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Thirteen new polymorphic microsatellite markers in Fenneropenaeus penicillatus were isolated and characterized. The polymorphism of the thirteen microsatellite markers was tested by 30 individuals from Lianjiang, China. It showed that the number of al-leles per locus ranged from 3 to 6 and the Polymorphism Information Content (PIC) was from 0.324 to 0.706. The observed and expected heterozygosities were 0.3217-0.8023 and 0.1977-0.6783, respectively. Only one loci (LJ-19) deviated significantly from Hardy-Weinberg equilibrium (HWE) (P < 0.00385) after Bonferroni correction, while the other twelve markers were in HWE after Bonferroni correction (P > 0.00385). The thirteen polymorphic microsatellite markers could pro-vide more genetic data for further research on cultivation and recovery of F. penicillatus.

The marbled rockfish, Sebastiscus marmoratus, is an important commercially near-shore fish that inhabits the beach rocky bottom from Japan to the South China Sea. Eleven polymorphic microsatellite loci were developed from S. marmoratus and were used to identify polymorphisms in 30 samples from a wild population. The allele locus number ranged from 2 to 7. Polymorphism data content ranged from 0.032 to 0.751. The observed and expected heterozygosity levels were 0.0333-0.9667 and 0.0328-0.7675, respectively. Two loci, Smd1-112 and Smd2-80, deviated from Hardy-Weinberg equilibrium. These polymorphic microsatellite markers will facilitate further studies of genetic diversity and genetic differentiation of S. marmoratus.

Holothuria leucospilota is a tropical holothurian species that is widely distributed in the tropical and sub-tropical India-Western Pacific Region. Eight polymorphic microsatellite loci were developed from H. leucospilota by using the protocol fast isolation by amplified fragment length polymorphism of sequences containing repeats and tested in 30 individuals from Hainan Island in China. The number of alleles was 2-6 and polymorphism information content ranged from 0.371-0.694. The levels of expected and observed heterozygosities varied from 0.3913-0.6701 and from 0.1154-0.7000, respectively. No significant linkage disequilibrium was detected for any pairwise combination of loci. Only loci YZHS1-42 deviated from Hardy-Weinberg equilibrium. These polymorphic microsatellite loci may be useful for germplasm conservation of H. leucospilota.

Summary: We tested whether two genetic variants were associated with BMD at multiple clinically relevant skeletal sites in Caucasians. We found that variant rs7776725 is consistently associated with hip, spine, wrist and whole-body BMD, which highlights the potential importance of this variant or linked variants for osteoporosis., Introduction: A recent genome-wide association study identified two single nucleotide polymorphisms (SNPs), rs7776725 and rs1721400, that were associated with bone mineral density (BMD) variation at the radius, tibia and calcaneus in a Korean population. In this study, we aimed to test whether the association of these two genetic variants can be replicated in Caucasians and whether their association with BMD can be extended to other clinically relevant skeletal sites., Methods: We performed this study in two large cohorts of unrelated US Caucasians. Area BMD at the hip, spine, wrist (ultra-distal radius) and whole body were measured with Hologic dual-energy X-ray absorptiometer. SNPs were genotyped with Affymetrix human genome-wide genotyping arrays. Association analyses were performed using PLINK., Results: We detected highly significant association (combined p = 1.42 × 10(-16)) of rs7776725 with wrist BMD but only borderline association signal (combined p = 0.017) for rs1721400 with wrist BMD. In addition, we found that rs7776725 was associated with BMD at the hip, spine and whole body. At the FAM3C gene locus where rs7776725 was located, we identified several other SNPs (rs4727922, rs1803389, rs718766 and rs7793554) that were also associated with BMD., Conclusions: This is the first follow-up association study of rs7776725 and rs1721400 with BMD. The rs7776725 showed consistent association with BMD at multiple clinically important skeletal sites, which highlighted the potential importance of rs7776725 or linked SNPs for risk of osteoporosis. Further in-depth re-sequencing studies and functional assays are necessary to elucidate the underlying mechanisms.

Summary: Given the biological function of SOX6 and recent genome-wide association finding, we performed a fine-mapping association analyses to investigate the relationship between SOX6 and BMD both in Caucasian and Chinese populations. We identified many single-nucleotide polymorphisms (SNPs) within or near the SOX6 gene to be significantly associated with hip bone mineral density (BMD)., Introduction: SOX6 gene is an essential transcription factor in chondrogenesis and cartilage formation. Recent genome-wide association studies (GWAS) detected a SNP (rs7117858) located at the downstream of SOX6 significantly associated with hip BMD., Methods: Given the biological function of SOX6 and the GWAS finding, we considered SOX6 as a new candidate for BMD and osteoporosis. Therefore, in this study, we performed a fine-mapping association analyses to investigate the relationship between SNPs within and near the SOX6 gene and BMD at both hip and spine. A total of 301 SNPs were tested in two independent US Caucasian populations (2,286 and 1,000 unrelated subjects, respectively) and a Chinese population (1,627 unrelated Han subjects)., Results: We confirmed that the previously reported rs7117858-A was associated with reduced hip BMD, with combined P value of 2.45 × 10(-4). Besides this SNP, we identified another 19 SNPs within or near the SOX6 gene to be significantly associated with hip BMD after false discovery rate adjustment. The most significant SNP was rs1347677 located at the intron 3 (P = 3.15 × 10(-7)). Seven additional SNPs in high linkage disequilibrium with rs1347677 were also significantly associated with hip BMD. SNPs in SOX6 showed significant skeletal site specificity since no SNP was detected to be associated with spine BMD., Conclusion: Our study identified many SNPs in the SOX6 gene associated with hip BMD even across different ethnicities, which further highlighted the importance of the SOX6 gene influencing BMD variation and provided more information to the understanding of the genetic architecture of osteoporosis.

Unlabelled: This study aimed to delineate the mechanism involved in type 1 diabetes-induced bone loss. The results revealed the alteration of vitamin D metabolic enzyme expression and the downregulation of renal calcium transporter abundance in type 1 diabetic mice., Introduction: The purpose of this study was to investigate the changes of the expression of vitamin D metabolic enzymes and transcellular calcium-transporting proteins in kidneys from mice with experimentally induced diabetes., Methods: Male DBA/2J mice were injected with either vehicle (control) or streptozotocin (STZ) daily for five consecutive days. Bone mineral density was measured by peripheral quantitative computerized tomography, and bone histomorphology was analyzed by Safranin O staining. Real-time PCR and Western blotting were applied to determine the expression of target genes and proteins., Results: Type 1 diabetes produced high urinary calcium excretion and loss of trabecular bone measured at the proximal metaphysis of the tibia and the distal femur. Bone loss was associated with deterioration of trabecular bone microstructure. Quantified PCR results showed that mRNA expression level in the kidney of diabetic mice for 25-hydroxyvitamin D-24-hydroxylase was downregulated at week 10, while those for 25-hydroxyvitamin D-1α-hydroxylase were upregulated at week 20. In addition, mRNA expression levels for renal transient receptor potential V6, plasma membrane Ca-ATPase (PMCA)1b, and vitamin D receptor (VDR) genes were decreased in STZ-treated mice. Western blot analysis showed that protein expression of PMCA1b and VDR was significantly decreased in kidneys from STZ-treated mice compared to that of controls., Conclusions: The limitation in this study is the lack of vitamin D, parathyroid hormone, and phosphorus levels in serum. However, the present study supports the conclusion that the underlying mechanism contributing to type 1 diabetes-associated bone loss may be alterations of vitamin D metabolic enzyme expression and associated decreases in expression of renal calcium transporters.

Unlabelled: This study aims at investigating the effects of age, sex, and ethnicity on five femoral neck geometric parameters (FNGPs): femoral neck periosteal diameter, cross-sectional area, cortical thickness, sectional modulus, and buckling ratio and found that the three factors would influence the FNGPs., Introduction: Bone geometry is one of the most important predictors of bone strength and osteoporotic fractures. This study aims at investigating the effects of age, sex, and ethnicity on five femoral neck geometric parameters (FNGPs): femoral neck periosteal diameter (W), cross-sectional area (CSA), cortical thickness (CT), sectional modulus (Z), and buckling ratio (BR)., Methods: In the studied 861 Caucasian subjects and 3,021 Chinese individuals, CSA, CT, and Z displayed trends of decrease with age, but W and BR showed increasing trends with age in both Chinese and Caucasian females and males (p < 0.05). W, CSA, CT, and Z were significantly higher (p

Unlabelled: Osteoporosis is a major public health problem characterized by low bone mineral density (BMD). This replication study confirmed 38 single-nucleotide polymorphisms (SNPs) out of 139 SNPs previously reported in three recent genome-wide association studies (GWASs) in an independent US white sample. Ten SNPs achieved combined p < 3.6 x 10(-4)., Introduction: BMD is under strong genetic control. This study aims to verify the potential associations between BMD and candidate genes/loci reported by GWAS of FHS100K, Icelandic deCODE, and UK-NL., Methods: Eight promising (at the genome-wide significant level after Bonferroni correction) and 131 available sub-promising (at the most stringent p value, p < 5.5 x 10(-5) in the three GWASs reports) SNPs were selected. By using genotypic information from Affymetrix 500 K SNP arrays, we tested their associations with BMD in 1,000 unrelated US whites. Fisher's combined probability method was used to quantify the overall evidence of association. BMD was measured by dual energy X-ray absorptiometry., Results: Two promising SNPs, rs3762397 and rs3736228, were replicated in the current study with p < 0.05. Besides, 36 sub-promising SNPs were replicated at the same significant level. Ten SNPs achieved significant combined p < 3.6 x 10(-4) (0.05/139 SNPs, corrected for multiple testing)., Conclusions: Osteoporosis susceptibility of 38 SNPs was replicated in 1,000 unrelated US whites. This study showed promise for replication of some initial genome-wide association signals.

Osteoporosis is a major health problem, mainly characterized by low bone mineral density (BMD). Osteocalcin (also known as BGP, for bone Gla protein) is a significant biomarker of bone turnover and thus the BGP gene has been considered as an important candidate gene for osteoporosis. A few studies on the relationship between variants of the BGP gene and BMD variation, via traditional association and/or linkage methods, have yielded conflicting results. In the present study, we simultaneously tested linkage and/or association of the BGP HindIII polymorphism with BMD in a large cohort of pre-menopausal Chinese women. A total of 1,263 subjects from 402 Chinese nuclear families were examined. Each family consists of both parents and at least one daughter aged between 20-45 years. BMDs at the lumbar spine and hip were measured by dual-energy X-ray absorptiometry (DXA). Using the QTDT (quantitative transmission disequilibrium test) program, we did not detect significant evidence of linkage or association between the BGP HindIII polymorphisms and the BMD variation at any skeletal site. Our data do not support the BGP gene having a major effect on BMD variation in pre-menopausal Chinese women.

Summary: The characterization of deleterious genomic mutation (DGM) is of central significance for evolutionary biology and genetic studies. Fitness moment method has been developed to efficiently characterize DGM from natural population directly. In order to enable researchers to employ this method for theoretical and empirical research on characterizing DGM, we here present a distributed Java Application for DGM Estimation (JADE)., Availability: http://orclinux.creighton.edu/DGM/index.htm.

Osteoporosis is a serious health problem in both Caucasians and Asians. Caucasians and Asians are two distinct major ethnic groups, which may have differential genetic determination underlying complex genetic diseases such as osteoporosis. However, to date, there has been no systematic review focusing on the aspect of ethnic difference in risk to osteoporosis and its potential underlying genetic determination between Asians and Caucasians. Here, we firstly review diverse aspects of osteoporosis-related differences, including the differences of epidemiology of osteoporotic fractures, peak bone mass, bone loss, bone area, bone geometry and drug treatment response between Asians and Caucasians. Then, we provide some potential genetic evidence on the different heritability and inheritance mode of bone phenotypes, the different osteoporosis candidate genes and the differential results in related molecular studies between them, to explain the above osteoporosis-related phenotypic differences. The results suggest that the osteoporosis-related phenotypic differences between Asians and Caucasians may be partially the result of the different ethnic genetic background. The present review may increase our understanding of potential different mechanisms related to ethnicity in pathogenesis of osteoporosis for effective and potentially customized treatments in different major ethnic groups.

Background: In both familial and sporadic atypical haemolytic-uraemic syndrome (aHUS), mutations have been reported in regulators of the alternative complement pathway including factor H (CFH), membrane cofactor protein (MCP), and the serine protease factor I (IF). A characteristic feature of both MCP and CFH associated HUS is reduced penetrance and variable inheritance; one possible explanation for this is that functional changes in complement proteins act as modifiers., Objective: To examine single nucleotide polymorphisms in both CFH and MCP genes in two large cohorts of HUS patients (Newcastle and Paris)., Results: In both cohorts there was an association with HUS for both CFH and MCP alleles. CFH and MCP haplotypes were also significantly different in HUS patients compared with controls., Conclusions: This study suggests that there are naturally occurring susceptibility factors in CFH and MCP for the development of atypical HUS.

A database application has been developed for phenotype data management employing the Entity-Attribute-Value (EAV) model. By applying the EAV model, this application allows users to manage arbitrary phenotypes and customize data entry forms; therefore, it is suitable for different and multi-center projects.

Background: The haplotype based association method offers a powerful approach to complex disease gene mapping. In this method, a few common haplotypes that account for the vast majority of chromosomes in the populations are usually examined for association with disease phenotypes. This brings us to a critical question of whether rare haplotypes play an important role in influencing disease susceptibility and thus should not be ignored in the design and execution of association studies., Methods: To address this question we surveyed, in a large sample of 1873 white subjects, six candidate genes for osteoporosis (a common late onset bone disorder), which had 29 SNPs, an average marker density of 13 kb, and covered a total of 377 kb of the DNA sequence., Results: Our empirical data demonstrated that two rare haplotypes of the parathyroid hormone (PTH)/PTH related peptide receptor type 1 and vitamin D receptor genes (PTHR1 and VDR) with frequencies of 1.1% and 2.9%, respectively, had significant effects on osteoporosis phenotypes (p = 4.2 x 10(-6) and p = 1.6 x 10(-4), respectively). Large phenotypic differences (4.0 approximately 5.0%) were observed between carriers of these rare haplotypes and non-carriers. Carriers of the two rare haplotypes showed quantitatively continuous variation in the population and were derived from a wide spectrum rather than from one extreme tail of the population phenotype distribution., Conclusions: These findings indicate that rare haplotypes/variants are important for disease susceptibility and cannot be ignored in genetics studies of complex diseases. The study has profound implications for association studies and applications of the HapMap project.

Background: The vitamin D receptor (VDR) gene is important to human stature, as it mediates metabolic pathways, calcium homeostasis, and phosphate homeostasis, which influence growth., Methods: We examined the relationship between VDR and adult height in 1873 white subjects from 406 nuclear families. Four SNPs, namely -4817A/G at intron 1, FokI C/T at exon 2 start codon, BsmI A/G at intron 8, and TaqI T/C at exon 9 in VDR were tested for linkage and association with adult height variation by the program QTDT (quantitative transmission disequilibrium test). The bT haplotype of the BsmI and TaqI loci was further tested for its association with height in unrelated samples randomly chosen from the 406 nuclear families by traditional population association methods., Results: All four tested SNPs were linked to adult height. Within family associations with height were detected at BsmI and TaqI loci (p = 0.048 and 0.039, respectively). Analyses based on BsmI/TaqI haplotypes also revealed evidence for linkage (p = 0.05) and association (p = 0.001) with height. The bT haplotype was significantly associated with higher adult height (p = 0.033, within family association test). Such an association might be female specific and influenced by menstrual status., Conclusions: Our results strongly suggest that VDR may be linked to and associated with adult height variation in white populations.

Background: Osteoporosis is a major public health problem, mainly quantified by low bone mineral density (BMD). The majority of BMD variation is determined by genetic effects. A pilot whole genome linkage scan (WGS) was previously reported in 53 white pedigrees with 630 subjects. Several genomic regions were suggested to be linked to BMD variation., Objective: To substantiate these previous findings and detect new genomic regions., Methods: A WGS was conducted on an extended sample where the size was almost tripled (1816 subjects from 79 pedigrees). All the subjects were genotyped with 451 microsatellite markers spaced approximately 8.1 cM apart across the human genome. Two point and multipoint linkage analyses were carried out using the variance component method., Results: The strongest linkage signal was obtained on Xq27 with two point LOD scores of 4.30 for wrist BMD, and 2.57 for hip BMD, respectively. Another important region was 11q23, which achieved a maximum LOD score of 3.13 for spine BMD in multipoint analyses, confirming the results on this region in two earlier independent studies. Suggestive linkage evidence was also found on 7p14 and 20p12., Conclusions: Together with the findings from other studies, the current study has further delineated the genetic basis of bone mass and highlights the importance of increasing sample size to confirm linkage findings and to identify new regions of linkage.

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture. To identify genomic regions harboring quantitative trait loci (QTLs) contributing to BMD variation, we performed a two-stage genome screen. The first stage involved genotyping of a sample of 53 pedigrees with 630 individuals using 400 microsatellite markers spaced at approximately 10-cM intervals throughout the genome. Ten genomic regions with multi- and/or two-point LOD scores greater than 1.5 were observed. In the present second-stage study, 60 microsatellite markers, with a mean spacing of about 5 cM, were genotyped in these regions in an expanded sample of 79 pedigrees that contained 1816 subjects. Each pedigree was ascertained through a proband with extreme BMD at the hip or spine. BMD at the spine (L1-4), hip (the femoral neck, trochanter, and intertrochanteric region), and wrist (the ultradistal region) was measured by dual-energy X-ray absorptiometry (DXA) and was adjusted for age, sex, height, and weight. Two-point and multipoint linkage analyses were performed for each BMD site using statistical genetic methods that are implemented in the computer package SOLAR. Several regions (7q11, 10q26, 12q13, and 12q24) achieved LOD scores in excess of 1 in the second-stage followup study. The current results replicate some of our previous linkage findings and also highlight some of the difficulties facing microsatellite linkage mapping for complex human diseases.

Osteoporosis is a disease characterized by fragile bones and high susceptibility to low trauma fractures. It is a serious health problem, especially in elderly women. Bone mineral density (BMD) has been employed most commonly as the index for defining and studying osteoporosis. In this presentation, we use examples of our studies in both Caucasians and Chinese to illustrate the approaches used and some main results obtained on 1) characterizing the degree and the inheritance mode of genetic determination of a complex trait such as BMD; 2) identifying and mapping genes for osteoporosis. The purpose of the presentation is to introduce to the medical researchers how and what modern genetics can do to disentangle the mist of an array of genetics factors, the major determinants, for BMD.

In the present study, we simultaneously test linkage and/or association of the collagen type I alpha 2 (COL1A2) gene with bone mineral density (BMD) and bone area. A total of 1280 subjects from 407 Chinese nuclear families (including both parents and their daughters) were genotyped for an intragenic marker MspI in the COL1A2 gene. BMD and bone area at the lumbar spine and hip were measured by dual-energy X-ray absorptiometry. Applying the QTDT (quantitative transmission disequilibrium test) program, we performed tests for population stratification, within-family association (via transmission disequilibrium test), total association, linkage, and linkage while modeling association. Significant or marginal within-family associations were found with BMD at the lumbar spine (P = 0.013), trochanter (P = 0.004), and total hip (P = 0.053) and with bone area at the intertrochanteric region (P = 0.024) and total hip (P = 0.048). The positive associations were confirmed in permutations except for bone area at total hip (P > 0.10). A small proportion (<1%) of the population variance of bone phenotypes can be explained by the MspI polymorphism; however, it may be underestimated given the significant population stratification detected in our sample. Due to the limited number of sib pairs in this sample, we did not find evidence of linkage. In summary, the MspI polymorphism is likely to be in linkage disequilibrium with a nearby functional mutation affecting BMD and bone area.

Osteoporosis is one of the most common bone-related disorders in the elderly. It is a complex disease, and largely determined genetically. Traditional gene expression studies have shown that osteoporosis has complex regulating mechanisms which are controlled by multiple inherent factors, such as hormones, cytokines, various receptors, etc. The complex nature of osteoporosis, and the large number of genes involved in its onset and development, suggest the use of the state of the art microarray technique as a powerful tool to unravel mechanisms underlying etiology of osteoporosis.

The transmission disequilibrium test (TDT) customarily uses affected children and their parents (often case-parent trios, TDTD). Control-parent trios are necessary to guard against spurious significant results due to segregation distortion but are not generally utilized in the identification of disease susceptibility loci (DSL). Controls are often easy to recruit and the TDT can easily be extended to include control-parent trios into the analyses with unrelated case-parent trios. We present an extension of the TDT (TDTDC) that incorporates unrelated cases and controls and their parents into a single analysis. We develop a simple and accurate analytical method for computing the statistical power of various TDT (e.g. the TDTD, TDTDC, TDTDC and TDTC that employ control-parent trios only) under any genetic model. We investigated the power of these TDT, and particularly compared the relative power of the TDTD and TDTDC. We found that the TDTDC is almost always more powerful than the TDTC and TDTD. The relative power of the TDTDC and TDTD depends largely upon a number of parameters identified in the study. This study provides a basis for efficient use of control-parent trios in DSL identification.

In this study, we simultaneously characterized genetic and lifestyle factors (exercise, smoking, and alcohol consumption) in determining variation in body mass index (BMI), fat mass, percentage of fat mass (PFM), and lean mass while adjusting for the effects of age and sex. Six hundred fifty-eight Caucasian individuals from 48 pedigrees were studied for BMI. Among these individuals, 289 from 38 pedigrees were studied for fat mass, PFM, and lean mass measured by dual X-ray absorptiometry (DXA). After adjusting for age, sex, and lifestyle factors, the heritabilities (h(2)) of BMI, fat mass, PFM, and lean mass ranged from 0.52 to 0.57 with associated standard errors ranging from 0.09 to 0.14. After accounting for significant sex and age effects, exercise had significant effects for all the phenotypes studied, and the effects of smoking and alcohol consumption were not significant. Therefore, significant proportions of variation in BMI, fat mass, PFM, and lean mass were under genetic control, and exercise had a significant effect in reducing BMI, fat mass, and PFM and in increasing lean mass. This study warrants further genetic linkage analyses to search for genes for the obesity-related phenotypes measured by DXA in our population.

Association studies using random population samples are increasingly being applied in the identification and inference of genetic effects of genes underlying complex traits. It is well recognized that population admixture may yield false-positive identification of genetic effects for complex traits. However, it is less well appreciated that population admixture can appear to mask, change, or reverse true genetic effects for genes underlying complex traits. By employing a simple population genetics model, we explore the effects and the conditions of population admixture in masking, changing, or even reversing true genetic effects of genes underlying complex traits.

The transmission disequilibrium test (TDT), originally developed for mapping disease genes, has recently been extended to identify quantitative trait loci (QTL). For quantitative traits important for human health, generally multiple QTLs are involved. In the investigation of the statistical properties of the TDT, background polygenes (QTLs other than the QTL under test) generally have not been explicitly considered. The effects of background polygenes on the statistical properties of the TDT are thus largely unknown. Investigation of these effects will provide more realistic analyses of the statistical properties of the TDT under biologically plausible situations, and thus provide more accurate guidelines on the application of the TDT in practice. A general TDT (TDT(G)) has been developed to test linkage of a QTL in nuclear families that may be composed of more than one heterozygous parent and multiple children. Using the TDT(G) as an example, we develop an analytical method to investigate the effects of background polygenes on the power of the TDT. The accuracy of our analytical method is validated by computation simulations. We found that the power of the TDT(G) is increased with background polygenes when more than one child is employed in nuclear families, and the effect is stronger with more children per family recruited for study. The power of the TDT(G) increases dramatically when the number of children recruited from each nuclear family increases from one to two or from two to three. The type one error rate is not affected by the presence of background polygenes. The results of this study should be of theoretical significance in generalizing the investigation of the TDT to biologically plausible situations with background polygenes. They should also be of practical values in providing guidance on the recruitment of nuclear families with multiple children with the TDT(G)., (Copyright 2001 Wiley-Liss, Inc.)

1. The purpose of the present study was to determine the relationship between endothelial dysfunction and the endogenous inhibitor of nitric oxide synthase NG,NG'-asymmetric dimethylarginine (ADMA) in aged rats. 2. Studies were performed in male adult Sprague-Dawley rats (6 months old; n = 8) and in aged rats (20 months old; n = 8). Serum levels of ADMA and L-arginine were measured by high-performance liquid chromatography and responses of endothelium-intact aortic rings to acetylcholine (ACh) were tested. Nitric oxide synthase activity in kidney tissue and serum concentrations of nitrite, a stable end-product of nitric oxide, were assayed and serum contents of malondialdehyde, derived from lipid peroxidation and serum lipid and creatinine level were determined. 3. Serum levels of ADMA increased significantly in aged rats compared with adult rats (P < 0.01), whereas serum levels of L-arginine were similar in both groups (P = NS). Accordingly, the ratio of L-arginine/ADMA in old rats was lower than that in young rats (P < 0.01). Endothelium-dependent relaxation responses to ACh in aortic rings from aged rats were impaired and these impaired responses were improved by pre-incubation of aortic rings with L-arginine. 4. Nitric oxide synthase activity in the kidney, together with serum concentration of nitrite, was significantly decreased and serum contents of malondialdehyde, cholesterol and triglycerides were increased in old compared with young rats. However, the serum creatinine level was not significantly different between adult and aged rats. 5. Endogenous ADMA may be a contributor to age-related endothelial dysfunction and increases in endogenous ADMA may be linked to lipid peroxidation in aged rats.

Aim: To study the role of calcitonin gene-related peptide (CGRP) in nitric oxide (NO)-mediated myocardial delayed preconditioning induced by heat stress., Methods: The isolated rat heart was perfused in a Langendorff model. Hearts for all groups were subjected to 4 h hypothermia (4 degrees C) and 40 min reperfusion (37 degrees C). In the hyperthermia-treated group, rats were subjected to whole-body hyperthermia (rectal 42 degrees C, 15 min) 24 h before the experiment. Heart rate, coronary flow, left ventricular pressure, and its derivative (+/- dp/dtmax) were recorded, and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in plasma and the activity of creatine kinase (CK) in the coronary effluent were measured., Results: Pretreatment with hyperthermia significantly improved the recovery of cardiac protection, reduced the release of CK, and increased plasma concentrations of CGRP. Pretreatment with L-NAME, an inhibitor of NOS, or capsaicin, which selectively depleted sensory neurotransmitter content, abolished the protective effects and the increased level of CGRP elicited by hyperthermia., Conclusion: Endogenous NO is involved in the cardioprotection afforded by heat stress, and the beneficial effects of NO are mediated by CGRP in the rat.

Transmission disequilibrium test (TDT) is a nuclear family-based analysis that can test linkage in the presence of association. It has gained extensive attention in theoretical investigation and in practical application; in both cases, the accuracy and generality of the power computation of the TDT are crucial. Despite extensive investigations, previous approaches for computing the statistical power of the TDT are neither accurate nor general. In this paper, we develop a general and highly accurate approach to analytically compute the power of the TDT. We compare the results from our approach with those from several other recent papers, all against the results obtained from computer simulations. We show that the results computed from our approach are more accurate than or at least the same as those from other approaches. More importantly, our approach can handle various situations, which include (1) families that consist of one or more children and that have any configuration of affected and nonaffected sibs; (2) families ascertained through the affection status of parent(s); (3) any mixed sample with different types of families in (1) and (2); (4) the marker locus is not a disease susceptibility locus; and (5) existence of allelic heterogeneity. We implement this approach in a user-friendly computer program: TDT Power Calculator. Its applications are demonstrated. The approach and the program developed here should be significant for theoreticians to accurately investigate the statistical power of the TDT in various situations, and for empirical geneticists to plan efficient studies using the TDT., (Copyright 2001 Wiley-Liss, Inc.)

1. It has been suggested that calcitonin gene-related peptide (CGRP) is involved in the protection provided by ischaemic preconditioning in rat hearts and that ischaemic preconditioning is absent in diabetic rat hearts. 2. In the present study, we tested the relationship between sensory nerve function and ischaemic preconditioning in diabetic rats. 3. In 4- and 8-week diabetic rats and age-matched non- diabetic controls, 30 min global ischaemia and 40 min reperfusion caused a significant decrease in cardiac function and a marked increase in creatine kinase (CK) release. Ischaemic preconditioning, by three cycles of 5 min ischaemia and 5 min reperfusion, improved the recovery of cardiac function and decreased CK release during reperfusion in 4-week diabetic rat hearts. However, the cardioprotection afforded by ischaemic preconditioning was lost in 8-week diabetic rat hearts. Pretreatment with CGRP for 5 min also significantly improved the recovery of cardiac function and decreased CK release in rats subjected to 4 or 8 weeks of diabetes. 4. The content of CGRP in the coronary effluent during ischaemic preconditioning was significantly increased in 4-week diabetic rat hearts (P < 0.05). However, only a slight increase in the release of CGRP was shown in 8-week diabetic rat hearts (P > 0.05). 5. In summary, the present results suggest that the protection afforded by ischaemic preconditioning is attenuated in diabetic rats and that the change may be related to the reduction in CGRP release in diabetic rat hearts.

Previous investigations have shown that nitric oxide donors and nicorandil can suppress allergic reaction. In the present study, the protective effects of nitroglycerin and nicorandil on cardiac anaphylaxis were examined. Presensitized guinea-pig hearts challenged with specific antigen caused a marked decrease in coronary flow (CF), left ventricular pressure (LVP) and its derivatives (+/-dp/dtmax), increase in heart rate, and prolongation of P-R interval. Nitroglycerin (300 nM) or nicorandil (100 microM) markedly increased the content of calcitonin gene-related peptide (CGRP) concomitant with a significant improvement of the cardiac dysfunction and alleviation of the extension of P-R interval. Nicorandil at a concentration of 100 microM also inhibited the sinus tachycardia and histamine release. The protection afforded by nitroglycerin was abolished by glibenclamide, a blocker of ATP-sensitive potassium channels, or by CGRP8-37, the selective CGRP receptor antagonist, or by pretreatment with capsaicin, which depletes endogenous CGRP. The inhibitory effect of nicorandil on cardiac anaphylaxis was abolished only by glibenclamide but not by pretreatment with capsaicin. These results suggest that nitroglycerin and nicorandil possess a protection of cardiac anaphylactic injury. The present study also suggests that the protective effect of nitroglycerin may be related to stimulation of CGRP release and opening the KATP channel, and that the effect of nicorandil is mainly due to the activation of the KATP channel.

The silkworm (Bombyx mori L.) is a lepidopteran insect with a long history of significant agricultural value. We have constructed the first amplified fragment length polymorphism (AFLP) genetic linkage map of the silkworm B. mori at a LOD score of 2.5. The mapping AFLP markers were genotyped in 47 progeny from a backcross population of the cross no. 782 x od100. A total of 1248 (60.7%) polymorphic AFLP markers were detected with 35 PstI/TaqI primer combinations. Each of the primer combinations generated an average of 35.7 polymorphic AFLP markers. A total of 545 (44%) polymorphic markers are consistent with the expected segregation ratio of 1:1 at the significance level of P = 0.05. Of the 545 polymorphic markers, 356 were assigned to 30 linkage groups. The number of markers on linkage groups ranged from 4 to 36. There were 21 major linkage groups with 7-36 markers and 9 relatively small linkage groups with 4-6 markers. The 30 linkage groups varied in length from 37.4 to 691.0 cM. The total length of this AFLP linkage map was 6512 cM. Genetic distances between two neighboring markers on the same linkage group ranged from 0.2 to 47 cM with an average of 18.2 cM. The sex-linked gene od was located between the markers P1T3B40 and P3T3B27 at the end of group 3, indicating that AFLP linkage group 3 was the Z (sex) chromosome. This work provides an essential basic map for constructing a denser linkage map and for mapping genes underlying agronomically important traits in the silkworm B. mori L.

Aim: To study improvement of preservation with cardioplegic solution induced by nitroglycerin was related to stimulation of calcitonin gene-related peptide (CG RP) release., Methods: The isolated rat heart was arrested using St Thomas Hospital cardioplegic solution and then was reperfused with normothermic Krebs-Henseleit solution for 40 min after the 4-h hypothermic ischemic period. Hear t rate, coronary flow, left ventricular pressure (LVP), and its first derivative (+/- dp/dtmax) were recorded, and the calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and the release of creatine kinase (CK) were measured., Results: Nitroglycerin (0.1 or 1 micro;mol/L) or CGRP (5 or 10 nmol/L) caused an improvement of cardiac function (LVP and +/- dp/dtmax) and a decrease in the release of creatine kinase during reperfusion. The protection induced by nitroglycerin was abolished by CGRP(8-37), the selective CGRP receptor antagonist, or pretreatment wit h capsaicin to deplete sensory nerves neurotransmitter content, but was unaltered by treatment w ith glibenclamide, the blocker of the ATP-sensitive potassium channel (KATP). The protection induced by exogenous CGRP was not also blocked by glibenclamide. Levels of CGRP-LI in the coronary effluent were significantly increased in the hearts treated with nitroglycerin. However, the elevated level of CGRP-LI by nitroglycerin was abolished by pretreatment with capsaicin., Conclusion: The improvement of preservation with cardioplegic solution induced by nitroglycerin was related to stimulation of CGRP release in the rat heart, and the effect is not related to the activation of the KATP channel.

In association studies searching for genes underlying complex traits, the results are often inconsistent, and population admixture has been recognized qualitatively as one major potential cause. Hardy-Weinberg equilibrium (HWE) is often employed to test for population admixture; however, its power is generally unknown. Through analytical and simulation approaches, we quantify the power of the HWE test for population admixture and the effects of population admixture on increasing the type I error rate of association studies under various scenarios of population differentiation and admixture. We found that (1) the power of the HWE test for detecting population admixture is usually small; (2) population admixture seriously elevates type I error rate for detecting genes underlying complex traits, the extent of which depends on the degrees of population differentiation and admixture; (3) HWE testing for population admixture should be performed with random samples or only with controls at the candidate genes, or the test can be performed for combined samples of cases and controls at marker loci that are not linked to the disease; (4) testing HWE for population admixture generally reduces false positive association findings of genes underlying complex traits but the effect is small; and (5) with population admixture, a linkage disequilibrium method that employs cases only is more robust and yields many fewer false positive findings than conventional case-control analyses. Therefore, unless random samples are carefully selected from one homogeneous population, admixture is always a legitimate concern for positive findings in association studies except for the analyses that deliberately control population admixture.

Aim: To examine the role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning induced by prostaglandins in isolated guinea pig hearts., Methods: The isolated guinea pig hearts were perfused in a Langendorff model. The heart rate, coronary flow, left ventricular pressure, and its first derivatives (+/-dp/dt(max)) were recorded and the calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and 6-keto-PGF(1 alpha) were measured., Results: Endothelin-1 (200 pmol in 1 mL K-H buffer) reduced the left ventricular developed pressure and its first derivatives (+/-dp/dt(max)), heart rate, and coronary flow. Preconditioning with two cycles of 5-min global ischemia and 5-min reperfusion attenuated endothelin-1 induced myocardial injury, and concentrations of both CGRP and 6-keto-PGF(1alpha) in the coronary effluent were markedly raised in the preconditioning periods. Pretreatment with capsaicin, which depletes endogenous CGRP, abolished the elevated level of CGRP concomitantly with loss of the cardioprotection induced by ischemic preconditioning. CGRP(8-37) (100 nmol/L), a selective CGRP1 receptor antagonist, also abolished the protective effects of ischemic preconditioning. After pretreatment with indometacin (10 micromol/L), an inhibitor of cyclooxygenase, the protective effects of ischemic preconditioning were abolished and the release of 6-keto-PGF1alpha was no longer elevated. Pretreatment with indometacin abolished the elevated level of CGRP in the coronary effluent., Conclusion: Endogenous prostaglandins are involved in the protective effects of ischemic preconditioning, and the beneficial effects of prostaglandins are mediated by CGRP in the guinea pig heart.

In 40 human pedigrees with 563 subjects, we evaluated the contribution of genetic and life-style factors (exercise, smoking, and alcohol consumption) and the interactions between non-genetic factors in determining bone mineral density (BMD) of the hip and spine. In our analysis, we adjusted for age, weight, height, menopausal status in females, life-style factors, and the significant interactions among these factors. For the spine and hip BMD, heritabilities (h(2)) (+/- SE) were, respectively, 0.68 (0.21) and 0.86 (0.28) in males and 0.64 (0.13) and 0.67 (0.14) in females. Exercise had significant beneficial effects for male spine BMD and female hip BMD. Alcohol consumption experienced in our sample had significant beneficial effects on hip BMD in both sexes. Although the main effect of smoking was not significant, there were significant interaction effects between smoking and other important factors (e.g., exercise, weight, alcohol consumption). For example, for female spine BMD, exercise had significant beneficial effects in smokers; however, its effect in non-smokers was non-significant. This result indicates that exercise may reduce deleterious effects of smoking (if any) on BMD, but may have minor effects in increasing BMD in non-smokers. The various interaction effects among risk factors explicitly revealed here for the first time indicate that the detailed effects and direction of individual risk factors may depend on the presence and magnitude of other factors. Weight invariably affected BMD of the hip and spine in both sexes. Age effects were significant for hip BMD, but not for male spine BMD., (Copyright 2000 Wiley-Liss, Inc.)

Several lines of evidence suggest the importance of the estrogen receptor (ER) in determining body mass index (BMI). Our purpose was to investigate whether genetic polymorphisms at the restriction enzyme PvuII site of the ER-alpha gene locus are associated with BMI variation. Data on BMI, age, and ER-alpha genotypes were obtained from 108 healthy midwestern U.S. postmenopausal Caucasian women. The study subjects were unrelated and aged 65 yr and over (mean age +/- SD, 73.4 +/- 5.1 yr), with an average BMI of 25.25 (SD, 4.04). The ER-alpha genotypes were obtained by PCR followed by restriction enzyme PvuII digestion. We found that in our study subjects the ER-alpha genotypes are significantly associated with BMI (by ANOVA, P = 0.04), explaining about 6.2% of the BMI variation in our study sample. The allelic effects of this locus on BMI are approximately additive. In our sample, individuals of the PP and Pp genotypes have, respectively, 11.4% and 4.8% higher BMI than individuals of the pp genotype. There is a significant ER-alpha genotype by age interaction, so that in our sample PP individuals tend to gain weight with age, whereas Pp and pp individuals tend to lose weight with age. Therefore, the ER-alpha polymorphisms are associated with BMI variation in healthy postmenopausal Caucasian women aged 65 yr and over. Our result is consistent with some recent findings suggesting the potential effects of the ER on BMI. The importance of the ER-alpha genotypes in other populations and other age groups needs to be demonstrated. Although the results of the ER-alpha genotype by age interaction are obtained here from cross-sectional data, direct confirmation may come from longitudinal studies in which individuals are measured multiple times over several years. The importance of the ER-alpha genotypes on BMI should be confirmed by further studies using methods robust to the potential problem of population substructuring that may confound the conclusions of population association studies.

Osteoporotic fractures (OFs) are a major public health problem. Direct evidence of the importance and, particularly, the magnitude of genetic determination of OF per se is essentially nonexistent. Colles' fractures (CFs) are a common type of OF. In a metropolitan white female population in the midwestern United States, we found significant genetic determination of CF. The prevalence (K) of CF is, respectively, 11.8% (+/- SE 0.7%) in 2471 proband women aged 65.55 years (0.21), 4.4% (0.3%) in 3803 sisters of the probands, and 14.6% (0.7%) in their mothers. The recurrence risk (K0), the probability that a woman will suffer CF if her mother has suffered CF is 0.155 (0.017). The recurrence risk (Ks), the probability that a sister of a proband woman will suffer CF given that her proband sister has suffered CF is 0.084 (0.012). The relative risk lambda (the ratio of the recurrence risk to K), which measures the degree of genetic determination of complex diseases such as CF, is 1.312 (0.145; lambda 0) for a woman with an affected mother and 1.885 (0.276; lambda s) for a woman with an affected sister. A lambda-value significantly greater than 1.0 indicates genetic determination of CF. The terms lambda 0 and lambda s are related to the genetic variances of CF. These parameters translate into a significant and moderately high heritability (0.254 [0.118]) for CF. These parameters were estimated by a maximum likelihood method that we developed, which provides a general tool for characterizing genetic determination of complex diseases. In addition, we found that women without CF had significantly higher bone mass (adjusted for important covariates such as age, weight, etc.) than women with CF.

The Deng-Lynch method was developed to estimate the rate and effects of deleterious genomic mutations (DGM) in natural populations under the assumption that populations are either completely outcrossing or completely selfing and that populations are at mutation-selection (M-S) balance. However, in many plant and animal populations, selfing or outcrossing is often incomplete in that a proportion of populations undergo inbreeding while the rest are outcrossing. In addition, the degrees of deviation of populations from M-S balance are often not known. Through computer simulations, we investigated the robustness and the applicability of the Deng-Lynch method under different degrees of partial selfing or partial outcrossing and for nonequilibrium populations approaching M-S balance at different stages. The investigation was implemented under constant, variable, and epistatic mutation effects. We found that, generally, the estimation by the Deng-Lynch method is fairly robust if the selfing rate (S) is <0.10 in outcrossing populations and if S > 0.8 in selfing populations. The estimation may be unbiased under partial selfing with variable and epistatic mutation effects in predominantly outcrossing populations. The estimation is fairly robust in nonequilibrium populations at different stages approaching M-S balance. The dynamics of populations approaching M-S balance under various parameters are also studied. Under mutation and selection, populations approach balance at a rapid pace. Generally, it takes 400-2000 generations to reach M-S balance even when starting from homogeneous individuals free of DGM. Our investigation here provides a basis for characterizing DGM in partial selfing or outcrossing populations and for nonequilibrium populations.

It has recently been demonstrated that fine-scale mapping of a susceptibility locus for a complex disease can be accomplished on the basis of deviations from Hardy-Weinberg (HW) equilibrium at closely linked marker loci among affected individuals. We extend this theory to fine-scale localization of a quantitative-trait locus (QTL) from extreme individuals in populations, by means of HW and linkage-disequilibrium (LD) analyses. QTL mapping and/or linkage analyses can establish a large genomic region ( approximately 30 cM) that contains a QTL. The QTL can be fine mapped by examination of the degree of deviation from HW and LD at a series of closely linked marker loci. The tests can be performed for samples of individuals belonging to either high or low percentiles of the phenotype distribution or for combined samples of these extreme individuals. The statistical properties (the power and the size) of the tests of this fine-mapping approach are investigated and are compared extensively, under various genetic models and parameters for the QTL and marker loci. On the basis of the results, a two-stage procedure that uses extreme samples and different tests (for HW and LD) is suggested for QTL fine mapping. This two-step procedure is economic and powerful and can accurately narrow a genomic region containing a QTL from approximately 30-1 cM, a range that renders physical mapping feasible for identification of the QTL. In addition, the relationship between parameterizations of complex diseases, by means of penetrance, and those of complex quantitative traits, by means of genotypic values, is outlined. This means that many statistical genetic methods developed for searching for susceptibility loci of complex diseases can be directly adopted and/or extended to QTL mapping for quantitative traits.

Anaphylactic events occurring in cardiac tissues can result in cardiac dysfunction via vasoconstriction and arrhythmias. Calcitonin gene-related peptide (CGRP) is the most potent vasodilator and possesses anti-arrhythmic action. We examined the influence of CGRP on cardiac anaphylaxis in guinea-pigs. In the Langendorff-perfused heart of passively sensitized guinea-pigs, antigen challenge evoked a decrease in coronary flow, left ventricular pressure and its maximum first derivatives (+/-dP/dtmax) and an increased heart rate. Antigen challenge also induced atrioventricular conduction block. Treatment with CGRP (1 or 3 nM) significantly improved the recovery of cardiac function and reduced the incidence and duration of atrioventricular block without influencing the increased heart rate. Pretreatment with capsaicin caused effects similar to those of CGRP and markedly elevated the content of CGRP in coronary effluent. Ischaemic preconditioning, induced by two cycles each of 5 min global ischaemia and 5 min reperfusion, also improved cardiac function and raised the level of CGRP in coronary effluent. The protective effects of ischaemic preconditioning were abolished in the presence of the CGRP receptor antagonist CGRP8-37. Histamine release did not differ significantly during any of the interventions. The findings of the present study indicate that, in guinea-pig hearts, CGRP protects against cardiac anaphylaxis and that the cardioprotection by CGRP is independent of histamine release.

Aim: To study early or delayed cardioprotection afforded by pretreatment with capsaicin., Methods: The isolated rat heart was perfused in a Langendorff model. Heart rate, coronary flow, left ventricular pressure, and its first derivative (+/- dp/dtmax) were recorded, and the calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and the release of creatine kinase (CK) were measured., Results: Capsaicin (50 mg.kg-1, s.c.) improved the recovery of cardiac function and decreased the release of CK. CK was (2.12 +/- 0.40) and (0.26 +/- 0.04) u.min-1.g-1(wet wt) for ischemia-reperfusion (I/R) and capsaicin + I/R, respectively (P < 0.05). Capsaicin treatment caused an increase in the concentration of CGRP-LI in plasma. CGRP-LI was (135 +/- 12) and (304 +/- 45) ng.L-1 for vehicle + I/R and capsaicin + I/R, respectively (P < 0.05). After pretreatment with capsaicin to deplete the sensory nerve transmitter content, the cardioprotection and the increased level of CGRP by capsaicin were abolished. A delayed protection was shown in the hearts obtained from the rats pretreated with capsaicin 24 h or 48 h before the experiments., Conclusion: Pretreatment with capsaicin induces the early and delayed cardioprotection, which may be related to stimulation of CGRP release in the rat.

The likelihood of low trauma fracture in the elderly is highly predictable by peak bone mass (PBM) at age approximately 25-50 yr. We estimated the magnitude of genetic determination of the variation and covariation of PBM of the spine and hip (adjusted by age, gender, and ethnicity) in 47 independent healthy full-sib pairs and 27 healthy mother-offspring pairs. For the spine and hip, the narrow-sense heritabilities (h(2)) (mean +/- SE) were 0.76 +/- 0.34 and 0.84 +/- 0.36, respectively, when estimated from full sibs, and 0.86 +/- 0.38 and 0.84 +/- 0.39, respectively, when estimated from parent-offspring. Some genetic loci underlying PBM variation at the hip and spine are the same or closely linked, as is reflected by the high genetic correlation of 0.95 +/- 0.05 between them when estimated from full sibs, and 0.57 +/- 0.27 when estimated from parent-offspring, respectively. Generally, common familial environmental effects shared by relatives may bias these estimates. However, these effects may be small, since our results reported herein and those in other earlier studies indicate that common familial environmental effects are probably negligible in causing similarity of bone mass among family members. The correlation of bone mass among randomly sampled couples living in the same household is small and nonsignificant as measured either by densitometry at the radius and ulna or by quantitative ultrasound at the patella. The problem of shared environmental effects notwithstanding, we conclude that much of the PBM variation and covariation at the hip and spine is determined genetically.

Brief ischaemia or heat stress protects the myocardium against ischaemia-reperfusion injury. Heat stimulus evokes release of sensory nerve transmitters, including calcitonin gene-related peptide (CGRP). Since CGRP has been shown to play an important role in the mediation of ischaemic preconditioning, the present study examined whether early or delayed preconditioning induced by retrograde hyperthermic perfusion in vitro or by whole-body hyperthemia in vivo also involves endogenous CGRP. Isolated rat hearts were perfused in the Langendorff mode and subjected to 30 min global ischaemia and 30 min reperfusion. Heart rate, coronary flow, left ventricular pressure and its first derivatives (+/-dp/dt) were recorded and the CGRP-like immunoreactivity (CGRP-LI) content and the release of creatine kinase (CK) during reperfusion were measured. Retrograde hyperthermic perfusion (42 degrees C) for 5 min improved the recovery of cardiac function, decreased the release of CK and elevated the content of CGRP-LI in the coronary effluent. CGRP(8-37) (10(-7 mol/l), a selective CGRP receptor antagonist, abolished the cardioprotection by heat stress. Pretreatment with capsaicin (50 mg/kg s.c.), which specifically depletes sensory nerve transmitter content, abolished both the cardioprotection and the increased release of CGRP-LI. Whole-body hyperthermia (42 degrees C for 15 min) caused an increase in the plasma concentration of CGRP-LI. Early or delayed protection was shown in the hearts obtained from the animals subjected to whole-body hyperthermia 10 min or 48 h before the experiments. The early or delayed protection by heat stress was also abolished by pretreatment with capsaicin. The present study suggests that, in the rat, the early and delayed cardioprotection induced by heat stress involves endogenous CGRP.

Characterizing deleterious genomic mutations is important. Most of the few current estimates come from the mutation-accumulation (M-A) approach, which has been extremely time- and labour-consuming. There is a resurgent interest in implementing this approach. However, its estimation properties under different experimental designs are poorly understood. By simulations we investigate these issues in detail. We found that many of the previous M-A experiments could have been more efficiently implemented with much less time and expense while still achieving the same estimation accuracy. If more than 100 lines are employed in M-A and if each line is replicated at least 10 times during each assay, an experiment of 10 M-A generations with two assays (at the beginning and at the end of M-A) may achieve at least the same estimation quality as a typical M-A experiment. The number of replicates per M-A line necessary for each assay largely depends on the magnitude of environmental variance. While 10 replicates are reasonable for assaying most fitness traits, many more are needed for viability, which has an exceptionally large environmental variance. The investigation is mainly carried out using Bateman-Mukai's method of moments for estimation. Estimation using Keightley's maximum likelihood is also investigated and discussed. These results should not only be useful for planning efficient M-A experiments, but also may help empiricists in deciding to adopt the M-A approach with manageable labour, time and resources.

Early cardioprotection can be achieved by a brief ischaemia of noncardiac tissues. Our study examined whether a brief ischaemia of the small intestine induces both early and delayed cardioprotection in the rabbit and assessed the possible mechanism involved in the activation of capsaicin-sensitive sensory nerves. The plasma concentration of creatine kinase (CK) and infarct size (necrotic zone/left ventricular zone) after 30 min coronary artery occlusion and 180 min reperfusion were determined in rabbits. Infarct size was 35.5+/-6.8% in the control non-preconditioned group. Preconditioning induced by a brief period of 10-min small intestine ischaemia significantly reduced infarct size (6.5+/-1.9%, P<0.01 vs. the control non-preconditioned group) and decreased CK release (3092+/-236 and 1094+/-117 U/l for myocardial ischaemia-reperfusion and preconditioning plus myocardial ischemia-reperfusion, respectively, P<0.01), and the protection was partly abolished by pretreatment with capsaicin (50 mg/kg, s.c.) 4 days before the experiments. A brief period of anterior mesenteric artery occlusion caused an increase in the plasma level of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI), an effect which was abolished by pretreatment with capsaicin. Similar protection was shown in the animals subjected to a brief period of anterior mesenteric artery occlusion 24 h before coronary artery occlusion, and this delayed protection was also abolished partly by pretreatment with capsaicin. Capsaicin treatment (50 mg/kg, s.c.) alone also protected the ischaemic myocardium. The results suggest that brief ischaemia of the small intestine induces both early and delayed protection against reperfusion-induced myocardial injury, and the effects are, at least partly, related to the activation of capsaicin-sensitive sensory nerves.

Alternatives to the mutation-accumulation approach have been developed to characterize deleterious genomic mutations. However, they all depend on the assumption that the standing genetic variation in natural populations is solely due to mutation-selection (M-S) balance and therefore that overdominance does not contribute to heterosis. Despite tremendous efforts, the extent to which this assumption is valid is unknown. With different degrees of violation of the M-S balance assumption in large equilibrium populations, we investigated the statistical properties and the robustness of these alternative methods in the presence of overdominance. We found that for dominant mutations, estimates for U (genomic mutation rate) will be biased upward and those for h (mean dominance coefficient) and s (mean selection coefficient), biased downward when additional overdominant mutations are present. However, the degree of bias is generally moderate and depends largely on the magnitude of the contribution of overdominant mutations to heterosis or genetic variation. This renders the estimates of U and s not always biased under variable mutation effects that, when working alone, cause U and s to be underestimated. The contributions to heterosis and genetic variation from overdominant mutations are monotonic but not linearly proportional to each other. Our results not only provide a basis for the correct inference of deleterious mutation parameters from natural populations, but also alleviate the biggest concern in applying the new approaches, thus paving the way for reliably estimating properties of deleterious mutations.

Aim: To study the mediation of calcitonin gene-related peptide (CGRP) in the cardioprotective effect of bradykinin-induced preconditioning in heart., Methods: The isolated rat hearts were perfused in a Langendorff mode. The cardiac function and creatine kinase (CK) were measured., Results: Pretreatment with bradykinin for 5 min caused an improvement of heart function and a decrease of CK release during reperfusion [CK was (0.18 +/- 0.06), (1.07 +/- 0.14), and (0.37 +/- 0.15) U.min-1.g-1/(wet wt) for control, ischemia-reperfusion, and bradykinin, respectively, P < 0.01], and the effect of bradykinin was abolished in the presence of icatibant acetate (Hoe140 1 mumol.L-1) or CGRP8-37 (0.1 mumol.L-1) [CK was (0.37 +/- 0.15), (1.01 +/- 0.23), and (1.07 +/- 0.23) U.min-1.g-1 (wet wt) for bradykinin, Hoe140, and CGRP8-37, respectively, P < 0.01]. Pretreatment with capsaicin also abolished the protection of bradykinin [CK was (0.30 +/- 0.04) and (1.14 +/- 0.12) U.min-1.g-1 (wet wt) for vehicle and capsaicin, respectively, P < 0.01]., Conclusion: The cardioprotective effect of bradykinin-induced preconditioning was related to stimulation of CGRP release in the rat.