Analysis of casein alpha S1 & S2 proteins from different mammalian species

Nowadays, the quality of any food used for human consumption is, to a considerable extent, considered by its possible contribution to the maintenance or improvement of the consumer's health. In developed countries there is increasing interest in goat milk and its derivates, the quality of which is considered of special importance in the light of current tendencies favouring healthy eating. In particular, goat's milk is a hypoallergenic alternative to cow's milk in the human diet. In the present work, we studied the casein alpha S1 and S2 proteins of cow, goat and sheep for comparative analysis. We found that the amino acid sequence of these proteins is almost same in goat and sheep but there are several changes at different base pairs when these two sequences are compared with cow. Prediction of secondary structures (GOR) was performed for alpha s1 and s2 proteins to gain functional insights. Our in silico study revealed considerable identity in chemical properties between goat and sheep but a considerable dissimilarity in cow with goat and sheep casein proteins. Moreover, the effect amino acid change on secondary structures in the three species is discussed. There was no significant difference found between goat and sheep alpha S1 and S2 proteins, so naturally both will be having same properties. The study concludes that sheep milk is another convenient alternative for the cow milk allergic children.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种。为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们针成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞（试验组）,移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6－二甲基氨基嘌呤－DMAP）激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移入同期发情羊子宫内。妊娠早期作B超诊断,确立妊娠的观察至足月。同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞（对照组）,按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内。结果：试验组,波尔羊颗粒粒细胞与耳皮肤成纤维2细胞的融合率分别为78．2％（115／147）,57．4％（116／202）,重构胚卵裂率为85．8％（115／134）,桑椹胚,囊胚的发育率38．8％（52／134）,早期妊娠三头,分别于妊娠40,60,60日龄终止妊娠。对照组,融合率为89．5％（136／152）,早期妊娠率为42．9％（6／14）,四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症。经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系。以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育。     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种.为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们将成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞(试验组),移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6-二甲基氨基嘌呤(6-DMAP)激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移人同期发情羊子宫内.妊娠早期作B超诊断,确立妊娠的观察至足月.同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞(对照组),按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内.结果试验组,波尔羊颗粒粒细胞与耳皮肤成纤维细胞的融合率分别为78.2%(115/147)、57.4%(116/202),重构胚卵裂率为85.8%(115/134),桑椹胚、囊胚的发育率38.8%(52/134),早期妊娠三头,分别于妊娠40、60、60日龄终止妊娠.对照组,融合率为89.5%(136/152),早期妊娠率为42.9%(6/14),四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症.经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系.以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育.     

Infection of small ruminants with Ehrlichia spp. in Sicily

In 1991, the experimental infection of a goat with pooled blood from goats that were positive for anti-Ehrlichia canis, E. risticii, E. equi and E. phagocytophila antibodies was monitored (physical examination, cell blood count, microscopical examination of blood smears, serology) for 180 days. The infection produced a clinical condition characterized by intermittent fever, anaemia and leukopenia with neutropenia during the first 40 days. Recurrent leukocytosis with lymphocytosis was noticed afterwards. A permanent high-level thrombocytosis appeared after the 18th day. During the first week, cytoplasmic basophilic inclusion bodies were seen in smears of peripheral venous blood stained with May-Grunwald-Giemsa, first in mononuclear cells and then in neutrophils (in max 3% of circulating leukocytes). Seroconversion occurred during the 2nd week and the highest antibody titre (IFAT) was registered vs E. equi (10,240) at the 19th day, vs E. canis (320) at the 24th and vs E. risticii (80) at the 30th day. At the end of the observation period the infected goat was still positive for E. equi (titre 160) and E. canis (titre 10) only. The preinoculation serum of the infected goat was reactive with E. phagocytophila antigen (serum was tested for IF antibodies to E. phagocytophila at 1:200 dilution only, because of the limited quantities of antigen available), but the qualitative evaluation of fluorescence showed an increase from the 7th day, maximum intensity between the 14th and the 40th day and passed to negative from the 74th day. Although it was based on microscopy and serology only and not carried out in a SPF goat, the above experiment gave evidence of the existence of species of the E. phagocytophila genogroup in Italy for the first time.     

Allogeneic bone marrow transplantation for leukaemia and aplastic anaemia. Results of the Leipzig BMT Group

In acute leukaemias there was a stable plateau in the survival curve at 45% after two years if grafted in first complete remission (n = 20) but only 13% of the patients are disease-free alive if grafted in a more advanced stage of the disease (n = 8). In 16 patients transplanted for chronic myeloid leukaemia the overall survival is 40%, in cases with graft-versus-host disease (GVHD) prevention by cyclosporine survival rate could be improved. Only 8 patients with severe aplastic anaemia, partially in low performance status were able to be transplanted; three died of infections, another by acute GVHD. The fatal complications in our study characterize the international well-known major problems in BMT: GVHD, interstitial pneumonitis, infections, graft failure in aplastic anaemia and recurrence of leukaemia, especially in more advanced leukaemia stage.     

Seroprevalence and Risk Factors for Coxiella burnetii (Q Fever) Seropositivity in Dairy Goat Farmers' Households in The Netherlands, 2009-2010

Community Q fever epidemics occurred in the Netherlands in 2007-2009, with dairy goat and dairy sheep farms as the implicated source. The aim of the study was to determine the seroprevalence and risk factors for seropositivity in dairy goat farmers and their household members living or working on these farms. Sera of 268 people living or working on 111 dairy goat farms were tested for Coxiella burnetii IgG and IgM antibodies using immunofluorescence assay. Seroprevalences in farmers, spouses and children (12-17 years) were 73.5%, 66.7%, and 57.1%, respectively. Risk factors for seropositivity were: performing three or more daily goat-related tasks, farm location in the two southern provinces of the country, proximity to bulk milk-positive farms, distance from the nearest stable to residence of 10 meters or less, presence of cats and multiple goat breeds in the stable, covering stable air spaces and staff not wearing farm boots. Goat farmers have a high risk to acquire this occupational infection. Clinicians should consider Q fever in this population presenting with compatible symptoms to allow timely diagnosis and treatment to prevent severe sequelae. Based on the risk factors identified, strengthening general biosecurity measures is recommended such as consistently wearing boots and protective clothing by farm staff to avoid indirect transmission and avoiding access of companion animals in the goat stable. Furthermore, it provides an evidence base for continuation of the current vaccination policy for small ruminants, preventing spread from contaminated farms to other farms in the vicinity. Finally, vaccination of seronegative farmers and household members could be considered.     

Treatment of severe aplastic anaemia with antilymphocyte globulin or bone-marrow transplantation.

Fifty-three patients with severe aplastic anaemia were admitted to this hospital between January 1976 and June 1980, of whom three arrived in terminal condition and died before treatment for their basic disease could be given. Thus 50 patients were treated and evaluated in a prospective study according to one protocol. Eighteen patients with an HLA-identical sibling underwent bone-marrow transplantation with the aim of achieving haematopoietic chimerism. Thirty-two patients without an HLA-identical sibling were given antilymphocyte globulin with or without an infusion of HLA-haplotype-identical marrow. All these 32 patients received low-dose androgens after the procedure. In the first group eight patients (44%) survived. In the two other groups, 22 patients survived (69%), of whom 20 were completely self-sustaining (63%). Engraftment and graft-versus-host disease did not occur in the group who received antilymphocyte globulin and haploidentical marrow, and the haematopoietic reconstitutions in these patients were all autologous. These results confirm the efficacy of antilymphocyte globulin in the treatment of severe aplastic anaemia and show that such treatment is at least as good as bone-marrow transplantation. Its mechanism of action remains unknown, but most patients with aplastic anaemia have a pool of haematopoietic stem cells able to repopulate the marrow after this type of treatment.     

Ascaris co-infection does not alter malaria-induced anaemia in a cohort of Nigerian preschool children

Background

Co-infection with malaria and intestinal parasites such as Ascaris lumbricoides is common. Malaria parasites induce a pro-inflammatory immune response that contributes to the pathogenic sequelae, such as malarial anaemia, that occur in malaria infection. Ascaris is known to create an anti-inflammatory immune environment which could, in theory, counteract the anti-malarial inflammatory immune response, minimizing the severity of malarial anaemia. This study examined whether Ascaris co-infection can minimize the severity of malarial anaemia.

Methods

Data from a randomized controlled trial on the effect of antihelminthic treatment in Nigerian preschool-aged (6–59 months) children conducted in 2006–2007 were analysed to examine the effect of malaria and Ascaris co-infection on anaemia severity. Children were enrolled and tested for malaria, helminths and anaemia at baseline, four, and eight months. Six hundred and ninety subjects were analysed in this study. Generalized linear mixed models were used to assess the relationship between infection status and Ascaris and Plasmodium parasite intensity on severity of anaemia, defined as a haemoglobin less than 11 g/dL.

Results

Malaria prevalence ranged from 35-78% over the course of this study. Of the malaria-infected children, 55% were co-infected with Ascaris at baseline, 60% were co-infected four months later and 48% were co-infected eight months later, underlining the persistent prevalence of malaria-nematode co-infections in this population. Over the course of the study the percentage of anaemic subjects in the population ranged between 84% at baseline and 77% at the eight-month time point. The odds of being anaemic were four to five times higher in children infected with malaria compared to those without malaria. Ascaris infection alone did not increase the odds of being anaemic, indicating that malaria was the main cause of anaemia in this population. There was no significant difference in the severity of anaemia between children singly infected with malaria and co-infected with malaria and Ascaris.

Conclusion

In this cohort of Nigerian preschool children, malaria infection was the major contributor to anaemia status. Ascaris co-infection neither exacerbated nor ameliorated the severity of malarial anaemia.     

Accidental percutaneous hexachlorophane intoxication in children.

Eighteen children with normal skin were accidentally intoxicated by a talc powder containing 6% hexachlorophane. Four died and two remained paraplegic. The clinical picture was intracranial hypertension, eight patients developing signs of spinal cord damage. The condition seemed to result from massive intramyelinic oedema. In the spinal cord vascular disturbances may occur as mechanical complications of oedema, giving rise to permanent sequelae.     

Marrow transplantation for pancytopenia in dyskeratosis congenita

A 33-year-old man with dyskeratosis congenita received a marrow transplant to treat severe pancytopenia. The graft was successful-, but the patient developed severe acute graft-versus-host-disease grade IV and died 51 days postgrafting. - The outcome of transplantation in dyskeratosis congenita is compared to that in Fanconi's anaemia due to the resemblance of both diseases in some aspects.     

Acquired refractory anaemia with excess of blasts of promegakaryoblastic type

A case of acquired refractory anaemia with excess of blasts (RAEB) of the promegakaryoblastic type is described. The promegakaryoblastic origin was demonstrated by means of ultrastructural cytochemistry (platelet peroxidase) and immunological methods. The blasts were also 5'-Nucleotidase positive. After a stable course over 2.5 years the patient died from hematological failure, his marrow demonstrating a high degree of fibrosis.     

Clinical importance of acquired cystic disease of the kidney in patients undergoing dialysis.

From 1976 to 1982 five patients undergoing haemodialysis at Oxford Renal Unit suffered serious complications from acquired cystic disease of the kidney and two died as a direct result. Clinical features seen were pain, haematuria, palpable renal enlargement, massive haemorrhage, resolution of anaemia, and metastatic malignancy. The clinical histories emphasise the features of a disease that is likely to assume increasing importance in patients undergoing haemodialysis.     

乳腺特异高表达人促红细胞生成素转基因奶山羊的制备

目的制备乳腺特异性高表达人促红细胞生成素（hEPO）转基因奶山羊。方法采用牛β-乳球蛋白基因（BLG）调控元件和hEPO全长编码序列基因组DNA构建真核表达载体,应用受精卵原核注射的方法制备hEPO转基因山羊。结果在原核注射获得的188头羔羊中,经Southern blot法检测有4头羊含有hEPO基因,其中3头为母羊,1头公羊于出生后20d死亡;3头转基因母羊hEPO基因的拷贝数分别为1、10、2;Western blot检测结果显示转基因羊乳中的hEPO分子质量为32kDa;MTT法检测结果表明,在泌乳10d的3只转基因羊乳汁中,每毫升乳汁中hEPO活性分别达到1.17×10^2IU、1.90×10^4IU、1.91×10^4IU。结论牛BLG能够调控hEPO基因在山羊乳腺中高表达,为实现其他药用蛋白在山羊乳腺中表达奠定了基础。     

Morbidity from Acute Carbon Monoxide Poisoning at Three-year Follow-up

Seventy-four survivors of acute carbon monoxide poisoning were followed up for an average of three years. In eight patients gross neuropsychiatric damage was directly attributable to the poisoning. Three patients had committed suicide and eight had died from other causes. Morbidity and mortality in those deliberately and accidentally poisoned was approximately equal.Of 63 patients alive at follow-up eight showed an improvement and 21 (33.3%) a deterioration of personality after poisoning, and 27 (43%) reported a subsequent impairment of memory. Deterioration of personality and memory impairment were highly correlated. The level of consciousness on admission to hospital in the acute phase of poisoning correlated significantly with the development of gross neuropsychiatric sequelae. These findings emphasize the importance of prompt and efficient treatment of carbon monoxide poisoning and the need to follow-up all cases in the anticipation of a relapsing course or the development of sequelae.     

Effect of murine cytomegalovirus on the in vitro responses of T and B cells to mitogens.

Both in vitro and in vivo murine cytomegalovirus (MCMV) infection depressed the responses of lymphocytes to both B and T cell mitogens. The possibilities that macrophages or nonspecific T cell inhibition of B cells might account for the depressed responses were eliminated. In vitro data suggested that B cell responses are more susceptible to this depression than T cell responses. The possibility that the depression of T cell responses is not a direct effect of viral infection of lymphocytes is discussed. To investigate further the interaction between B and T lymphocytes and MCMV, mice with B and T cell deficiences were studied. A comparison of the susceptibility of athymic Nu/Nu mice and T cell competent Nu/+ littermates to MCMV showed that the LD50 for Nu/Nu mice is 10-fold lower than that for Nu/+ mice, but Nu/+ mice given an LD50 of virus died much sooner after infection than Nu/Nu mice given an LD50. Pathogenic mechanisms responsible for death may be different in these two groups of mice. Similarly the MCMV LD50 for B cell-deficient mice (treated with goat anti-mouse IgM serum) was 10-fold lower than the LD50 for mice treated with normal goat serum, but given an LD50 of virus, the latter died sooner after infection than the former. In contrast, there was little difference between the LD50 or time of death after MCMV infection of CBA x DBA F1 male mice (which are deficient in their response to thymic independent antigens) and their normal littermates, the CBA x DBA F1 female mice.     

Cloned kids derived from caprine mammary gland epithelial cells

The use of nucleus transfer techniques to generate transgenic dairy goats capable of producing recombinant therapeutic proteins in milk could have a major impact on the pharmaceutical industry. However, transfection or gene targeting of nucleus transfer donor cells requires a long in vitro culture period and the selection of marker genes. In the current study, we evaluated the potential for using caprine mammary gland epithelial cells (CMGECs), isolated from udders of lactating F1 hybrid goats (Capra hircus) and cryopreserved at Passages 24 to 26, for nucleus transfer into enucleated in vivo-matured oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 31 recipient goats. Twenty-three (74%), 21 (72%), and 14 (48%) recipients were confirmed pregnant by ultrasonography on Days 30, 60, and 90, respectively. Four recipients aborted between 35 and 137 d of gestation. Five recipients carried the pregnancies to term and delivered one goat kid each, one of which subsequently died due to respiratory difficulties. The remaining four goat kids were healthy and well. Single-strand conformation polymorphism analysis confirmed that all kids were clones of the donor cells. In conclusion, the CMGECs remained totipotent for nucleus transfer.     

Status of allogeneic bone marrow transplantation in childhood in the GDR

Of 25 HLA-identical, MLC negative transplants 10 patients had acute lymphoblastic leukaemia (ALL), 8 acute nonlymphoblastic leukaemia (ANLL), 3 severe aplastic anaemia, 2 malignant histiocytosis, 1 patients neuroblastoma and 1 Fanconi anaemia. 3 HLA nonidentical, MLC positive transplants were performed, two children had malignant infantile osteopetrosis and 1 child had a severe combined immunodeficiency disease. Patients with ALL and ANLL received cyclophosphamide and single dose total body irradiation. 3 patients received fractionated TBI. The results for the allogeneic group overall indicate that the actuarial disease free survival rate is 0.62. 16 of 25 patients are in continuous complete remission (CCR) periods of 3-78 months posttransplant. All three transplanted children with severe aplastic anaemia alive disease-free for periods of 21-81 months. 10 patients with ALL were transplanted (2 in first remission for high risk ALL, 8 in second remission). 7 of 10 patients are alive and disease-free (CCR rate 0.67). 8 patients underwent BMT for ANNL while in first remission in 7 patients and in third partial remission in 1 patient. 4 of 8 patients are alive and disease-free for periods of 25-56 months (CCR rate 0.50). 1 patient with neuroblastoma stage IV survives 24 months, 1 child with Fanconi anemia died on day +25 of GVHD and septicaemia. 1 of the 2 patients transplanted for malignant histiocytosis relapsed 3 months posttransplant, 1 patient is alive and disease-free 5 months posttransplant. In none of the HLA-nonidentical and MLC positive transplantations T-cell depleted marrow engrafted.     

First birth of an animal from an extinct subspecies (Capra pyrenaica pyrenaica) by cloning

Two experiments have been performed to clone the bucardo, an extinct wild goat. The karyoplasts were thawed fibroblasts derived from skin biopsies, obtained and cryopreserved in 1999 from the last living specimen, a female, which died in 2000. Cytoplasts were mature oocytes collected from the oviducts of superovulated domestic goats. Oocytes were enucleated and coupled to bucardo's fibroblasts by electrofusion. Reconstructed embryos were cultured for 36 h or 7 d and transferred to either Spanish ibex or hybrid (Spanish ibex male ×  domestic goat) synchronized recipients. Embryos were placed, according to their developmental stage, into the oviduct or into the uterine horn ipsilateral to an ovulated ovary. Pregnancy was monitored through their plasmatic PAG levels. In Experiment 1, 285 embryos were reconstructed and 30 of them were transferred at the 3- to 6-cells stage to 5 recipients. The remaining embryos were further cultured to day 7, and 24 of them transferred at compact morula/blastocyst stage to 8 recipients. In Experiment 2, 154 reconstructed embryos were transferred to 44 recipients at the 3- to 6-cells stage. Pregnancies were attained in 0/8 and 7/49 of the uterine and oviduct-transferred recipients, respectively. One recipient maintained pregnancy to term, displaying very high PAG levels. One morphologically normal bucardo female was obtained by caesarean section. The newborn died some minutes after birth due to physical defects in lungs. Nuclear DNA confirmed that the clone was genetically identical to the bucardo's donor cells. To our knowledge, this is the first animal born from an extinct subspecies.     

Ascite chyleuse à Mycobacterium tuberculosis : un cas clinique

Chylous ascites is a rare manifestation of tuberculosis. We report the case of a 63-year-old man admitted with ascites and bilateral lower limb edema developed two years previously, with progressive increase. He had a history of microbiologically confirmed and properly treated pleuropulmonary tuberculosis, 20 years before. Although Mycobacterium tuberculosis could not be detected in the ascitic fluid, chest, abdominal and pelvic computed tomography showed findings very suggestive of tuberculosis sequelae. A lymphoscintigraphy was performed and also supported our diagnostic hypothesis of chylous ascites and lymphedema due to tuberculosis sequelae associated or not with active infection. The intention was to further investigate if any form of active tuberculosis was present due to reactivation of the infection. Unfortunately, the patient died before these assessments could be completed.     

Long-term Carotid Access in the Goat: Observations on Application of a Totally Implantable Catheter System

The investigation of metabolism of individual organs and tissues in vivo requires simultaneous measurements of arterio-venous concentration differences. Long-term metabolic studies require frequent and convenient access to veins and arteries of the relevant organs or tissues, and if blood samples are obtained by repeated veni — or arteriopuncture, difficulties may arise due to vessel thrombosis and an increasing apprehension of the goat during time. Arterial punctures are considered more painful than venipuncture, according to human patients. In the conscious goat, arteriopuncture may induce bleating, dilatation of the pupil, increased heart rate, blood glucose and free fatty acid levels, which may bias the results of a metabolic study (Linzell 1963). Furthermore, it is important to avoid situations which contribute negatively to the welfare of the goat. Establishment of permanent exteriorized carotid loops does not eliminate these problems and has a disadvantage of being vulnerable to injury. Jha et al. (1961) reported that 3 out of 10 goats died from haemorrhages, due to loop injury. The carotid artery may surgically be relocated to a subcutaneous position, which makes it less vulnerable, but also less accessible for percutaneous puncture (Tavenor 1969). Our experience with such subcutaneous relocated artery loops is that scar tissue formation is so strong after being catheterized 3-4 times, that further catheterization requires specialized assistance. The artery is eventually closed by fibrous tissue. An alternative to the repeated percutaneous puncture of subcutaneously relocated artery loops is the establishment of chronic indwelling arterial catheters, where the tip of the catheter is exteriorized to the skin. This method is well described (Huntington et al. 1989), but requires a dressing of the exteriorisation site, and restrain of the goat, in order to protect the tip from being pulled out. Therefore, we describe a modification of a totally implantable catheter system, developed for human intra-arterial long-term delivery of drugs, for permanent catheterization of the common carotid artery of goats. The entire access system of this catheter is under the skin.