Estrogen-binding properties of rat serum alpha 1-fetoprotein and its isoforms. Investigation of the apparent non-integrality of sites on the unfractionated protein.

Rat fetal serum alpha 1-fetoprotein (AFP), a heterogeneous glycoprotein, binds estrogens with high affinity but at a fractional number of sites even after treatment with charcoal (n = 0.6), which may mean 60% of the protein has 1 site and the remainder none. To investigate the origin of this fractional number of sites the "native" protein (purified by negative affinity chromatography) was further purified (step 1) and fractionated (step 2) into its two main charge variants (electrophoretically "slow" and "fast") by a two-step fast-protein liquid chromatography method. The binding parameters for estrone and estradiol-17 beta of the "native" and "repurified" proteins and of each charge variant were determined by equilibrium microdialysis. The molar extinction coefficient at 278 nm of each sample was also determined. (1) The "repurified" AFP and each charge variant had a number of binding sites for estrogens close to unity. This increase in the number of sites could neither be explained by the loss of a non-binding isoform (corresponding to 40% of the protein) during chromatography, nor by the existence of complex negative modulatory interactions between isoforms. (2) The affinities for estrogens of the "repurified" protein and the two charge variants were slightly decreased compared to that of "native" AFP, except that the "fast" form had the "native" protein's high affinity for estrone--but not for estradiol-17 beta. (3) The molar extinction coefficients at 278 nm of the "repurified" AFP and the isoforms were much lower than that of the "native" protein. These results suggest that the presence of (an) inhibitor(s) of estrogen binding on the "native" protein which is/are removed by the ion-exchange fast protein liquid chromatography (FPLC) column. A ligand absorbing at 278 nm, which may or may not be the inhibitor, is also removed. The isoform heterogeneity with respect to estrone binding is discussed.     

Cellular source of soluble interleukin 2 receptors in serum of mice after recombinant interleukin 2 administration.

Previous studies have shown that peripheral blood mononuclear cells activated in vitro not only express cell-associated interleukin 2 receptors (IL2R) but also release a soluble form of this receptor. In this study, we demonstrate that administration of human recombinant IL 2 (rIL 2) to mice results in increased spleen weights, splenic natural killer (NK) cell cytolytic activity, and serum levels of soluble IL2R. However, compared with rIL 2-treated heterozygote controls, beige mice treated with rIL 2 displayed similar elevations in serum soluble IL2R but significantly less splenic NK activity. Likewise, administration of anti-asialo GM1 antiserum to rIL 2-treated mice resulted in a dramatic reduction in splenic NK cytolytic activity, but no reduction in serum soluble IL2R. Conversely, while rIL 2 treatment of BALB/c mice produced increased splenic NK activity and serum soluble IL2R, similar treatment of BALB/c nude mice resulted in elevation of only splenic NK activity. These studies demonstrate that administration of rIL 2 to normal mice can elevate both serum IL2R levels and splenic NK cytolytic activity. However, the results suggest that T cells are likely to be the source of elevated serum IL2R after rIL 2 administration.     

Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

Low multiplicity infection of insect cells with a recombinant baculovirus: The cell yield concept

In vitro infection of insect cells with baculoviruses is increasingly considered a viable means for the production of biopesticides, recombinant veterinary vaccines, and other recombinant products. Batch fermentation processes traditionally employ intermediate to high multiplicities of infection necessitating two parallel scale-up processes-one for cells and one for virus. In this study, we consider the use of multiplicities of infection as low as 0.0001 plaque-forming units per cell, a virus level low enough to enable infection of even large reactors (e.g., 10 m(3)) directly from a frozen stock. Using low multiplicities in the Sf9/beta-gal-AcNPV system, recombinant protein titers comparable with the maximum titer observed in high multiplicity infections were achieved. Cultures yielding the maximum titer were characterized by reaching a maximum cell density between 3 and 4 x 10(9) cell L(-1). This optimal cell yield did not depend on the multiplicity of infection, supporting the existing view that batch cultures are limited by availability of substrate. Up to a certain cell density, product titer will increase almost linearly with availability of biocatalyst, that is, cells. Beyond this point any further cell formation comes at the expense of final product titer. Low multiplicity infections were found not to cause any significant dispersion of the protein production process. Hence, product stability is not a major issue of concern using low multiplicities of infection. The sensitivity to initial conditions and disturbances, however, remains an issue of concern for the commercial use of low multiplicity infections. (c) 1996 John Wiley & Sons, Inc.     

Characterization of a Kinesin-Related Gene ATSV, within the Tuberous Sclerosis Locus (TSC1) Candidate Region on Chromosome 9Q34

In the search for candidate genes for the tuberous sclerosis (TSC1) disease locus on chromosome 9q34, we have isolated an overlapping series of 22 plasmid and phage cDNA clones covering nearly 7 kb and with an open reading frame of 5070 bp encoding a protein of 1690 amino acids. The putative protein product is a member of the kinesin superfamily and is homologous to the mouse KIF1A and theCaenorhabditas elegansunc-104 genes. Both KIF1A and unc-104 function in the anterograde axonal transport of synaptic vesicles. The human homolog is therefore termed H-ATSV (axonal transporter of synaptic vesicles, HGMW-approved nomenclature ATSV) Screening of DNA from 107 tuberous sclerosis patients and 80 unaffected individuals with H-ATSV cDNA probes by pulsed-field gel electrophoresis/Southern blotting following digestion by rare-cutting methylation-sensitive restriction enzymes showed variant banding patterns in three patients with tuberous sclerosis. However, further analysis indicated that these variant fragments represent a rare polymorphism probably associated with methylation of clustered restriction sites. There is no evidence to support H-ATSV as a candidate gene for TSC1.     

Conformational maturation of measles virus nucleocapsid protein.

We have obtained a polyclonal antiserum, N-BE, against the denatured, amino-terminal half of the measles virus (MV) nucleocapsid (N) protein and a monoclonal antibody (MAb), N46, which recognizes a conformation-dependent epitope in the same region. Amino acid residues 23 to 239 were required and sufficient for the formation of the conformational epitope. Using these antibodies, we show that the N protein of MV is synthesized as a relatively unfolded protein which first appears in the free-protein pool. This nascent N protein undergoes a conformational change into a more folded mature form. This change does not require the participation of other viral proteins or genomic RNA. The mature N protein does not accumulate in the free-protein pool but is quickly and selectively incorporated into the viral nucleocapsids. The mature N protein is a target for interaction with the phosphoprotein (P protein) of MV. This interaction interferes with the recognition of the N protein by the N46 MAb. This suggests that the association with the P protein may mask the binding site for the N46 MAb or that it induces a conformational change in the N protein.     

Identification of Products from Oxidation of Uric Acid Induced by Hydroxyl Radicals

The aim of the present study was to separate and characterise products formed by oxidation of uric acid by hydroxyl radicals with a view to probing for these products in vivo in clinical contexts. Aerated solutions of 200 μM uric acid, or its oxidation products, allantoin or parabanic acid, were exposed to gamma radiolysis, (52.0 Gy/min), as a source of HO- radicals, at pH 3.4 and 7.4. Aliquots were taken every 5 minutes for 20 minutes and oxidation products were separated by HPLC and analysed with a diode array detector. Identities of oxidation products were confirmed on the basis of similarity of retention times and absorbance spectra and peak purity parameters of known standards. Hydroperoxides were measured by tri-iodide formation in the 20 minute sample. Exposure of uric acid to such HO fluxes produced a net loss of the parent compound with formation of a complex mixture of products with allantoin and parabanic acid being the predominant products at pH 3.4. The rate of uric acid degradation at physiological pH was slower and the distribution of oxidation products was different. A small but significant amount of uric acid hydroperoxide was detected at both pHs. A mechanism for uric acid oxidation under these conditions is presented.     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.