Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep / Mutações no gene da proteína morfogenética óssea 15 causadoras de defeitos na secreção proteica em ovelhas inférteis Cholistani

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep

Abstract Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Resumo Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep.

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.

Development of the intestinal SGLT1 transporter in rats.

Glucose absorption from the small intestine is largely mediated via the sodium-coupled glucose transporter (SGLT1). The goal of this study was to investigate the ontogenesis of the SGLT1, using the rat as an animal model at three stages of development: during lactation, at weaning, and at physiologic maturity. The techniques involved upper small intestinal perfusions with solutions containing 200 mM glucose and 50 mM NaCl, with or without 1 mM phloridzin (Phl), as an inhibitor of SGLT1. Molecular expression of the SGLT1 was also investigated via Western blot analysis from intestinal specimens of the three growth periods. Glucose absorption in weanling rats, in the absence of Phl, was several times higher than in sucklings and approximately double that of mature animals, and the effects of Phl were the greatest in weanlings. Furthermore, the physiologic data correlate to the molecular analysis of the SGLT1 which showed an increase in expression of the SGLT1 in both the weanlings and the adults compared to the sucklings. At all three stages of development Phl abolished Na absorption, and in sucklings there was a net outflow of Na. Due to the coupling between Na and water transport, net water absorption and the influx/efflux ratio, a more sensitive indicator of changes in unidirectional fluid movement, were similarly affected by Phl at the three stages of development. Net water absorption was highest in weanling animals. These findings are consistent with an early development of SGLT1 in rat small intestine and an apparent burst of activity at weaning. Less than complete maturity of other absorptive mechansims is occurring at this time.

A rapid whole-blood immunoassay system.

A new immunoassay system has been developed which allows the detection of circulating antigens, antibodies or drugs in whole blood without specialized personnel or equipment. This is achieved by the use of bispecific reagents, which comprise specific antibodies or antigens that are coupled to a non-agglutinating antierythrocyte antibody. Within two minutes, these reagents cause specific agglutination of a patient's own red cells in samples that contain the relevant analyte. Levels of low molecular weight haptens also can be measured by the use of an indirect, agglutination-inhibition assay. This simple immunoassay method would fulfil the needs of many physicians and Third-World countries and also has mass-screening and veterinary applications.

Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

Suppression by serum of immunoreactivity to a common tumour antigen in cancer patients.

Leucocytes from 4 cancer patients showed cellular reactivity in the leucocyte adherence inhibition (LAI) assay in the presence of the synthetic encephalitogenic peptide of human myelin basic protein. All patients exhibited reactivity at a peptide concentration of 500 ng/ml. Leucocytes from 4 non-cancer patients failed to react. Suppression of LAI was detected in all 4 cancer patients by adding their serum to reactive mixtures containing peptide and autologous leucocytes. Each serum was subjected to column chromatography on Sephacryl S-200 to determine the molecular weight distribution of suppressive (blocking) factors. The greatest suppression was found in all cases within the range 90-155 kdalton.

Leukocyte adherence inhibition: tumor specificity of cellular and serum-blocking reactions in human melanoma, breast cancer, and colorectal cancer.

Blood samples were obtained from hosptial patients suspected of having cancer (colorectal carcinoma, breast carcinoma, or melanoma) or from patients after surgical treatment for these cancers. Leukocytes were tested for reactivity with appropriate tumor extracts by leukocyte adherence inhibition (LAI), without knowledge of the diagnosis. Leukocytes from each patient were tested with the specific related extract corresponding to the suspected tumor type and with at least one unrelated extract. Each patient's serum was tested for its effect on the adherence of autologous leukocytes with specific tumor extract. Detailed leukocyte adherence data are presented for each of the 110 patients. Of the 75 patients eventually diagnosed as having cancer of one of the above types, 67 (89.3%) gave positive specific LAI reactions and only 5 (6.7%) reacted nonspecifically. Of the 35 patients with benign, unrelated, or unidentified disease, 10 (28.6%) gave positive reactions; most of these were patients with benign breast disease reacting with breast tumor extract. Leukocyte reactions with related extracts were almost always blocked by autologous serum (only two exceptions). When tested with allogeneic leukocytes reacting with their corresponding extracts, sera never blocked leukocytes of a different tumor type; in some cases they also failed to block leukocytes of the same tumor type although autologous leukocytes were blocked.