The ASIP gene in the llama (Lama glama): Alternative transcripts, expression and relation with color phenotypes.

The Agouti gene (ASIP) is one of the most important genes for coat color determination in mammals. It has a complex structure with several promoters and alternative non-coding first exons that are transcribed into mRNAs with different 5'UTR. These mRNA isoforms regulate the temporal and spatial expression of the gene, producing diverse pigmentation patterns. Here, we studied ASIP transcriptional variants and their expression in the skin of llamas with different coat color phenotypes. We also described the ASIP locus, including promoter usage and the splicing events that originate each transcript variant. Using 5'RACE-PCR we isolated seven ASIP transcripts with alternative 5'UTR, where exons 1A, 1A', 1C, 1D, and a novel non-coding exon 1A" were identified. Additionally, new alternative spliced forms were found. The diversity of ASIP 5'UTRs is originated by a complex pattern of alternative promoter usage, multiple transcription start sites and splicing events that include exon skipping and alternative 3' splicing site selection. We found that ASIP was highly expressed in llamas with white and brown phenotypes while black animals presented very low expression. The main responsible for this difference was a fusion transcript between ASIP and NCOA6 genes, which was present in the skin of white and brown llamas but not in the black ones. The rest of ASIP transcripts presented very low expression in the skin, indicating that the main regulation point for ASIP gene expression is at the transcriptional level. Nevertheless, the characteristics of the 5'UTRs sequences suggest that alternative transcripts could be regulated differently at the protein synthesis level.

Characterization and expression analysis of KIT and MITF-M genes in llamas and their relation to white coat color.

The llama (Lama glama) is a fiber-producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto-oncogene receptor tyrosine kinase (KIT) and microphthalmia-associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF-M, the melanocyte-specific isoform, and the two transcriptional variants MITF-M(-) and MITF-M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF-M. Interestingly, white llamas expressed less MITF-M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF-M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown.

Molecular characterization of the llama FGF5 gene and identification of putative loss of function mutations.

Llama, the most numerous domestic camelid in Argentina, has good fiber-production ability. Although a few genes related to other productive traits have been characterized, the molecular genetic basis of fiber growth control in camelids is still poorly understood. Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that controls hair growth in humans and other mammals. Mutations in the FGF5 gene have been associated with long-hair phenotypes in several species. Here, we sequenced the llama FGF5 gene, which consists of three exons encoding 813 bp. cDNA analysis from hair follicles revealed the expression of two FGF5 alternative spliced transcripts, in one of which exon 2 is absent. DNA variation analysis showed four polymorphisms in the coding region: a synonymous SNP (c.210A>G), a single base deletion (c.348delA), a 12-bp insertion (c.351_352insCATATAACATAG) and a non-sense mutation (c.499C>T). The deletion was always found together with the insertion forming a haplotype and producing a putative truncated protein of 123 amino acids. The c.499C>T mutation also leads to a premature stop codon at position 168. In both cases, critical functional domains of FGF5, including one heparin binding site, are lost. All animals analyzed were homozygous for one of the deleterious mutations or compound heterozygous for both (i.e. c.348delA, c.351_352insCATATAACATAG/c.499T). Sequencing of guanaco samples showed that the FGF5 gene encodes a full-length 270-amino acid protein. These results suggest that FGF5 is likely functional in short-haired wild species and non-functional in the domestic fiber-producing species, the llama.

Genetic diversity and conservation status of managed vicuña (Vicugna vicugna) populations in Argentina.

The vicuña (Vicugna vicugna) was indiscriminately hunted for more than 400 years and, by the end of 1960s, it was seriously endangered. At that time, a captive breeding program was initiated in Argentina by the National Institute of Agricultural Technology (INTA) with the aim of preserving the species. Nowadays, vicuñas are managed in captivity and in the wild to obtain their valuable fiber. The current genetic status of Argentinean vicuña populations is virtually unknown. Using mitochondrial DNA and microsatellite markers, we assessed levels of genetic diversity of vicuña populations managed in the wild and compared it with a captive population from INTA. Furthermore, we examined levels of genetic structure and evidence for historical bottlenecks. Overall, all populations revealed high genetic variability with no signs of inbreeding. Levels of genetic diversity between captive and wild populations were not significantly different, although the captive population showed the lowest estimates of allelic richness, number of mitochondrial haplotypes, and haplotype diversity. Significant genetic differentiation at microsatellite markers was found between free-living populations from Jujuy and Catamarca provinces. Moreover, microsatellite data also revealed genetic structure within the Catamarca management area. Genetic signatures of past bottlenecks were detected in wild populations by the Garza Williamson test. Results from this study are discussed in relation to the conservation and management of the species.

Estudio experimental con Ecografía Doppler en uropatía obstructiva parcial crónica / Experimental study with Doppler Ultrasound in partial chronic obstructive uropathy

Objetivo: La valoración de los parámetros hemodinámicos índice de resistencia renal (IR), velocidad pico sistólica (PSV), velocidad mínima diastólica (EDV) y flujo de la arteria renal (FR) mediante ecografía Doppler para el diagnóstico y monitorización posquirúrgica de la uropatía obstructiva parcial crónica. Material y métodos: Se emplean 50 animales de la especie porcina. El estudio se divide en 3 fases. La fase I consta de la valoración ecográfica dúplex-Doppler de ambos riñones, determinando los parámetros objeto de estudio. La ratio de cada índice se calcula como la diferencia entre el valor del riñón en estudio y su contralateral. El examen termina con la realización de cistografía de compresión, urografía excretora y ureteropielografía retrógrada. Seguidamente, se procede a la creación de un modelo de obstrucción parcial del uréter derecho. Transcurridas 6 semanas, comienza la fase II mediante la realización de las pruebas descritas anteriormente y posterior resolución endourológica de la obstrucción. La fase III consiste en un seguimiento a los 6 meses del tratamiento mediante las pruebas realizadas en las fases previas. Resultados: De los parámetros estudiados la EDV y su ratio son los que mayor sensibilidad y especificidad muestran como marcador diagnóstico de uropatía obstructiva. En el seguimiento posquirúrgico se observa como el IR y principalmente la EDV retornan a los valores basales. Conclusiones: La ΔEDV es el parámetro que mayor eficacia muestra para el diagnóstico de uropatía obstructiva parcial crónica; pero esta es insuficiente para relegar las técnicas de diagnóstico convencionales. Todos los parámetros, principalmente la EDV, han mostrado ser útiles como pruebas complementarias de monitorización y pronóstico tras la resolución endourológica de la uropatía obstructiva (AU)

Introduction: This study has aimed to assess the hemodynamic parameters, Renal Resistive Index (RI), Peak Systolic Velocity (PSV), End-Diastolic Velocity (EDV) and Blood Flow of the Renal Artery (FR) by Doppler Ultrasound for diagnosis and monitoring postsurgical partial chronic obstructive uropathy. Material and methods: Fifty pigs were used. The experiment was divided into three phases. Phase I consisted of a duplex-Doppler evaluation of the both kidneys to determine the parameters under study. The ratio of each index is calculated as the difference between the value of study kidney and the contralateral. After, a fluoroscopic examination was performed by compressive cystography, excretory urography and retrograde ureteropyelography. Finally, a model of partial right ureteral obstruction was created. After six weeks of the obstructive model, Phase II was begun with the diagnosis of the uropathy, by means of the aforementioned diagnostic methods and the endourological treatment was completed. Phase III is a follow-up performed at 6 months of treatment using the same methods as in the previous phases. Results: Of the parameters studied, the EDV and its ratio showed greater sensitivity and specificity as a diagnostic marker of obstructive uropathy. In the postoperative monitoring, it was observed that the RI and the EDV returned to baseline levels, with the baseline values. Conclusions: The ΔEDV and its ratio is the parameter that shows the greater efficacy for the diagnosis of chronic partial obstructive uropathy, however, it is insufficient to avoid conventional diagnostic techniques. All the parameters, mainly the EDV, have proven useful as complementary tests for monitoring after endourologic resolution of obstructive uropathy (AU)

[Experimental study with Doppler ultrasound in partial chronic obstructive uropathy]. / Estudio experimental con Ecografía Doppler en uropatía obstructiva parcial crónica.

INTRODUCTION: This study has aimed to assess the hemodynamic parameters, Renal Resistive Index (RI), Peak Systolic Velocity (PSV), End-Diastolic Velocity (EDV) and Blood Flow of the Renal Artery (FR) by Doppler Ultrasound for diagnosis and monitoring postsurgical partial chronic obstructive uropathy. MATERIAL AND METHODS: Fifty pigs were used. The experiment was divided into three phases. Phase I consisted of a duplex-Doppler evaluation of the both kidneys to determine the parameters under study. The ratio of each index is calculated as the difference between the value of study kidney and the contralateral. After, a fluoroscopic examination was performed by compressive cystography, excretory urography and retrograde ureteropyelography. Finally, a model of partial right ureteral obstruction was created. After six weeks of the obstructive model, Phase II was begun with the diagnosis of the uropathy, by means of the aforementioned diagnostic methods and the endourological treatment was completed. Phase III is a follow-up performed at 6 months of treatment using the same methods as in the previous phases. RESULTS: Of the parameters studied, the EDV and its ratio showed greater sensitivity and specificity as a diagnostic marker of obstructive uropathy. In the postoperative monitoring, it was observed that the RI and the EDV returned to baseline levels, with the baseline values. CONCLUSIONS: The ΔEDV and its ratio is the parameter that shows the greater efficacy for the diagnosis of chronic partial obstructive uropathy, however, it is insufficient to avoid conventional diagnostic techniques. All the parameters, mainly the EDV, have proven useful as complementary tests for monitoring after endourologic resolution of obstructive uropathy.

South American camelid illegal traffic detection by means of molecular markers.

South American camelids comprise the wild species guanaco and vicuña and their respective domestic relatives llama and alpaca. The aim of the present study was to determine by DNA analysis to which of these species belong a herd of camelids confiscated from a llama breeder but alleged to be alpacas by the prosecution, and to evaluate the usefulness of mitochondrial and autosomal DNA markers to solve judicial cases involving camelid taxa. Cytochrome b and cytochrome oxidase I mitochondrial genes and 7 STR were analyzed in 25 confiscated samples. Mitochondrial results were inconclusive because 18 of the sequestered samples presented haplotypes that corresponded to the guanaco haplogroup and the remaining seven belonged to a vicuña linage. Microsatellite data of casework samples and llama reference samples revealed different genetic profiles by the presence of private alleles at two microsatellites suggesting that the confiscated animals could be alpaca, or at least alpaca hybrids instead of pure llama.

Identification of camelid specific residues in mitochondrial ATP synthase subunits.

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.

2006 GEP-ISFG collaborative exercise on mtDNA: reflections about interpretation, artefacts, and DNA mixtures.

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate ( approximately 77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing.

Can systemic lupus erythematosus be the cause of collapsing glomerulopathy?

Collapsing glomerulopathy is a severe form of glomerular injury, closely associated with HIV infection, characterized by the collapsing feature of glomerular damage with frequent tubulointerstitial involvement and rapid progression to terminal renal failure. The etiopathogenesis in non-HIV infected patients remains obscure. We reported a patient whose diagnosis of collapsing glomerulopathy (CG) and systemic lupus erythematosus (SLE) was done simultaneously and described the diseases characteristics suggesting that SLE could be an etiologic factor for the induction of this glomerulopathy, clinical evolution and treatment.

Mutation rates at Y chromosome specific microsatellites.

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) to estimate Y-STR mutation rates. Seventeen Y chromosome STR loci (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, DYS635 [GATA C4], GATA H4, and GATA A10) were analyzed in a sample of 3,026 father/son pairs. Among 27,029 allele transfers, 54 mutations were observed, with an overall mutation rate across the 17 loci of 1.998 x 10(-3) (95% CI, 1.501 x 10(-3) to 2.606 x 10(-3)). With just one exception, all of the mutations were single-step, and they were observed only once per gametogenesis. Repeat gains were more frequent than losses, longer alleles were found to be more mutable, and the mutation rate seemed to increase with the father's age. Hum Mutat 26(6), 520-528, 2005. (c) 2005 Wiley-Liss, Inc.

Genetic diversity and differentiation of guanaco populations from Argentina inferred from microsatellite data.

Genotype data from 14 microsatellite markers were used to assess the genetic diversity and differentiation of four guanaco populations from Argentine Patagonia. These animals were recently captured in the wild and maintained in semi-captivity for fibre production. Considerable genetic diversity in these populations was suggested by the finding of a total of 162 alleles, an average mean number of alleles per locus ranging from 6.50 to 8.19, and H(e) values ranging from 0.66 to 0.74. Assessment of population differentiation showed moderate but significant values of F(ST)=0.071 (P=0.000) and R(ST)=0.083 (P=0.000). An amova test showed that the genetic variation among populations was 5.6% while within populations it was 94.4%. A number of 6.6 migrants per generation may support these results. Unambiguous individual assignment to original populations was obtained for the Pilcaniyeu, Las Heras and La Esperanza populations. The erroneous assignment of 18.75% Rio Mayo individuals to the Las Heras population can be explained by the low genetic differentiation found between these two populations. Thirty-nine of 56 loci per population combinations were in Hardy--Weinberg disequilibrium because of guanaco heterozygote deficiency, which may be explained by population subdivision. The high level of genetic diversity of the guanacos analysed here indicates that the Patagonian guanaco constitutes an important genetic resource for conservation or economic utilization programmes.

Significance of protein p53 overexpression in the clinical course of high-risk superficial bladder cancer.

OBJECTIVES: This is a retrospective study in which the long-term biological behavior of 67 "high-risk" superficial bladder tumors and the prognostic relevance (prediction of disease recurrence and progression) of the determination of the p53 phenotype in these cases were studied. MATERIAL AND METHODS: 67 tumors with a "high-risk" of progression were selected from the 1,103 transurethral resections for bladder cancer carried out in 640 patients in this center between 1987 and 1992. These included 39 T1G3, 14 Tis (isolated or associated with Ta-T1, non-G3 tumors), and 14 Ta-T1, non-G3 tumors with submucosal lymphatic affection (L+). The median follow-up of these cases was 69.7 months. An immunohistochemical technique with monoclonal antibodies (DO-7) was used to detect the p53 phenotype in paraffin-fixed material. RESULTS: Tumor recurrence occurred in 31 patients (46.3%) and local or distant progression in 14 (20.9%). Radical cystectomy was carried out in 16 (23.9%) cases. p53 overexpression of > or =20% ("p53+") was detected in 40 tumors (59.7%). The rate of recurrence and progression, the disease and progression-free intervals, cancer-specific survival, disease-free survival and progression-free survival were similar in the 3 tumor groups (in all cases, p > 0.05). There were no significant differences in the overexpression of protein p53, using the standard cutoff point of 20% stained nuclei, on comparing the same variables in the whole group of 67 patients (in all cases, p > 0.05). CONCLUSION: The detection of protein p53 was not found to be of use in the retrospective prediction of disease progression or survival in "high-risk" superficial bladder cancer.

Marcadores biológicos en el cáncer de próstata / Biological markers of prostate cancer

En los últimos 20 años se han producido muchos adelantos en el uso clínico de marcadores biológicos en el cáncer de la próstata. El antígeno prostático específico (PSA) se ha consolidado como el más importante de todos los marcadores tumorales humanos. En este artículo se describen las formas moleculares de PSA y sus características, los factores que influyen en los valores séricos de PSA, y los métodos para aumentar la sensibilidad y/o especificidad de PSA total (especialmente en valores entre 4-10 ng/ml), como rangos de PSA específicos de edad, velocidad de PSA, PSA libre, densidad de PSA, densidad de PSA de la zona de la transición; así como el papel de PSA en la monitorización de las diversas formas de tratamiento del cáncer de próstata. Se revisa también el papel de otras formas de calicreínas de las direcciones futuras respecto a marcadores biológicos en el cáncer de la próstata (AU)

Phylogenetic relationships among Robertsonian karyomorphs of Graomys griseoflavus (Rodentia, Muridae) by mitochondrial cytochrome b DNA sequencing.

Graomys griseoflavus (Waterhouse 1837) is a phyllotine murid rodent with a Robertsonian autosomal polymorphism, having been described 2n = 42, 41, 38, 37, 36, 35 and 34 karyomorphs, and proposed a chromosomal divergence pathway accounted by four sequential Robertsonian fusions. Sequences of a fragment (422 bp long) of the cytochrome b (cyt b) mitochondrial gene and its 5' flanking region (tRNA Glu) were obtained for 19 Graomys griseoflavus from different karyomorphs to infer phylogenetic relationships by using maximum parsimony. Outgroups considered for this analysis were the phyllotine rodents Phyllotis xanthopygus and Eligmodontia typus cyt b sequences. Three trees were produced showing the 2n = 38-34 karyomorphs grouped in a single clade while the 2n = 42-41 animals formed a different one. This is in agreement with a hypothesis of a single origin for 2n = 38-34 Robertsonian karyomorphs from the ancestral 2n = 42.

Long-term follow-up of a randomized prospective trial comparing a standard 81 mg dose of intravesical bacille Calmette-Guérin with a reduced dose of 27 mg in superficial bladder cancer.

OBJECTIVES: To determine the efficacy of a three-fold reduced dose (RD, 27 mg) of intravesical bacille Calmette-Guérin (BCG) against the standard dose (81 mg) in patients with superficial bladder cancer, assessing recurrence, progression and differences in toxicity. PATIENTS AND METHODS: Five hundred patients with superficial bladder cancer (Ta, T1, Tis) were enrolled and randomly assigned to be treated after transurethral resection of all visible lesions with intravesical BCG Connaught strain (weekly x six and thereafter fortnightly x six) either with the standard or RD instillation. RESULTS: All but one of the 500 patients were evaluable for efficacy and toxicity (252 in the standard arm and 247 in the RD arm). The median follow-up was 69 months (maximum 104); 71 (28%) patients in the standard arm and 76 (31%) in the RD arm developed recurrences; the median time to recurrence has not yet been attained, but at 5 years the mean (sd) percentage of recurrence-free patients was 70.5 (3.12) and 70.4 (3.1) for the standard and RD arms, respectively. In patients presenting with multifocal tumours, the standard dose was more effective against recurrences than the RD (P=0.0151). In those with G3 and high-risk tumours overall, the superiority of the standard dose was marginal (P=0.060 and P=0.082). Twenty-nine (11.5%) tumours in the standard arm and 33 (13.3%) in the RD arm progressed to invasive disease; the median time to progression has not yet been attained, but the percentage of progression-free patients at 5 years was 88.8 (2.23) and 86.9 (2.31) for the standard and RD arms, respectively. The standard dose was more effective than the RD against progression only in patients with multifocal disease (P=0.048). Twelve (4.8%) cystectomies were performed in the standard and 15 (6.1%) in the RD arm. Currently, 106 (21.2%) patients have died, but only 38 (7.6%) from bladder cancer, i.e. 20 (7.9%) in the standard and 18 (7.5%) in RD arm. Overall the disease-specific death rate was lower for those patients who completed the scheduled treatment. The cause-specific survival at 5 years did not differ between the arms (P=0.76) but there was a trend toward better cause-specific survival for patients with multifocal tumours in the standard arm. Toxicity differed between the arms, significantly more patients having no toxicity in the RD arm, and fewer having delayed instillations or withdrawing. However, severe systemic toxicity occurred even in patients treated with the RD, in a similar proportion to those receiving the standard dose. CONCLUSION: Overall, the RD gave similar results for recurrence and progression but with significantly less toxicity. However, patients with multifocal tumours fared better with the standard dose and there was a trend towards better recurrence rates in patients with high-risk tumours. We recommend continuing to use the standard dose for high-risk tumours, while we consider the reduced dose safe and effective for intermediate-risk lesions and for maintenance schedules.

The use of neural networks and logistic regression analysis for predicting pathological stage in men undergoing radical prostatectomy: a population based study.

PURPOSE: Clinical under staging occurs in 40% to 60% of patients who undergo radical prostatectomy for prostate cancer. To decrease under staging several methods of predicting pathological stage preoperatively have been developed based on statistical logistic regression analysis and neural networks. To our knowledge none has been validated in our homogeneous regional patient population to date. We created logistic regression and neural network models, and implemented and adapted them into our practice. We also compared the 2 methods to determine their value and practicality in daily clinical practice. We present the results of our novel approach for predicting pathological staging of prostate adenocarcinoma. MATERIALS AND METHODS: Between 1986 and 1999, 600 white men from the Aragon region of Spain underwent surgery for prostate cancer; of whom 468 were selected for study. Predictive study variables included patient age, clinical stage, biopsy Gleason score and preoperative prostate specific antigen (PSA). The predicted result included in analysis was organ confined or nonorgan confined disease. Data were analyzed by multivariate logistic regression and a supervised neural network (multilayer perceptron and radial basis function). Results were compared by comparing the areas under the receiver operating characteristics curves. RESULTS: We generated 5 logistic regression models. The model created with clinical staging, Gleason biopsy score and PSA distributed in 5 categories (p <0.001) with an area under the receiver operating characteristics curve of 0.840 proved to be most predictive of pathological stage. Similarly of the 6 neural network models evaluated the radial basis function model, which included age, clinical stage, Gleason biopsy score and preoperative PSA distributed in 5 categories with an area under the curve of 0.882, proved the most predictive but not superior to the logistic regression model. The difference in the area under the curves in the 2 chosen models was 0.042 (p = 0.1). CONCLUSIONS: It is possible to generate useful predictive models of organ confined disease using logistic regression or neural networks with high indexes of clinical and statistical validity. However, using these variables neural networks did not prove to be better than logistic regression analysis. Therefore, better predictive variables must be identified, preferably nonlinear characteristics with respect to the probability of organ confined tumor, to generate better predictive models using neural networks.

Variability of the F13B locus in South American populations.

A population study for the F13B locus was carried out in a total of 396 South American individuals. The analysis comprised new data from 5 Amerindian populations, existing data from 3 Amerindian populations, and I urban sample from La Plata, Argentina. In both pooled Amerindian and La Plata samples, 6 alleles were found. The individual Amerindian samples showed a lower number of alleles, changes in modal alleles, and restricted variability. Interpopulation comparisons revealed significant differences among samples from distinct geographical regions. Differences among the groups were also corroborated by the F(ST) statistic. Data support the hypothesis that genetic drift and gene flow influence Amerindian differentiation.