Apoptosis and necrosis of polymorphonuclear leukocytes in goat milk with high and low somatic cell counts

The purpose of the present trial was to compare the percentages of necrotic and apoptotic polymorphonuclear leukocytes (PMNL) in goat milk with low and high somatic cell count (SCC). Twenty eight milk samples were collected from 20 lactating goats, determined to be negative in bacteriological examination, and divided in three groups, according to their SCC: samples with SCC lower than 500 x 10(3) cells/mL; between 500 and 1500 x 10(3) cells/mL; and higher than 1500 x 10(3) cells/m L. SCC was performed in an automatic somatic cell counter. Apoptosis and necrosis were quantified using dual-color flow cytometry with fluorescein labeled annexin-V and propidium iodide (PI). Results of the present study showed a significant positive correlation between the percentage of the viable PMNL and milk SCC(r = 0.495, P=0.008), as well as a significant negative correlation between apoptotic PMNL and milk SCC(r = -0.486, P = 0.009). Results also pointed out lower PMNL viability rates due to higher apoptosis rates in milk samples with SCC lower than 5 x 10(5) cells/mL. (C) 2011 Elsevier B.V. All rights reserved.

Microbial and sensory changes throughout the ripening of Prato cheese made from milk with different levels of somatic cells

The objective of this research was to evaluate the effects of 2 levels of raw milk somatic cell count (SCC) on the composition of Prato cheese and on the microbiological and sensory changes of Prato cheese throughout ripening. Two groups of dairy cows were selected to obtain low-SCC (<200,000 cells/mL) and high-SCC (>700,000 cells/mL) milks, which were used to manufacture 2 vats of cheese. The pasteurized milk was evaluated according to the pH, total solids, fat, total protein, lactose, standard plate count, coliforms at 45 degrees C, and Salmonella spp. The cheese composition was evaluated 2 d after manufacture. Lactic acid bacteria, psychrotrophic bacteria, and yeast and mold counts were carried out after 3, 9, 16, 32, and 51 d of storage. Salmonella spp., Listeria monocytogenes, and coagulase-positive Staphylococcus counts were carried out after 3, 32, and 51 d of storage. A 2 x 5 factorial design with 4 replications was performed. Sensory evaluation of the cheeses from low- and high-SCC milks was carried out for overall acceptance by using a 9-point hedonic scale after 8, 22, 35, 50, and 63 d of storage. The somatic cell levels used did not affect the total protein and salt: moisture contents of the cheeses. The pH and moisture content were higher and the clotting time was longer for cheeses from high-SCC milk. Both cheeses presented the absence of Salmonella spp. and L. monocytogenes, and the coagulase-positive Staphylococcus count was below 1 x 10(2) cfu/g throughout the storage time. The lactic acid bacteria count decreased significantly during the storage time for the cheeses from both low- and high-SCC milks, but at a faster rate for the cheese from high-SCC milk. Cheeses from high-SCC milk presented lower psychrotrophic bacteria counts and higher yeast and mold counts than cheeses from low- SCC milk. Cheeses from low- SCC milk showed better overall acceptance by the consumers. The lower overall acceptance of the cheeses from high-SCC milk may be associated with texture and flavor defects, probably caused by the higher proteolysis of these cheeses.

Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Evaluation of somatic cell count thresholds to detect subclinical mastitis in Gyr cows

The objectives of this study were (1) to determine the sensitivity (Se) and specificity (Sp) of somatic cell count (SCC) thresholds to identify subclinical mastitis in Gyr cows caused by major and minor pathogens; (2) to study the effects of month of sampling, rear or front mammary quarters, herd, intramammary infection (IMI), and bacterial species on SCC at quarter level; and (3) to describe the prevalence of IMI in Gyr cows in commercial dairy herds. In total, 221 lactating Gyr cows from 3 commercial dairy farms were selected. Milk samples were collected from individual quarters once a month for 1 yr from all lactating cows for SCC and bacteriological analysis. Mammary quarters were considered the experimental units and the SCC results were log(10)-transformed. Four SCC thresholds (100, 200, 300 and 400 x 10(3) cells/mL) were used to determine Se and Sp to identify infected mammary quarters. The overall prevalence of IMI in quarter milk samples of Gyr cows was 49.8%, and the prevalence of minor pathogens was higher (31.9%) than that of major pathogens (17.8%). Quarter samples with microbial isolation presented higher SCC compared with negative samples. Sensitivity and Sp of selected SCC thresholds varied according to the group of pathogen (major and minor) involved in the IMI definition. Sensitivity increased and Sp decreased when mammary quarters with only major pathogens isolation were considered positive. The use of a single SCC analysis to classify quarters as uninfected or infected in Gyr cows may not be a useful test for this breed because Se and Sp of SCC at the studied thresholds were low. The occurrence of IMI and the bacterial species are the main factors responsible for SCC variation in mammary quarters of Gyr cows. Milk samples with major pathogens isolation elicited higher SCC than those with minor pathogens.

Saethre-Chotzen Syndrome, Pro136His TWIST Mutation, Hearing Loss, and External and Middle Ear Structural Anomalies: Report on a Brazilian Family

Objective: To describe the clinical, speech, hearing, and imaging findings in three members of a Brazilian family with Saethre-Chotzen syndrome (SCS) who presented some unusual characteristics within the spectrum of the syndrome. Design: Clinical evaluation was performed by a multidisciplinary team. Direct sequencing of the polymerase chain reaction amplified coding region of the TWIST1 gene, routine and electrophysiological hearing evaluation, speech evaluation, and imaging studies through computed tomography (CT) scan and magnetic resonance imaging (MRI) were performed. Results: TWIST1 gene analysis revealed a Pro136His mutation in all patients. Hearing evaluation showed peripherial and mixed hearing loss in two of the patients, one of them with severe unilateral microtia. Computed tomography scan showed structural middle ear anomalies, and MRI showed distortion of the skull contour as well as some of the brain structures. Conclusions: We report a previously undescribed TWIST1 gene mutation in patients with SCS. There is evidence that indicates hearing loss (conductive and mixed) can be related both with middle ear (microtia, high jugular bulb, and enlarged vestibules) as well as with brain stem anomalies. Here we discuss the relationship between the gene mutation and the clinical, imaging, speech, and hearing findings.

Novel mutation in the adiponectin (ADIPOQ) gene is associated with hypoadiponectinaemia in Japanese-Brazilians

P>Objective Adiponectin is an important mediator of insulin sensitivity, encoded by the ADIPOQ gene. Here we describe two Japanese-Brazilian families with hypoadiponectinaemia due to a novel mutation in ADIPOQ. Design and patients In this study, we examined the entire translated regions of adiponectin in Japanese-Brazilians, a population with one of the highest prevalence rates of diabetes worldwide. We screened 200 patients with type 2 diabetes (DM) and 240 age-matched subjects with normal glucose tolerance. Results A novel heterozygous T deletion at position 186 in exon 2 of ADIPOQ, causing a frameshift at codon 62 and leading to a premature termination at codon 168 (p.Gly63ValfsX106), was found in two individuals with diabetes. This mutation was not found in 240 nondiabetic control subjects. In addition, we screened the mutation in an expanded set of 100 nondiabetic subjects from the general Brazilian population, but we found no mutations. In addition, six family members of the probands were identified as mutation-carriers. Individuals who were mutation-carriers had markedly low plasma adiponectin concentrations compared with those without the mutation [DM: 0 center dot 65 (0 center dot 59-1 center dot 34) mu g/ml vs. 5 center dot 30 (3 center dot 10-8 center dot 55) mu g/ml, P < 0 center dot 0001; normal glucose tolerance: 0 center dot 95 (0 center dot 76-1 center dot 48) mu g/ml vs. 8 center dot 50 (5 center dot 52-14 center dot 55) mu g/ml, P = 0 center dot 003]. All individuals carrying the p.Gly63ValfsX106 mutation and older than 30 years were found to be diabetic. Conclusions We describe for the first time a frameshift mutation in exon 2 of the ADIPOQ gene, which modulates adiponectin levels and may contribute to the genetic risk of late-onset diabetes in Japanese-Brazilians.

Relationship between the somatic cell count in raw milk and the casein fractions of UHT milk

The relationship between somatic cell count (SCC) in raw milk and casein fractions of 15 batches of the corresponding ultra-high-temperature (UHT) milk was examined. Raw milk was collected, pasteurised and submitted to UHT treatment. Samples of the UHT milk were taken on days 8, 30, 60, 90 and 120 of storage at room temperature and their casein fractions analysed by high performance liquid chromatography. SCC ranged from 197,000 to 800,000 cells/mL. No correlation (p>0.05) was found between SCC and K-casein concentrations in raw or UHT milks. The alpha(s2) and P-casein concentrations in raw milk were negatively correlated with SCC (p<0.05). In UHT milk, negative correlations were observed for a,1-casein (p<0.05) and beta-casein (p<0.05) on the 8th day, and for alpha S-2-casein (p<0.01) on the 60th day of storage. Results indicate that higher SSC in raw milk is associated with substantial degradation of beta-casein and alpha(s)-casein, which may lead to quality defects in UHT milk during storage. Aust. J. Dairy Technol. 63, 45-49

Loss of Methylation at H19 DMD Is Associated with Biallelic Expression and Reduced Development in Cattle Derived by Somatic Cell Nuclear Transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.

Effect of somatic cell counts on lipolysis, proteolysis and apparent viscosity of UHT milk during storage

In this work, lipolysis, proteolysis and viscosity of ultra-high temperature (UHT) milk containing different somatic cell counts (SCC) were investigated. UHT milks were analysed on days 8, 30, 60, 90 and 120 of storage. Lipolysis as measured by free fatty acids increase, casein degradation and viscosity of UHT milk were not affected by SCC but increased during storage. A negative relationship was observed between SCC and casein as a percentage of true protein on the 120th day of storage, hence indicating that high SCC increases the proteolysis of UHT milk by the end of its shelf life.

Viable Calves Produced by Somatic Cell Nuclear Transfer Using Meiotic-Blocked Oocytes

Somatic cell nuclear transfer (SCNT) has had an enormous impact on our understanding of biology and remains a unique tool for multiplying valuable laboratory and domestic animals. However, the complexity of the procedure and its poor efficiency are factors that limit a wider application of SCNT. In this context, oocyte meiotic arrest is an important option to make SCNT more flexible and increase the number of cloned embryos produced. Herein, we show that the use of butyrolactone I in association with brain-derived neurotrophic factor (BDNF) to arrest the meiotic division for 24 h prior to in vitro maturation provides bovine (Bos indicus) oocytes capable of supporting development of blastocysts and full-term cloned calves at least as efficiently as nonarrested oocytes. Furthermore, the procedure resulted in cloned blastocysts with an 1.5- and twofold increase of POU5F1 and IFNT2 expression, respectively, which are well-known markers of embryonic viability. Mitochondrial DNA (mtDNA) copy number was diminished by prematuration in immature oocytes (718,585 +/- 34,775 vs. 595,579 +/- 31,922, respectively, control and treated groups) but was unchanged in mature oocytes (522,179 +/- 45,617 vs. 498,771 +/- 33,231) and blastocysts (816,627 +/- 40,235 vs. 765,332 +/- 51,104). To our knowledge, this is the first report of cloned offspring born to prematured oocytes, indicating that meiotic arrest could have significant implications for laboratories working with SCNT and in vitro embryo production.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

Variable phenotypic manifestations of a K44N mutation in the TGIF gene

The etiologies and clinical spectra of HPE are extremely heterogeneous. Here, we report a Brazilian boy with lobar holoprosencephaly who was ascertained in a sample of 60 patients with HPE and HPE-like phenotypes and screened for molecular analysis of the major HPE causative genes: SHH, PTCH, SIX3, GLI2, and TGIF This boy presented a p.K44N (c.132G > T) mutation in exon 2 of the TGIF gene which was inherited from his phenotypically normal mother. This mutation leads to lysine to arginine amino acid change and is predicted to be a damaging mutation. Clinical aspects involving variable phenotypical manifestations in different mutations of TGIF are discussed. (c) 2007 Elsevier B.V. All rights reserved.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Changes in soluble carbohydrates and starch amounts during somatic and zygotic embryogenesis of Acca sellowiana (Myrtaceae)

Comparative analysis of zygotic and somatic embryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing minor variation in each embryo stage. Despite the presence of sucrose in the culture medium, its levels conspicuously diminished in somatic embryos compared with the zygotic ones. Raffinose enhanced parallel to embryo development, regardless of its zygotic or somatic origin. Analysis of the soluble carbohydrate composition of mature zygotic cotyledon used as explant pointed out fructose, glucose, myo-inositol, sucrose, and raffinose as the most important. Similar composition was also found in the corresponding somatic cotyledon. Total soluble carbohydrates varied inversely, decreasing in zygotic embryos and increasing in somatic embryos until the 24th d, at which time they increased rapidly about sixfold in zygotic embryos until the 27th d, a period coinciding with the zygotic proembryos formation. Such condition seems to reflect directly the variation of endogenous sucrose level, mainly because glucose and fructose diminished continuously during this time period. This means that, in terms of soluble sugars, zygotic embryo formation occurred under a situation represented by high sucrose amounts, simultaneously with low fructose and glucose levels, while in contrast, somatic embryo formation took place under an endogenous sugar status characterized by a substantial fructose enhancement. Starch levels increased continuously in zygotic embryos and decreased in somatic ones, the reverse to what was found in fructose variation. Starch accumulation was significantly higher in somatic torpedo and cotyledonary embryos than in the corresponding zygotic ones.

Carpenter Syndrome: Extended RAB23 Mutation Spectrum and Analysis of Nonsense-mediated mRNA Decay

Carpenter syndrome, a rare autosomal recessive disorder characterized by a combination of craniosynostosis, polysyndactyly, obesity, and other congenital malformations, is caused by mutations in RAB23, encoding a member of the Rab-family of small GTPases. In 15 out of 16 families previously reported, the disease was caused by homozygosity for truncating mutations, and currently only a single missense mutation has been identified in a compound heterozygote. Here, we describe a further 8 independent families comprising 10 affected individuals with Carpenter syndrome, who were positive for mutations in RAB23. We report the first homozygous missense mutation and in-frame deletion, highlighting key residues for RAB23 function, as well as the first splice-site mutation. Multi-suture craniosynostosis and polysyndactyly have been present in all patients described to date, and abnormal external genitalia have been universal in boys. High birth weight was not evident in the current group of patients, but further evidence for laterality defects is reported. No genotype-phenotype correlations are apparent. We provide experimental evidence that transcripts encoding truncating mutations are subject to nonsense-mediated decay, and that this plays an important role in the pathogenesis of many RAB23 mutations. These observations refine the phenotypic spectrum of Carpenter syndrome and offer new insights into molecular pathogenesis. (C) 2011 Wiley-Liss, Inc.