Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.     

Thymosin beta 4 induced phenotypic changes in Molt-4 leukemic cell line

Thymosin beta 4 was tested for its ability to induce phenotypic changes in the human T-cell line Molt-4. Cells were cultured with nanogram concentrations of thymosin beta 4 for up to 16 days and were analyzed with a panel of monoclonal antibodies, sheep erythrocyte rosetting, peanut agglutinin binding (PNA) and an antibody to the enzyme, terminal deoxynucleotidyl transferase (TdT). Thymosin beta 4 induced Molt-4 cells to reduce the expression of a T-cell lineage specific antigen, with preferential expression on T blast-cells, detected by WT 1 monoclonal antibody. Thymosin beta 4 also induced an increase in sheep erythrocyte rosettes and PNA binding as well as an increased expression of OKT 11 A and OKT 8 in Molt-4 cells. TdT was found to be unchanged, however. Analysis of thymosin beta 4-treated cells with other monoclonal antibodies (OKT 3, OKT 6, OKT 9) showed no change when compared to controls. These results showed that thymosin beta 4 is capable of inducing phenotypic changes in Molt-4 cells. Such changes may represent a differentiation process of these cells through the early stages of the maturation process of thymus-dependent lymphocytes, albeit not to the stage of mature T cells.     

Microtubules,chromosome movement,and reorientation after chromosomes are detached from the spindle by micromanipulation

The relationship between chromosome movement and mirotubules was explored by combining micromanipulation of living grasshopper spermatocytes with electron microscopy. We detached chromosomes from the spindle and placed them far out in the cytoplasm. Soon, the chromosomes began to move back toward the spindle and the cells were fixed at a chosen moment. The microtubules seen in three-dimensional reconstructions were correlated with the chromosome movement just prior to fixation. Before movement began, detached chromosomes had no kinetochore microtubules or a single one at most. Renewed movement was always accompanied by the reappearance of kinetochore microtubules; a single kinetochore microtubule appeared to suffice. Chromosome movements and kinetochore microtubule arrangements were unusual after reattachment, but their relationship was not: poleward forces, parallel to the kinetochore microtubule axis (as in normal anaphase), would explain the movement, however odd. The initial arrangement of kinetochore microtubules would have led to aberrant chromosome distribution if it persisted, but instead, reorientation to the appropriate arrangement always followed. Observations on living cells permitted us to place in sequence the kinetochore microtubule arrangements seen in fixed cells, revealing the microtubule transformations during reorientation. From the sequence of events we conclude that chromosome movement can cause reorientation to begin and that in the changes which follow, an unstable attachment of kinetochore microtubules to the spindle plays a major role.     

Molecular analysis of human Chromosome 16 cosmid clones containing NotI sites

To test the feasibility of using cloned NotI sites as markers for physical mapping, we have screened for cosmid clones spanning the NotI sites on human Chromosome (Chr) 16. Fluorescence in situ hybridization analysis of these clones confirms the previously reported cluster of NotI sites on 16p13.3. Methylation status of the cloned NotI sites on genomic DNA was established by hybridization of the cosmids to Southern blots containing EcoRI and EcoRI/NotI digest of genomic DNA. These results indicated that four of six clones included in our study can be used as linking clones for physical mapping. Two clones have NotI sites which are not cleavable in the cell lines tested. In one clone, the NotI site exists as an isolated rare-cutting restriction enzyme site, whereas in the other clone the NotI site appears to be island-related.     

Production of a full length Tat protein in E. coli and its purification

A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We then defined a rapid, three-step procedure for the purification of the Tat protein.     

Interaction of cotton tissue culture cells andVerticillium dahliae

Summary Elicitation of sesquiterpenoid aldehyde phytoalexins inGossypium arboreum cell suspension cultures was confirmed by thin layer chromatography, high performance reverse phase liquid chromatography, and an aniline-reaction assay after inoculation with heat-treated conidia ofVerticillium dahliae A 2.3X mean increase in total terpenoids was observed. Component phytoalexins varied, with either hemigossypol and gossypol being detected or the O-methylated terpenoids hemigossypol-6-methyl ether and related compounds. Long-termGossypium suspension cultures were mixoploid with an increase in chromosome number and mean DNA content. Addition ofV. dahliae elicitor(s) to the medium for embryo-proliferating callus ofG. hirsutum inhibited growth and embryo production with a linear correlation (r=−0.87;P<0.01) between the elicitor concentration and the number of embryos. Addition of¹⁴C-labeled NaOAc to suspension cells gave 30% incorporation, and from¹³C-NaOAc addition, labeled sesquiterpenoid aldehydes were recovered. The cotton-Verticillium system is another case of secondary metabolite elicitation in plant tissue culture and might be used for basic studies of hostpathogen interaction as well as for a selection tool to obtain resistance to an important disease.     

Psychiatric assessment of patients with “20th-century disease” (“total allergy syndrome”)

“Twentieth-century disease”, or “total allergy syndrome”, is a condition attributed to hypersensitivity to the environment that may sometimes be seen as so serious that the patient is incapable of living in the modern world. Although the popular media frequently carry stories about it, there is little scientific literature. It is diagnosed by clinical ecologists, who maintain, among other theories, that susceptible individuals experience an overload in assaults by artificial materials in the environment. The patients usually have multiple ill defined symptoms for which no organic cause can be found, but they vigorously resist psychiatric referral, as they attribute their symptoms to allergy. A group of 18 patients who were purportedly suffering from 20th-century disease were referred to a university psychiatric consultation liaison service. They virtually all had a long history of visits to physicians, and their symptoms were characteristic of several well known psychiatric disorders. The case histories and management of three of them are presented. Although this group of patients may have been atypical in that they had more severe psychologic symptoms, the experience indicates that a psychiatric diagnosis ought to be considered. The symptoms of 20th-century disease have much in common with other conditions known to physicians for centuries.     

<Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> mutations and clinical interpretation in 398 ovarian cancer patients: comparison with breast cancer variants in a similar population

Background

Ovarian cancer is the leading cause of death worldwide among gynecologic malignancies. The recent approval of inhibitors of poly (ADP-ribose) polymerase (iPARP) in the treatment of ovarian cancer in the presence of a BRCA1/2 mutation has sparked the analysis of women with such diagnosis, which can further benefit from the detection of carriers in the family. Germline sequence and large rearrangements for BRCA1/2 were tested in 398 consecutive epithelial ovarian cancer (EOC) patients.The aim of this study was to identify the frequency and spectrum of germline BRCA1/2 pathogenic alterations in a cohort of patients with ovarian serous carcinoma, with a view to adequately selecting patients for prevention through family counseling and correlating this frequency with platinum sensitivity as a guidance to identify patients eligible for iPARP in our population.

Results

A total of 96 patients carried a pathogenic germline mutation, accounting for an overall 24.1% mutation incidence. Among mutation carriers, BRCA1 showed 62.5% incidence, BRCA2 rendered 36.5%, and one patient exhibited a mutation in both genes. Three pathogenic mutations were recurrent mutations detected five, three, and four times and represented 12.5% of the mutated samples. Worth highlighting, a 50% mutation incidence was detected when breast and ovarian cancer coexisted in the same patient. Novel mutations amounted to 9.4% of the total mutations, as compared to 4.7% in breast cancer. Forty out of 60 BRCA1 mutations were beyond the ovarian cancer cluster region (OCCR), in stark contrast with 22 out of 36 BRCA2 mutations being inside the OCCR. Taken together, germline BRCA1/2 mutations in EOC patients showed a distinct mutational spectrum compared to our previously published data on breast cancer patients.

Conclusions

In sum, our study provides novel data on ovarian BRCA1/2 mutation prevalence worldwide, enhances adequate patient selection for family counseling and prevention, and sheds light on the benefits of iPARP treatment.