Estrogen Induction Of Biotin-Binding Protein In Immature Chicks - Kinetics, Hormonal Specificity And Modulation

Employing a specific radioimmunoassay for quantification, the kinetics of estrogen-induced elevation in the plasma concentration of biotin-binding protein (BBP) in immature male chicks was investigated. A single injection of the steroid hormone enhanced the plasma BBP content several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A 2-fold amplification of the response was evident during secondary stimulation with the hormone. The magnitude of the response was hormonal dose-dependent while the initial lag phase and the time of peak protein accumulation were unaltered within the hormonal doses tested. The circulatory half-life of the specific protein in normal and estrogenized birds was 10 h. Hyperthyroidism markedly decreased the hormonal response while the opposite effect was seen during hypothyroidism. The antiestrogens E- and Z-clomiphene citrate effectively blocked the protein induction whereas progesterone, either alone or in combination with estrogen, was ineffective in modulating the induction. Cycloheximide administration drastically inhibited the inductive response. The above observations clearly suggest that the genes corresponding to the two isofunctional proteins of chicken egg, viz. BBP and avidin, are differentially regulated.

Estrogen modulation of riboflavin carrier protein in the bonnet monkey (Macaca radiata)

Circulatory concentrations of riboflavin carrier protein (RCP) were quantitated in bonnet macaques by employing a heterologous radioimmunoassay involving 125I-labelled chicken RCP and its antiserum. The levels of monkey RCP in the serum seem to be governed by the estrogenic status of the animals. An increase in concentration of serum estradiol in the adult females during the menstrual cycle and early pregnancy could be correlated with enhanced serum RCP levels. Estadiol-17β administered to both immature female and male monkeys, specifically brought about elevated levels of RCP with a slower time course of response in males than in females. These results could be a reflection of a more rapid decline of both circulatory estrogen and RCP concentrations in male serum. Repeated administration of estradiol-17β to male animals led to prolonged elevated levels of RCP following estrogen administration. Thus, it would appear that the evolutionary conservation of RCPs from the aves to the primates encompasses not only their physicochemical similarities but also extends to the estrogenic modulation of their elaboration.

A simple procedure for purification of N-carbamoylputrescine: Application to assays of putrescine transcarbamoylase and agmatine iminohydrolase activities

The observation that N-carbamoylputrescine is quantitatively excluded on O-(carboxymethyl)-cellulose columns with simultaneous retention of putrescine and agmatine has been utilized to develop a sensitive radiometric assay for putrescine transcarbamoylase and a colorimetric assay for agmatine iminohydrolase. A simple procedure for obtaining bulk amounts of pure synthetic N-carbamoylputrescine by separation from putrescine and dicarbamoylputrescine on Dowex 50 (Na+) resin is described.

Purification of Antibodies Specific to a Dinucleotide Using the Hapten Bound to Deae Cellulose as an Affinity Column

The dinucleotide dpTpA held electrostatically on DEAE cellulose was used as an affinity column for the purification of dpTpA specific antibodies. Chromatography of the y-globulin fraction from dpTpA specific antisera on this column resulted in the retention of dpTpA specific antibodies which were later eluted along with the bound dpTpA using 1M NaC1. Dextran coated charcoal was the method of choice for the dissociation and removal of dpTpA bound to the antibodies. This method may extend itself to the purification of antibodies specific for other oligonucleotides.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

Cost effective formulation of vegetable-protein based diets

The project will provide information on the use of phytase in sorghum based diets so that producers and nutrionists will have confidence in vegetable protein based diets.

Palladin, a novel microfilament protein

Palladin is a novel actin microfilament associated protein, which together with myotilin and myopalladin forms a novel cytoskeletal IgC2 domain protein family. Whereas the expression of myotilin and myopalladin is limited mainly to striated muscle, palladin is widely expressed in both epithelial and mesenchymal tissues, including heart and the nervous system. Palladin has a complex genetic structure and it is expressed as several different sized and structured splice variants, which also display differences in their expression pattern and interactions. In muscle cells, all the family members localize to the sarcomeric Z-disc, and in non-muscle cells palladin also localizes to the stress-fiber-dense regions, lamellipodia, podosomes and focal adhesions. A common feature of this protein family is the binding to α-actinin, but other interactions are mostly unique to each member. Palladin has been shown to interact with several proteins, including VASP, profilin, Eps8, LASP-1 and LPP. Its domain structure, lack of enzymatic activity and multiple interactions define it as a molecular scaffolding protein, which links together proteins with different functional modalities into large complexes. Palladin has an important role in cytoskeletal regulation, particularly in stress fiber formation and stabilization. This assumption is supported by several experimental results. First, over-expression of palladin in non-muscle cells results in rapid reorganization of the actin cytoskeleton and formation of thick actin bundles. Second, the knock-down of palladin with anti-sense and siRNA techniques or knock-out by genetic methods leads to defective stress fiber formation. Furthermore, palladin is usually up-regulated in situations requiring a highly organized cytoskeleton, such as differentiation of dendritic cells, trophoblasts and myofibroblasts, and activation of astrocytes during glial scar formation. The protein family members have also direct disease linkages; myotilin missense mutations are the cause of LGMD1A and myofibrillar myopathy. Palladin mutations and polymorphisms, on the other hand, have been linked to hereditary pancreatic cancer and myocardial infarction, respectively. In this study we set out to characterize human palladin. We identified several palladin isoforms, studied their tissue distribution and sub-cellular localization. Four novel interaction partners were identified; ezrin, ArgBP2, SPIN90 and Src-kinase.The previously identified interaction between palladin and α-actinin was also characterized in detail. All the identified new binding partners are actin cytoskeleton associated proteins; ezrin links the plasma membrane to the cytoskeleton, ArgBP2 and SPIN90 localize, among other structures, to the lamellipodia and in cardiomyocytes to the Z-disc. Src is a transforming tyrosine kinase, which besides its role in oncogenesis has also important cytoskeletal associations. We also studied palladin in myofibroblasts, which are specialized cells involved in diverse physiological and pathological processes, such as wound healing and tissue fibrosis. We demonstrated that palladin is up-regulated during the differentiation of myofibroblasts in an isoform specific manner, and that this up-regulation is induced by TGF-β via activation of both the SMAD and MAPK signalling cascades. In summary, the results presented here describe the initial characterization of human palladin and offer a basis for further studies.

p53 and little brother p53/47: linking IRES activities with protein functions

The tumor suppressor p53 represents a paradigm for gene regulation. Its rapid induction in response to DNA damage conditions has been attributed to both increased half-life of p53 protein and also increased translation of p53 mRNA. Recent advances in our understanding of the post-transcriptional regulation of p53 include the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA. These IRES elements regulate the translation of the full length as well as the N-terminally truncated isoform, p53/47. The p53/47 isoform is generated by alternative initiation at an internal AUG codon present within the p53 ORF. The aim of this review is to summarize the role of translational control mechanisms in regulating p53 functions. We discuss here in detail how diverse cellular stress pathways trigger alterations in the cap-dependent and cap-independent translation of p53 mRNA and how changes in the relative expression levels of p53 isoforms result in more differentiated p53 activity.

Use of a volatile buffer at ambient temperature *1: Versatile approach to the purification of self-complementary synthetic deoxyoligonucleotides by reversed-phase high-performance liquid chromatography

Abstract is not available.