Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.

Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFR1) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFR1, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.     

Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus.

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.     

H-2RIIBP expressed from a baculovirus vector binds to multiple hormone response elements.

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex class I genes. Based on its homology with Drosophila XR2C/CF1, H-2RIIBP may play a role in development. By using a baculovirus expression system, a large amount of recombinant H-2RIIBP was produced. The recombinant protein accumulated in the nucleus of insect cells. A series of monoclonal antibodies reacting with the recombinant H-2RIIBP was then generated. A DNA-protein immunoprecipitation assay was developed with these antibodies, enabling the DNA-binding specificity of H-2RIIBP to be distinguished from that of an endogenous region II binding factor expressed in uninfected insect cells. We show that H-2RIIBP binds to estrogen response elements with an affinity comparable to that for the region II enhancer. H-2RIIBP also bound to some, but not all, thyroid hormone response elements and retinoic acid response elements, albeit at a lower affinity. Binding to these elements was demonstrated without exogenous addition of a ligand. The H-2RIIBP binding specificity determined by this assay was in agreement with the specificity assessed by Southwestern and gel mobility shift assays. Furthermore, methylation interference assays indicated that H-2RIIBP recognizes the conserved hormone response motif GG(T/A)CA. Taken together, these data demonstrate that H-2RIIBP is capable of binding to hormone response elements of a variety of genes. They suggest that H-2RIIBP may exert a pleiotropic function.     

The effect of Cr (VI) on different inbred lines ofZea mays

Summary Four inbred lines ofZea mays (33.16, B 68, N 7B, B 77) were grown in nutrient solution to which K₂Cr₂O₇ was added to give final concentration of 5 mg/l Cr (VI). The most evident differences in metal tolerance were observed between the B 68 and 33.16 line: in fact, even though the level of Cr (VI) was almost the same in the root tissues of both lines after 6 d of treatment, in the B 68 line, Cr induced marked alterations of nuclear structure and a progressive arrest of the cell cycle in G 1. In the 33.16 line, on the contrary, the integrity of the nuclei was well preserved and the progression of the cell cycle was only barely affected.Abbreviations Cr (VI) hexavalent chromium - CRBC chick red blood cells - DAPI 4,6-diamidino-2-phenylindole - FCM Flow cytometry - FM Fluorescence microscopy - TEM Transmission electron microscopy - Tris 10 mM Tris(hydroxymethyl)aminomethane     

Cytometry and flow cytometry of 4',6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants

Cytometry and flow cytometry were used to study characteristics of fluorescence of the DNA-DAPI complex in nuclei released from different fresh and formaldehyde-fixed pea ( Pisum sativum L. cv. Lincoln) tissues. The two methods of isolation are compared and discussed as well as their possible use for quantitative analysis of DNA in plant tissues. With fixed tissues it is possible to obtain a number of nuclei sufficient for the flow cytometric analysis, even using small amounts of plant tissue.     

A novel immunoassay with direct relevance to protection against organophosphate poisoning

Antiparaoxon immune sera were employed in a new immunoassay based on competition between acetylcholinesterase and antibodies for the binding ofparaoxon. Unlike radioimmunoassay, the new assay described herein can be extended to predict the feasibility of antibodies to confer in vivo protection of acetylcholinesterase against organophosphate poisoning. The toxicity of paraoxon was reduced in mice which were pre-injected with the immune sera.     

Microbiological dehydrogenation of polyfunctional Δ5-3β-acetoxy steroids by Corynebacterium mediolanum strain B-964

Summary A high-yield microbiological transformation of polyfunctional ⁵-3-acetoxy steroids, containing an additional ring E, by Corynebacterium mediolanum strain B-964 was carried out, resulting in the corresponding ⁴-3-ketones. It was shown that the type of transformation and the yield of the reaction depend on the degree of saturation of the ring E and on the position of the oxygen-containing substituents in it.     

Compartmentation of the inulin space in mouse brain slices

(1) Mouse cerebrum slices swell in tris-buffered Krebs-Ringer medium. Swelling is rapid at first, then slows to a more or less constant rate. Even after 3 hr incubation, water content/g of tissue dry wt. shows no sign of an asymptotic limit. Swelling is the same at 37 degrees and at 0 degree. (2) Tissue water measured by incubation with tritiated water is equal to total tissue water measured by drying slices. Equilibration between tritiated water and tissue water is complete within 2 min. (3) Tissue liquid can be divided into three phenomenologically distinguishable compartments: first inulin space, which is the compartment permeable to inulin at both 0 degree and 37 degrees; second inulin space, which is the compartment permeable to inulin at 37 degrees but not at 0 degree; and 37 degrees non-inulin space, which is the compartment impermeable to inulin at both 0 degree and 37 degrees. The evidence for this is: (a) Penetration of inulin into tissue is greater at 37 degrees than at 0 degree. After the first 20 min the rate of penetration at 0 degree is approximately equal to the rate of penetration at 37 degrees, and only slightly less than the rate of increase of total tissue water. Therefore the smaller inulin space observed at 0 degree cannot be due to slower entry of inulin. (b) The inulin content of slices incubated in inulin-containing medium at 37 degrees and cooled to 0 degree in the same medium is the same as the inulin content of tissue incubated at 37 degrees without subsequent cooling. In contrast, the inulin content of tissues preincubated in inulin-free medium at 37 degrees and then incubated in inulin-containing medium at 0 degree is the same as the inulin content of tissues incubated in inulin-containing medium at 0 degree without preincubation at 37 degrees. Therefore the smaller inulin space at 0 degree than at 37 degrees can be due neither to a reversible temperature-dependent change in the size of one single inulin space nor to an irreversible, greater swelling of a single inulin space at the higher temperature, but is due to some portion of the 37 degrees inulin space becoming impermeable to inulin at 0 degree. (c) Some inulin is retained by tissue incubated with inulin at 37 degrees, then transferred to inulin-free medium at 0 degree; the amount of retained inulin is equal to the difference between inulin content of tissue incubated with inulin at 37 degrees and tissue incubated with inulin at 0 degree This confirms 3b above and in addition shows that inulin which has entered the second inulin space at 37 degrees is trapped there when this space becomes impermeable to inulin at 0 degree. (4) The penetration of the amino acids, L-lysine and D-glutamate at 0 degree is equal to the penetration of inulin at 37 degrees. This confirms the real existence of the 37 degrees inulin space at 0 degree, and shows that the barrier at 0 degree between the first and second inulin spaces does not exist for these substances. (5) The amino acids L-leucine and glycine penetrate total tissue water at 0 degree. L-leucine is actively transported at this temperature. (6) The amino acids alpha-aminoisobutyric acid, L-leucine, and L-lysine at 2 mM have no effect at 37 degrees on either the inulin space or the non-inulin space. (7) The inulin space is insensitive at 37 degrees to physiologically significant changes in the medium. In contrast, the non-inulin space is quite sensitive to these changes. Addition of D-glutamate greatly increases the non-inulin space; addition of ouabain or cyanide, or omission of glucose, increases the non-inulin space slightly; and replacement of Na+ ion by choline+ ion greatly decreases this space. These changes are independent and roughly additive.