lifestyle factors to the parent-child fatness association remain largely unknown. No clear relationship is reported between socio-economic status (SES) in early life and childhood fatness. However, a strong consistent relationship is observed between low SES in early life and increased fatness in adulthood. Studies investigating SES were generally large but very few considered confounding by parental fatness. Women who change social class (social mobility) show the prevalence of obesity of the class they join, an association which is not present in men. The influence of other social factors such as family size, number of parents at home and childcare have been little researched. There is good evidence from large and reasonably long-term studies for an apparently clear relationship for increased fatness with higher birth weight, but in studies which attempted to address potential confounding by gestational age, parental fatness, or social group, the relationship was less consistent. The relationship between earlier maturation and greater subsequent fatness was investigated in predominantly smaller, but also a few large studies. Again, this relationship appeared to be consistent, but in general, the studies had not investigated whether there was confounding by other factors, including parental fatness, SES, earlier fatness in childhood, or dietary or activity behaviours. Studies investigating the role of diet or activity were generally small, and included diverse methods of risk factor measurement. There was almost no evidence for an influence of activity in infancy on later fatness, and inconsistent but suggestive evidence for a protective effect of activity in childhood on later fatness. No clear evidence for an effect of infant feeding on later fatness emerged, but follow-up to adulthood was rare, with only one study measuring fatness after 7y. Studies investigating diet in childhood were limited and inconc

With the discovery of the polymerase chain reaction (PCR) in the mid-1980's, the last in a series of critical molecular biology techniques (to include the isolation of DNA from human and non-human biological material, and primary sequence analysis of DNA) had been developed to rapidly analyze minute quantities of mitochondrial DNA (mtDNA). This was especially true for mtDNA isolated from challenged sources, such as ancient or aged skeletal material and hair shafts. One of the beneficiaries of this work has been the forensic community. Over the last decade, a significant amount of research has been conducted to develop PCR-based sequencing assays for the mtDNA control region (CR), which have subsequently been used to further characterize the CR. As a result, the reliability of these assays has been investigated, the limitations of the procedures have been determined, and critical aspects of the analysis process have been identified, so that careful control and monitoring will provide the basis for reliable testing. With the application of these assays to forensic identification casework, mtDNA sequence analysis has been properly validated, and is a reliable procedure for the examination of biological evidence encountered in forensic criminalistic cases., (Copyright © 1999 Central Police University.)

Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator.

The nucleic acid of a serologically distinct potyvirus, originally isolated out of sugar cane from Pakistan, was reverse transcribed and the 3' terminal 2000 bp was PCR amplified, cloned, and sequenced. Phylogenetic comparisons of viruses representing each genus of the Potyviridae show that the Pakistani isolate is most closely related to the rymoviruses wheat streak mosaic virus (WSMV) and brome streak mosaic virus. We therefore propose that this new virus species be named sugar cane streak mosaic virus to reflect its similarity to WSMV. The phylogenetic data also show that the genus Rymovirus contains at least two unique evolutionary lineages. Thus the current taxonomy, based on transmission vector, is paraphyletic. We present an analysis of the taxonomic relationships among members of the family and propose a classification that both resolves the paraphyly and more accurately represents the evolutionary history of the Potyviridae., (Copyright 1998 Academic Press.)

Molecular analysis of biological specimens usually requires extraction of high-molecular weight DNA free of foreign DNA contaminants. DNA was extracted from black flies at different life stages that had been preserved by 4 methods: larvae and adults in ethanol, larvae in Carnoy's solution, adults on card-points, and adults hand-swatted and sun-dried. Using specific primers for the mitochondrial ND4 gene, a 257-bp amplicon was obtained from specimens preserved by ethanol, card-point mounting, and sun-drying. Successful amplification often required DNA dilutions > or = 1:20 (< 1-10 ng). DNA from specimens preserved in Carnoy's solution (ethanol: acetic acid, 3:1) yielded degraded DNA, resulting in fewer successful amplications. Parasitic nematodes and, to a lesser extent, gut contents resulted in extra products when amplified with randomly amplified polymorphic DNA (RAPD) primers. Sufficient DNA was extracted from the head of a larva for a successful polymerase chain reaction (PCR), eliminating the need to remove the contaminating gut and parasites.

The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common "European" mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect.

This study investigated the effect of a macrobiotic (vegan-type) diet, low in calcium and vitamin D, consumed in early life, on bone mineral during adolescence. Bone mineral content (BMC) and bone area were measured in 195 adolescents (103 girls, 92 boys) aged 9-15 years, using dual-energy X-ray absorptiometry. Ninety-three adolescents (43 girls, 50 boys) had followed a macrobiotic diet in childhood, and 102 (60 girls, 42 boys) were control subjects. After adjustment for bone area, weight, height, percent body lean, age, and puberty, BMC was significantly lower in macrobiotic subjects, in boys and girls, respectively, at the whole body, -3.4% and -2.5%, spine, -8.5% and -5.0%, femoral neck, -8.0% and -8.2%, midshaft radius, -6.8% and -5.6%, and also in girls, at the trochanter, -5.8% (p < 0.05). No group differences were observed at the wrist. Group differences were not explained by current calcium adjusted bone mass at age 9-15 years, observations which may hold important implications for fracture risk in later life.

The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution and to forensic identity testing. Here, we report a direct measurement of the intergenerational substitution rate in the human CR. We compared DNA sequences of two CR hypervariable segments from close maternal relatives, from 134 independent mtDNA lineages spanning 327 generational events. Ten substitutions were observed, resulting in an empirical rate of 1/33 generations, or 2.5/site/Myr. This is roughly twenty-fold higher than estimates derived from phylogenetic analyses. This disparity cannot be accounted for simply by substitutions at mutational hot spots, suggesting additional factors that produce the discrepancy between very near-term and long-term apparent rates of sequence divergence. The data also indicate that extremely rapid segregation of CR sequence variants between generations is common in humans, with a very small mtDNA bottleneck. These results have implications for forensic applications and studies of human evolution.

In 1991, nine sets of skeletal remains were excavated from a mass grave near Yekaterinburg, Russia which were believed to include the Russian Tsar Nicholas II, the Tsarina Alexandra, and three of their daughters. Nuclear DNA testing of the remains verified such a family group, and mitochondrial DNA (mtDNA) sequences of the presumed Tsarina matched a known maternal relative, Prince Philip. mtDNA sequences from bone of the presumed Tsar matched two living maternal relatives except at a single position, where the bone sample had a mixture of matching (T) and mismatching (C) bases. Cloning experiments indicated that this mixture was due to heteroplasmy within the Tsar; nevertheless, the 'mismatch' fueled a lingering controversy concerning the authenticity of these remains. As a result, the official final report on the fate of the last Russian Royals has been postponed by Russian authorities pending additional, convincing DNA evidence. At the request of the Russian Federation government, we analysed the skeletal remains of the Tsar's brother Georgij Romanov in order to gain further insight into the occurrence and segregation of heteroplasmic mtDNA variants in the Tsar's maternal lineage. The mtDNA sequence of Georgij Romanov, matched that of the putative Tsar, and was heteroplasmic at the same position. This confirms heteroplasmy in the Tsar's lineage, and is powerful evidence supporting the identification of Tsar Nicholas II. The rapid intergenerational shift from heteroplasmy to homoplasmy, and the different heteroplasmic ratios in the brothers, is consistent with a 'bottleneck' mechanism of mtDNA segregation.

Forty males and 40 females, ages 18-21 years, were recruited into a prospective study to investigate the consolidation of bone mineral after cessation of linear growth and the influences of calcium intake, lifestyle factors, physical activity, and body composition on bone. Three sets of bone measurements were made annually at the spine, hip, and whole body (dual-energy X-ray absorptiometry) and at the wrist and midshaft radius (single photon absorptiometry). At baseline, the bone mineral content (BMC) was significantly greater in males than females at all sites (p < 0.004). After adjustment for scan area (BA), height, weight, and age, male BMC was significantly greater than female BMC at the midshaft radius (+7.4%; p < 0.01) but lower at the spine (-10.9%; p < 0.001) with no difference at the wrist, hip, or whole body. Positive effects of leanness on bone were observed in females but not in males. Dietary calcium, nutrient intakes, and physical activity levels were not size-independent determinants of BMC at any site. Significant increases in whole-body BMC were observed within individuals over the study period of 670 (SD 51) days, +1.3% in males and +2.1% in females (p < 0.01), mirrored by increases in BMC and BA at most skeletal sites. After allowing for changes in BA, significant increases (p < 0.01) were observed in adjusted BMC for the whole body (males, +1.1%; females, +0.6%), lumbar spine (males, +1.5%; females, +1.1%), and midshaft radius (males, +1.9%; females, +2.0%). No lifestyle or anthropometric factors were identified that influenced these longitudinal increases in bone mineral.

Several groups have reported an association between schizophrenia and the MscI polymorphism in the first exon of the dopamine D3 receptor gene (DRD3). We studied this polymorphism using a North American sample (117 patients plus 188 controls) and an Italian sample (97 patients plus 64 controls). In the first part of the study, we compared allele frequencies of schizophrenia patients and unmatched controls and observed a significant difference in the total sample (P = 0.01). The second part of the study involved a case control approach in which each schizophrenia patient was matched to a control of the same sex, and of similar age and ethnic background. The DRD3 allele frequencies of patients and controls revealed no significant difference between the two groups in the Italian (N = 53) or the North American (N = 54) matched populations; however, when these two matched samples were combined, a significant difference was observed (P = 0.026). Our results suggest that the MscI polymorphism may be associated with schizophrenia in the populations studied.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was applied to samples from widespread populations of the poorly characterized Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga species complex, and 4 genetically differentiated species were distinguished. A screen of 65 random decamer oligonucleotide primers identified 12 primers, which produced 19 reproducible species-specific genetic markers and 4 markers common to 2 or more species. These markers were correlated in nearly all individuals of each species throughout the ranges sampled, including populations as far apart as 2,500 km. Each individual analyzed was from a different isofemale progeny brood, with associated morphological specimens. These specimens will facilitate studies to relate these species to previously reported chromosomal and enzymatic variation as well as to their feeding behavior and potential as malaria vectors. We hypothesize that 3 of the species have recognized valid names: An. (Nys.) albitarsis Lynch-Arribálzaga, An. (Nys.) marajoara Galvão and Damasceno, and An. (Nys.) deaneorum Rosa-Freitas, whereas the 4th is undescribed.

Theory predicts that traits under positive selection can rapidly cross a hybrid zone in spite of a substantial barrier to neutral gene flow between hybridizing taxa. An avian hybrid zone between Manacus candei (white-collared manakin) and M. vitellinus (golden-collared manakin) is reported here that displays an unusual pattern of noncoincident clines. Male secondary sexual traits of M. vitellinus have spread into populations that are genetically and morphometrically like M. candei. These birds have a lek breeding system in which male mating success is highly skewed, suggesting that sexual selection is driving male sexual traits across the zone.

The usefulness of random amplified polymorphic DNA (RAPD) was examined as a potential tool to differentiate cryptic mosquito species. It proved to be a quick, effective means of finding genetic markers to separate two laboratory populations of morphologically indistinguishable African malaria vectors, Anopheles gambiae and An. arabiensis. In an initial screening of fifty-seven RAPD primers, 377 bands were produced, 295 of which differed between the two species. Based on criteria of interpretability, simplicity and reproducibility, thirteen primers were chosen for further screening using DNA from thirty individuals of each species. Seven primers produced diagnostic bands, five of which are described here. Some problematic characteristics of RAPD banding patterns are discussed and approaches to overcome these are suggested.

Overexpression and amplification of the neu (c-erbB2, ERBB2) protooncogene have been implicated in the development of aggressive human breast cancer. To directly assess the effect of mammary gland-specific expression of the neu protooncogene, transgenic mice carrying unactivated neu under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were established. By contrast to the rapid tumor progression observed in several transgenic strains carrying the activated neu transgene, expression of unactivated neu in the mammary epithelium resulted in the development of focal mammary tumors after long latency. The majority of the mammary tumors analyzed expressed elevated levels of neu-encoded mRNA and protein. Overexpression of neu in the mammary tumors was also associated with elevated neu intrinsic tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins. Interestingly, many of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. These observations suggest that overexpression of the unactivated neu protein can induce metastatic disease after long latency.

When the lower leaves of hybrid poplar trees are mechanically wounded, several novel mRNAs accumulate in the unwounded upper leaves (Parsons TJ, Bradshaw HD, Gordon MP: Systemic accumulation of specific mRNAs in response to wounding in poplar trees, Proc Natl Acad Sci USA, in press). A partial cDNA clone corresponding to a transcript from the wound-responsive gene designated win 3 (wound-inducible) has been cloned by differential hybridization to 32P-labelled cDNA from the leaves of wounded trees. Northern blots show a large accumulation of win 3 transcripts in the unwounded leaves of wounded trees. Southern blot analysis of poplar DNA suggests that win 3 is a member of a multigene family. The nucleotide sequences of several win 3 cDNA clones have been determined, indicating that at least three win 3 gene family members are transcribed. A genomic clone of a win 3 gene family member has been isolated and a 1.5 kb Hind III fragment containing the predicted protein-coding and 5' upstream regions has been sequenced. The putative win 3 gene product is similar to the major soluble proteins of sweet potato tubers, sporamin A and sporamin B. Both Win3 and the sporamins share significant amino acid sequence identity with Kunitz-type trypsin inhibitors from legume seeds. The Kunitz family of proteinase inhibitors thus joints three other proteinase inhibitor families which are systemically responsive to wounding.

Different types of collagen vary in their influence on platelet reactivity. Collagen Types III, IV, and V were obtained from human placental tissue, and Type I collagen was prepared from rat skin. Each collagen type was coated onto a plastic surface. Each collagen-coated surface or appropriate plastic surface control was studied using citrated human 51Cr-labeled platelet-rich plasma in both the presence and absence of 10 microM adenosine 5'-diphosphate (ADP). Both unstimulated and ADP-induced platelet adherence were: 1) reduced by Type V collagen coating in comparison to uncoated wells; and 2) increased by Types III and IV collagen coating in comparison to Type V coated or plastic surfaces. Addition of the fast-acting thrombin inhibitor dansylarginine (DAPA) had no significant effect on unstimulated and ADP-induced platelet adherence to Type III, IV or V collagen-coated surfaces. Type I collagen-coated surfaces, studied only in the presence of DAPA, caused greater platelet adherence than those coated with Types III, IV, or V collagen. We conclude that Type V collagen may be less thrombogenic than Types, I, III, or IV.

Damage to the endothelial surface of the vessel wall can result in exposure of circulating blood components to collagen and other subendothelial structures. Collagen types I, III, IV, and V have been demonstrated in the vessel wall by chemical and immunohistological methods; type V is thrombin-sensitive, and is present on the endothelial cell surface. In an earlier study using a rocking model, both unstimulated and ADP-induced platelet adherence was reduced on wells coated with type V collagen in comparison to uncoated wells; and increased on plastic surfaces coated with types III and IV collagen in comparison to those coated with type V collagen. The present study was designed to determine the effect of erythrocytes and shear rate on platelet adherence to these purified collagen types in a laminar flow system. With platelet-rich plasma, adherence of labeled platelets was much lower in the laminar flow system compared with the rocking model. Erythrocytes significantly enhanced platelet adherence to surfaces that were untreated or absorbed with collagen types I, III, and IV. However, this enhancement was not seen in the presence of type V collagen. These studies provide additional evidence for the selectively nonthrombogenic nature of type V collagen.

Human mononuclear phagocyte (M phi) populations were compared to adult human endothelial cells (HEC) for their respective abilities to influence the proliferative responses of purified human T lymphocytes to the mitogenic agents Na-m-periodate (IO-4), soybean agglutinin (SBA), or allogeneic cells. HEC and M phi were both capable of inducing proliferative responses of allogeneic T lymphocytes in mixed-lymphocyte culture. Under low cell density culture conditions, purified T-lymphocyte proliferative responses to IO-4 or SBA could be restored by addition of syngeneic M phi or HEC. At higher cell density culture conditions, proliferation of T cells to IO-4 could be amplified more by HEC than M phi. T-lymphocyte proliferative responses to SBA were amplified by addition of HEC but were suppressed by addition of M phi. These findings indicate that human adult HEC are unique and potent accessory cells for T lymphocytes. Furthermore, these findings demonstrate that accessory cell functions of HEC can be discriminated from those of M phi.

Consultation rates of medical house staff physicians were examined in a primary care setting in order to determine the factors that lead to the use of consultants. The factors that were studied in the regression analysis were clinical experience and teaching in medical school, intensity of use of services, residency year, Alpha Omega Alpha (AOA) membership, and future practice plans. The factors were marginally useful in predicting surgical consultations but were helpful in explaining the use of other consultations. House staff physicians with a pattern of intense ancillary service utilization also requested more consultations. Senior house staff physicians requested fewer consultations than junior house staff members. AOA members and future academic physicians used more consultations than other physicians. Consultation rates were highest for specialties requiring the lowest quantity of education in medical school. Consultation patterns reflect past educational experiences and future practice plans of medical house staff physicians and may be useful in the formulation of the curriculum of medical schools and residencies.

cDNAs for several transcripts that accumulate systemically in response to mechanical wounding in a hybrid poplar have been cloned. The corresponding mRNAs become abundant in the unwounded upper leaves of poplar trees whose lower leaves have been damaged. Two of the cDNA clones have been characterized by Northern and Southern blotting, and their nucleotide sequences have been determined. These clones, designated win6 (wound-inducible) and win8, are members of multigene families encoding proteins with a high degree of similarity to chitinases from bean, tobacco, and barley. This initial demonstration of a systemic response to wounding in trees provides an approach to study defense mechanisms in woody plants at the biochemical, physiological, and ecological levels.