Tubulin and tubulin-colchicine complex bind to brain microsomal membrane in vitro

Brain tubulin was labeled in vitro by post-translational incorporation of [14C]-tyrosine or in vivo by intra-cranial injection of [3H]-leucine. The labeled protein was purified by ion-exchange chromatography. After incubating at 37 degrees C with a microsomal membrane preparation from rat brain, part of the labeled soluble tubulin became sedimentable at high-speed centrifugation. This was independent of the native configuration of tubulin, the state of tyrosination of the COOH-terminus, or the presence of 100 microM colchicine in the mixture. In addition, the double-labeled tubulin-colchicine complex obtained from the binding of [3H]-colchicine to [14C]-tyrosinated tubulin, bound to the membrane preparation to the same extent as [14C]-tyrosinated tubulin. The data show that either tubulin or the complex resulting from its binding to colchicine distributed between the soluble and the membrane fractions when mixed at 37 degrees C with a microsome preparation. Seemingly, the site for colchicine binding to tubulin needs not to be free for the protein-membrane association.     

Production of Reference Enteroviruses

Forty-five human enterovirus reagents of certified purity and quality were prepared for use as seed viruses and as immunizing antigens. One of the reagents was ampouled as "untreated" seed virus, whereas 14 were ampouled as "MgCl(2)-stabilized" reagents. The remaining 30 reagents were ampouled as "untreated" seed viruses and as "MgCl(2)-stabilized" reagents. Thirty of the reagents were propagated on primary African green monkey kidney cells, 3 on primary baboon kidney cells, 3 on primary rhesus monkey kidney cells, and the remaining 9 on human amnion cells. Forty-two of the viral antigens were concentrated for use in the production of high-titered specific antisera in large animals.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

Relations between protein sequence and structure and their significance

The relation between amino acid sequence and local structure in proteins is investigated. The local structures considered are either the four classes of secondary structure (H, E, T and C) or four classes of local conformations defined using measures of conformational similarity based on distances between C alpha atoms. The classes are obtained by applying an automatic clustering procedure to short polypeptide fragments of uniform length from a database of 75 known protein structures. The thrust of our investigation consists of systematically searching the database for simple amino acid patterns of the type Gly-X-Ala-X-X-Val, where X denotes an arbitrary residue. Patterns that are nearly always associated with the same structure are retained. Finding many such associations, we then evaluate by a statistical approach how many among them are non-random and compare the results for different definitions of local structure. A similar comparison is made for the predictive value of retained associations, which is assessed using an internal test based on dividing the database into "learning" and "test" subsets. While we find that local structures defined by conformational similarity are not superior to secondary structure for prediction purposes, they help us gain insight into the factors that influence the predictive value of derived associations. A major conclusion is that the number of retained associations is in large excess over the number expected from a random correlation between sequence and structure, irrespective of how local conformation is defined. However, only a very small number of these associations can be earmarked as reliable using statistical criteria, due to the limited size of the database. We find, for instance, that the pattern Ala-Ala-X-X-Lys reliably characterizes helix, and the pattern Val-X-Val-X-X-X-Ala reliably characterizes extended structure and beta-strand. The possibility is discussed that these and other reliable associations correspond to regions of the polypeptide chain whose conformations are locally determined and that these regions may play a role in folding.     

Reduced productivity in adult yellowfever mosquito (Diptera: Culicidae) populations

Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.     

Molecular characterization by high resolution isoelectric focusing of the products encoded by the class II region loci of the MHC in humans. II. DP alpha and DP beta gene variants

To characterize the molecular polymorphism of the DP alpha and DP beta gene products, the HLA-DP molecules expressed by more than 200 cell lines were individually immunoprecipitated by using the mAb B7/21 and their neuraminidase-treated DP alpha and DP beta chains analyzed in IEF gels. These cell lines, most of them from members of 32 families, allowed the definition, by segregation analysis, of the IEF patterns of the DP polypeptide chains encoded by 129 distinct haplotypes. Both DP alpha and DP beta chains display polymorphic IEF-banding patterns. Two DP alpha (A and B) and seven DP beta (A, B, C, D, E, F, and G) IEF variants were characterized. The DP alpha B variant was found in linkage disequilibrium with both DP beta B and DP beta D. Linkage disequilibrium was also encountered with alleles at the DR and DQ loci. Finally, the correlations between the IEF DP alpha and DP beta variants and the primed lymphocyte test-defined HLA-DP specificities were determined by using a panel of 24 primed lymphocyte test-typed cell lines.