Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer. (C) 2002 Elsevier Science Ltd. All rights reserved.

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.

Characterization of microsatellite loci in Anopheles flavirostris, the principal malaria vector in the Philippines

Microsatellites were isolated and characterized from Anopheles flavirostris, the principal malaria vector in the Philippines. Fifty of the 150 positive clones sequenced contained mostly dinucleotide microsatellites and only 16 had trinucleotide repeats. We designed primers from the unique sequences flanking 18 microsatellite loci. Of these, 11 loci produced successful amplification and revealed high levels of polymorphism; 86 alleles were detected with allele number ranging from 2 to 16 at each locus. The high allelic variability will make these microsatellite loci very useful for taxonomic and population genetic studies.

Characterization of highly conserved microsatellite loci in Araucaria cunninghamii and related species

Ten microsatellite loci are described in Araucaria cunninghamii, the first reported in the Araucariaceae. Eight were tested in sections Eutacta and Bunya, which diverged more than 200 MYA, and to the sister genus Agathis. Specific amplification products within the expected size range were obtained for six to eight loci in section Eutacta (depending on species), five loci in section Bunya and three. loci in Agathis. Two of the loci (CRCAc1 and CRCAc2, both GA repeats) produced specific amplification products in all taxa, with orthology confirmed by sequence analysis. The repeats were perfect in all taxa. The flanking sequences were extremely conserved, with sequence divergence of 0% to 2.0% within Araucaria species and 2.9% to 7.5% between Araucaria and Agathis. These microsatellites represent some of the most conserved microsatellite loci reported in plants. This may be due to a low evolutionary rate in Araucariaceae genome or the loci may be closely associated with highly conserved, unreported genes.

Isolation and characterization of microsatellite loci in Bemisia tabaci

Bemisia tabaci (Hemiptera: Aleyrodidae) is a haplo-diploid species with a global distribution demonstrating strong geographical structure with eight recognizable genetic groups. Fifteen microsatellite loci (335 alleles, 6-44 alleles per locus) were derived from four of the eight groups and were then screened across 33 populations. These loci clearly differentiate the populations. The microsatellites amplified best in individuals from genetic groups representing the Mediterranean, Middle East, Asia (three groups) and Australasia/Oceania and amplified less well with populations from sub-Saharan Africa and the New World. This differential amplification pattern is a direct result of the relatedness to the microsatellite source material.

Isolation and characterization of nine microsatellite loci from the Hawaiian grouper Epinephelus quernus (Serranidae) for population genetic analyses

The availability of variable genetic markers for groupers (Serranidae) has generally been limited to mitochondrial DNA. For studies of population genetic structure, more loci are usually required; particularly useful are those that are nuclear in origin such as microsatellites. Here, we isolated and characterized 9 microsatellite loci from the endemic Hawaiian grouper Epinephelus quernus using a biotin-labeled oligonucleotide-streptavidin-coated magnetic bead approach. Of the 20 repeat-containing fragments isolated, 15 had sufficient flanking region in which to design primers. Among these, 9 produced consistent polymerase chain reaction product, and 6 were highly variable. These 6 loci were all composed of dinucleotide repeats, with the number of alleles ranging from 6 to 18, and heterozygosities from 33.3% to 91.7%. The high levels of variability observed should make these markers useful for population genetic studies of E. quernus, and potentially other epinephelines.

Testosterone is correlated with courtship but not aggression in the tropical buff-banded rail, Gallirallus philippensis

Past studies into the roles of testosterone in birds have focused on species that occur in temperate regions. In such species, plasma testosterone levels are high during the establishment of territories in the spring and are associated with increased aggression. In contrast to most temperate species, tropical birds frequently defend territories year-round, during which time territoriality often occurs in a nonsexual context. The few studies that have been carried out on tropical birds show lower levels of circulating testosterone than occur in their temperate counterparts. In some year-round territorial tropical species, testosterone and aggression are dissociated, while in other species testosterone still plays a role in regulating aggression. This study examined the relationship between aggression and plasma testosterone levels in a year-round territorial, subtropical population of the buff-banded rail with characteristics typical of tropical species. Peak testosterone levels were substantially lower than those found in temperate species. Males displayed a seasonal peak in plasma testosterone level when their partners were most likely to be fertile. At other times, testosterone levels were mostly undetectable, despite year-round territoriality. We found that T levels increased with courtship behavior but showed no relationship with aggression, supporting the hypothesis that dissociation between testosterone and territoriality may be widespread among tropical avian taxa. (C) 2003 Elsevier Science (USA). All rights reserved.

Fas ligand and tumour counter-attack in colorectal cancer stratified according to microsatellite instability status

Expression of membrane-bound Fas ligand (FasL) by colorectal cancer cells may allow the development of an immune-privileged site by eliminating incoming tumour-infiltrating lymphocytes (TILs) in a Fas-mediated counter-attack. Sporadic colorectal cancer can be subdivided into three groups based on the level of DNA microsatellite instability (NISI). High-level NISI (NISI-High) is characterized by the presence of TILs and a favourable prognosis, while microsatellite-stable (MSS) cancers are TIL-deficient and low-level MSI (MSI-Low) is associated with an intermediate TIL density. The purpose of this study was to establish the relationship between MSI status and FasL expression in primary colorectal adenocarcinoma. Using immunohistochemistry and a selected series of 101 cancers previously classified as 31 MSI-High, 30 NISI-Low, and 40 MISS, the present study sought to confirm the hypothesis that increased TIL density in MSI-High cancers is associated with low or absent membrane-bound FasL expression, while increased FasL in MSS cancers allows the killing of host TILs. TUNEL/CD3 double staining was also used to determine whether MSS cancers contain higher numbers of apoptotic TILs in vivo than MSI-High or MSI-Low cancers. Contrary to the initial hypothesis, it was found that MSI-High cancers were associated with higher FasL expression (p = 0.04) and a stronger intensity of FasL staining (p = 0.007). In addition, mucinous carcinomas were independently characterized by increased FasL expression (p = 0.03) and staining intensity (p = 0.0005). Higher FasL expression and staining intensity did not correlate with reduced TIL density or increased numbers of apoptotic TILs. However, consistent with the hypothesis that curtailment of the host anti-tumour immune response contributes to the poor prognosis in MSS cancers, it was found that apoptotic TILs were most abundant in MSS carcinomas and metastatic Dukes' stage C or D tumours (p = 0.004; p = 0.046 respectively). This study therefore suggests that MSS colorectal cancers are killing incoming TILs in an effective tumour counter-attack, but apparently not via membrane-bound FasL. Copyright (C) 2003 John Wiley Sons, Ltd.

A new multiplexed microsatellite tool for metapopulation studies in the overexploited endemic limpet Patella aspera (Röding, 1798)

Patellid limpets are ecologically important keystone grazers having a long history of overexploitation in the Macaronesian Archipelagos (NE Atlantic islands), where some species, such as Patella aspera, are under serious risk.[1, 2] Patella aspera is a protandric sequential hermaphrodite species with external fertilization, in which individuals start off as males but may undergo a sex reversal with age.[3] Hence, exploitation tends to focus on the larger females in the population as larger limpets (predominantly females) are selectively removed. Despite conservation legislation in Canaries, Madeira and Azores, limpets are under severe pressure and few individuals survive long enough to become females, a phenomenon that severely restricts the effective population size.[4] New conservation actions for the protection and sustainable use of limpets in Macaronesian Archipelagos are urgently needed and should be based on a multidisciplinary framework based on knowledge of the population dynamics and connectivity of this species.

Digit Ratio (2D:4D) in Newborns: Influences of Prenatal Testosterone and Maternal Environment

Introduction: The 2D:4D digit ratio is sexually-dimorphic, probably due to testosterone action through the perinatal period. We characterize the 2D:4D ratio in newborn (NB) infants, in between the pre- and postnatal surges of testosterone, and relate it to the mother's 2D:4D and to testosterone levels in the amniotic fluid (AF). Subjects and methods: Testosterone was assayed in samples of maternal plasma and AF collected at amniocentesis. Shortly after birth, 106 NBs and their mothers were measured for 2D:4D ratio. Results: NB males had lower mean 2D:4D ratios than females but this dimorphism was significant only for the left hand (males: 0.927; females: 0.950; p=0.004). Mothers who had sons had lower 2D:4D ratios than those who had daughters and the mother's 2D:4D were higher than those of NBs regardless of sex. Both hands of NB females were negatively correlated with AF testosterone and positively correlated with the mother's 2D:4D, but males showed no significant associations. Maternal plasma testosterone also showed a negative weak correlation with NB's digit ratio in both sexes. Conclusions: Sexual dimorphism at birth was only significant for the left hand, in contrast with reports of greater right hand dimorphism, suggesting that postnatal testosterone is determinant for 2D:4D stabilization. The lower 2D:4D ratios in mothers who had sons support claims that hormone levels in parents are influential for determining their children's sex. NB female's digit ratio, but not males', was associated to the level of AF testosterone. The mother's 2D:4D ratios were positively correlated with their daughters' 2D:4D, but the same was not observed for male NBs, suggesting that prenatal testosterone levels in male fetus lead their 2D:4D ratios to stray from their mothers' with high individual variability.

Microsatellite instability in solitary and sporadic gastric cancer

Recently, the presence of microsatellite instability (MSI) has been reported in gastric cancer and associated with older age of presentation, distal tumor location, early disease staging, and better overall prognosis. Different characteristics in presentation and in tumor behavior may be explained by different genetic alterations during carcinogenesis of gastric cancer. Identification of specific genetic pathways in gastric cancer may have direct impact on prognosis and selection of treatment strategies. PATIENTS AND METHODS: All 24 patients were treated by radical surgery. Fragments of normal and tumor tissues were extracted from the specimen and stored at -80ºC before DNA purification and extraction. PCR amplification utilizing microsatellite markers was performed. Tumors presenting PCR products of abnormal sizes were considered positive for microsatellite instability (MSI+). RESULTS: Five patients (21%) had tumors that were MSI+ in at least 1 marker. In the group of patients with Lauren's intestinal-type gastric carcinoma, 3 had tumors that were MSI+ (23%), while in the group of diffuse-type gastric cancer, 2 patients had tumors that were MSI+ (19%). The mean age of presentation and the male:female ratio was similar in both groups. Tumors that were MSI+ were more frequently located in proximal portion of the stomach compared to microsatellite-stable (MSS) tumors (40% vs. 16%). Although there was a trend of patients with MSI+ tumors towards a proximal gastric tumor location, early staging, and negative lymph node metastasis, there was no statistical significance compared to those with MSS tumors (P >.1). Comparison of overall and disease-free survival between gastric tumors that were MSI+ and those that were MSS found no statistically significant differences (P >.1). CONCLUSIONS: Microsatellite instability is a frequent event in gastric carcinogenesis and shows a trend towards distinct clinical and pathological characteristics of gastric cancer.

Identification of duplicates of cassava accessions sampled on the North Region of Brazil using microsatellite markers

Duplicates are common in germplasm banks and their identification is needed to facilitate germplasm bank management and to reduce maintenance costs. The aim of this work was to identify duplicates of cassava from a germplasm bank in Eastern Amazon, which had been previously characterized both morphological and agronomically. In order to be genotyped with 15 microsatellite loci, 36 accessions were selected. These accessions were classified into 13 groups of similar morpho-agronomical characteristics. All loci were polymorphic, and 75 alleles were identified, with an average of five alleles per loci and H E = 0.66. There were determined 34 pairs of genotypes with identical multiloci profiles and the probability of genetic identity was 1.1x10-12 with probability of exclusion of 99.9999%. Among these duplicates, there are accessions sampled on different years and places, but with different names and accessions with the same name sampled in different places and years. The study identified genotypes that are grown in different places and that have been maintained over the years by local farmers.

Testosterone Deficiency Increases Hospital Readmission and Mortality Rates in Male Patients with Heart Failure

Background:Testosterone deficiency in patients with heart failure (HF) is associated with decreased exercise capacity and mortality; however, its impact on hospital readmission rate is uncertain. Furthermore, the relationship between testosterone deficiency and sympathetic activation is unknown.Objective:We investigated the role of testosterone level on hospital readmission and mortality rates as well as sympathetic nerve activity in patients with HF.Methods:Total testosterone (TT) and free testosterone (FT) were measured in 110 hospitalized male patients with a left ventricular ejection fraction < 45% and New York Heart Association classification IV. The patients were placed into low testosterone (LT; n = 66) and normal testosterone (NT; n = 44) groups. Hypogonadism was defined as TT < 300 ng/dL and FT < 131 pmol/L. Muscle sympathetic nerve activity (MSNA) was recorded by microneurography in a subpopulation of 27 patients.Results:Length of hospital stay was longer in the LT group compared to in the NT group (37 ± 4 vs. 25 ± 4 days; p = 0.008). Similarly, the cumulative hazard of readmission within 1 year was greater in the LT group compared to in the NT group (44% vs. 22%, p = 0.001). In the single-predictor analysis, TT (hazard ratio [HR], 2.77; 95% confidence interval [CI], 1.58–4.85; p = 0.02) predicted hospital readmission within 90 days. In addition, TT (HR, 4.65; 95% CI, 2.67–8.10; p = 0.009) and readmission within 90 days (HR, 3.27; 95% CI, 1.23–8.69; p = 0.02) predicted increased mortality. Neurohumoral activation, as estimated by MSNA, was significantly higher in the LT group compared to in the NT group (65 ± 3 vs. 51 ± 4 bursts/100 heart beats; p < 0.001).Conclusion:These results support the concept that LT is an independent risk factor for hospital readmission within 90 days and increased mortality in patients with HF. Furthermore, increased MSNA was observed in patients with LT.