An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor

A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Monoclonal antibodies specific for oligosaccharides prepared by partial nitrous acid deamination of heparin

Monoclonal antibodies were raised against a conjugate between heparin oligosaccharides and human serum albumin. The oligosaccharides were prepared by partial nitrous acid degradation of heparin and were coupled to human serum albumin by reductive amination. Characterization of the antibodies secreted by one of the resulting clones showed that they recognize a determinant present in the oligosaccharide antigen, but not in intact heparin, nor in a variety of related polysaccharides. Degradation of heparin by nitrous acid generates a 2,5-anhydro-D-mannose residue at the reducing end of the resulting oligosaccharides, and it is concluded that this structure is essential for interaction with the antibodies. Reduced oligosaccharides (containing a terminal anhydromannitol residue) are also active. After gel chromatography of partially degraded heparin, the smallest components capable of binding to the antibodies were found in a tetrasaccharide fraction. Affinity chromatography on immobilized monoclonal antibodies separated this tetrasaccharide fraction into distinct populations of binding and nonbinding species. Structural analysis showed that the tetrasaccharide fraction that bound to the monoclonal antibodies contained one single component with the structure IdoA(2-OSO3)-GlcNSO3 (6-OSO3)-IdoA(2-OSO3)-aManR(6-OSO3), whereas the fraction that did not bind to the antibodies contained a mixture of different structures.     

An experimental evaluation of diversity indices as environmental discriminators

Seven diversity indices were calculated for each of fifty-eight microcosm communities. All fifty-eight communities were initiated from equal density inoculations of fourteen algal species. Each microcosm developed in one of six controlled experimental environments; the environments differing only in their temporal patterns of disturbance. Five linear discriminating techniques were used to evaluate the diversity index most useful for discriminating between these environments. The Shannon-Wiener index was best according to two of the discriminating methodologies and second best using the other techniques. Evenness was best when the Shannon-Wiener index was second best and vice versa.     

MORPHOLOGICAL VARIABILITY IN POPULATIONS OF CHRYSOPHYCEAN RESTING CYSTS. I. GENETIC (INTERCLONAL) AND ENCYSTMENT TEMPERATURE EFFECTS ON MORPHOLOGY1

Many chrysophycean species produce resting cysts (statospores) with purportedly species-specific morphology. I investigated variation in the cyst morphology of a single species that may result from genetic differences among the vegetative clones involved and from variation in the temperature of the environment during cyst development. Populations of Dinobryon cylindricum Imhof cysts were produced under defined conditions in vitro and then sampled for morphological analysis based on SEM micrographs. Morphological data is presented and then used in a multivariate discriminant analysis to determine the utility of each morphological character in distinguishing the six populations studied. Results suggest that some features of cyst morphology (i.e. cyst diameter) are invariant among the populations, while other features show distinctive variation. The density of spines covering the cyst body as well as the morphology of those spines appear correlated to the specific clones involved, and thus may represent useful phenotypic genetic markers. The length and definition of both the spines and the cyst collar, on the other hand, are markedly influenced by encystment temperature. The implications of these findings for paleoecological studies is discussed.     

Isolation and Biochemical Characterization of a Neural Proteoglycan Expressing the L5 Carbohydrate Epitope

The monoclonal L5 antibody reacts with an N-glycosidically linked carbohydrate structure which is present on the neural cell adhesion molecule L1, neural chondroitin sulfate proteoglycans, and other not yet identified glycosylated proteins. Using this antibody, we isolated and characterized proteoglycans from adult mouse brain and cultured astrocytes biosynthetically labeled with Na2 35SO4 and a 3H-amino acid mixture. Our data suggest that the L5 proteoglycans of both sources are identical in their biochemical properties. The apparent molecular mass of the L5 proteoglycan is approximately 500 kDa. Digestion of the iodinated L5 proteoglycan from mouse brain and of the [35S]methionine-labeled L5 proteoglycan from cultured astrocytes with proteinase-free chondroitinases ABC and AC revealed three major core proteins with apparent molecular masses of approximately 380, 360, and 260 kDa. These represent molecularly distinct protein cores.     

Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast.

Metallothionein-directed expression of TGF alpha in transgenic mice induced a spectrum of changes in the growth and differentiation of certain adult tissues. First, TGF alpha promoted a uniform epithelial hyperplasia of several organs without otherwise causing major alterations in tissue architecture. Second, in pancreas it promoted proliferation of both acinar cells and fibroblasts and focally altered acinar cell differentiation. The magnitude of this response was proportional to the level of local, tissue-specific TGF alpha expression and was reproduced when expression of TGF alpha was placed under the control of the elastase promoter, implying an autocrine or paracrine mechanism. Third, TGF alpha was oncogenic in vivo. It caused dramatic hyperplasia and dysplasia of the coagulation gland epithelium, which displayed evidence of carcinoma in situ, and in postlactational mammary gland it induced secretory mammary adenocarcinomas. Thus, TGF alpha displays characteristics of both a potent epithelial cell mitogen and an oncogenic protein in vivo.     

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice.

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.