Functional and morphological characteristics of isolated pancreatic islets in tissue culture

Islets were isolated from the pancreas of female rats by using the collagenase technique. After culturing for 4 days, the islets were taken for measurement of insulin release biosynthesis as well as glucose utilization in subsequent short-time incubations. A low glucose concentration was insufficient to maintain a glucose-stimulated insulin release in vitro. A high glucose concentration had a protecting and restoring effect on the insulin release: ultrastructurally, such islets showed signs of an active biosynthesis in the electron micrograph. The enhancement of Mg++ in the culture medium resulted in an improvement of insulin storage in the islets, accompanied by a well-preserved action of glucose in a subsequent incubation.     

Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/d-glucose cotransport system

To characterize further the Na⁺/d-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of d-glucose transport - has been chemically modified without affecting the d-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na⁺/d-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3′-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent d-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3′-mercuryphlorizin = phlorizin > 3-aminophlorizin > 3-bromoacetamidophlorizin > 3-azidophlorizin > 3-nitrophlorizin > 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na⁺/d-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).     

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI₂ as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to γ raidation results in an irreversible cessation of growth and enhanced production of PGI₂. The level of PGI₂ measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for γ radiation in the elevation of PGI₂ levels which is distinct from its effect on cell division. Result presented indicate that exposure to γ radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI₂ synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.     

Pancreatic grafts. Nuclear perfusion imaging to detect vascular complications and rejection crises

Perfusion studies with 99m-Tc-DTPA were used routinely to investigate renal grafts. Efforts were made to employ this technique in monitoring the perfusion of pancreatic grafts. A total perfusion failure is as reliably detectable as in renal grafts. Smaller perfusion alterations could be demonstrated by follow up studies. It appears to be feasible to differentiate the salivary edema, well known from animal experiments, from a rejection reaction with the help of other parameters (e.g. creatinine). Further clinical studies, however, are necessary to confirm these results.     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Interaction of CD2 with its ligand lymphocyte function-associated antigen-3 induces adenosine 3',5'-cyclic monophosphate production in T lymphocytes.

CD2 (T11, the T cell E receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, is a T cell-specific surface protein that plays an important role in T lymphocyte adhesion, signal transduction, and differentiation. A natural ligand of CD2 is lymphocyte function associated Ag-3 (LFA-3 (CD58)), a widely expressed glycoprotein of 50 to 70 kDa. The physiologic interaction of CD2 with LFA-3 functions to increase intercellular adhesion and plays a role in T cell activation. This interaction, however, in the absence of other stimuli, has not previously been shown to induce intracellular signals such as Ca2+ mobilization or IL-2 production. To investigate whether cAMP may play a role in ligand-triggered CD2-mediated signal transduction, we have studied the ability of purified LFA-3 and anti-CD2 mAb to induce changes in intracellular cAMP content in murine Ag-specific T cell hybridomas that stably express wild-type and mutated human CD2 molecules. By using a RIA sensitive to the femtomolar range and specific for cAMP, we demonstrate that purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3 alone to CD2-expressing hybridoma cells, however, did not stimulate phosphatidylinositol turnover nor IL-2 production. The cytoplasmic domain of CD2 is necessary for these ligand-induced cAMP changes, demonstrating that LFA-3 binding to CD2 transduces a signal to the cell. Experiments using the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine showed that CD2-mediated regulation of cAMP levels occurs primarily by the stimulation of cAMP production rather than by the inhibition of cAMP degradation. These results demonstrate that the interaction of LFA-3 with CD2, in the absence of other stimuli, is capable of initiating intracellular biochemical changes and suggest that CD2/LFA-3 interactions may regulate T cell function at least in part through the generation of intracellular cAMP.     

Tn5099, a xylE promoter probe transposon for Streptomyces spp.

Tn5099, a promoter probe transposon for Streptomyces spp., was constructed by inserting a promoterless xylE gene and a hygromycin resistance gene into IS493. Tn5099 transposed into different sites in the Streptomyces griseofuscus genome, and the xylE reporter gene was expressed in some of the transposition mutants. Strains containing Tn5099 insertions that gave regulated expression of the xylE gene were identified.     

RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.     

Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation.

Previous studies of the genetic and biologic characteristics of human immunodeficiency virus type 1 (HIV-1) have by necessity used tissue culture-derived virus. We recently reported the molecular cloning of four full-length HIV-1 genomes directly from uncultured human brain tissue (Y. Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we describe the biologic properties of these four clones and the complete nucleotide sequences and genome organization of two of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176 nucleotides in length, differed by 0.26% in nucleotide sequence, and except for a frameshift mutation in the pol gene in HIV-1YU-10, contained open reading frames corresponding to 5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long terminal repeats. HIV-1YU-2 was fully replication competent, while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU-32, were defective. All three defective clones, however, when transfected into Cos-1 cells in any pairwise combination, yielded virions that were replication competent and transmissible by cell-free passage. The cellular host range of HIV-1YU-2 was strictly limited to primary T lymphocytes and monocyte-macrophages, a property conferred by its external envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene sequences revealed this virus to be a member of the North American/European HIV-1 subgroup, with specific similarity to other monocyte-tropic viruses in its V3 envelope amino acid sequence. These results indicate that HIV-1 infection of brain is characterized by the persistence of mixtures of fully competent, minimally defective, and more substantially altered viral forms and that complementation among them is readily attainable. In addition, the limited degree of genotypic heterogeneity observed among HIV-1YU and other brain-derived viruses and their preferential tropism for monocyte-macrophages suggest that viral replication within the central nervous system may differ from that within the peripheral lymphoid compartment in significant and clinically important ways. The availability of genetically and biologically well characterized HIV-1 clones from uncultured human tissue should facilitate future studies of virus-cell interactions relevant to viral pathogenesis and drug and vaccine development.     

Isolation by ConA binding of haustoria from different rust fungi and comparison of their surface qualities

Summary Rust haustoria isolated from infected leaf tissue strongly bind to ConA. This property was exploited to purify them by affinity chromatography on a ConA-Sepharose macrobead column. Haustoria were obtained with more than 90% purity and yields of up to 50%. Binding of haustoria to the column was partially inhibited by a ConA-specific sugar, methyl -D-mannopyranoside. Compared to ConA,Lens culinaris agglutinin and wheat germ agglutinin were less efficient affinity ligands. Using ConA-Sepharose, rust haustoria from a variety of sources could be isolated with equal efficiency, indicating that they have similar carbohydrate surface properties. The haustoria maintained their typical shape after the isolation procedure, which suggests a rather rigid wall structure. The morphology of haustoria was characteristic both for a given species and the nuclear condition of the rust mycelium. Electron microscopy of isolated haustoria revealed an intact haustorial wall surrounded by a fibrillar layer presumably derived from the extrahaustorial matrix. The matrix thus appears to represent a layer with gel-like properties which is rich in ConA-binding carbohydrates and connected to the haustorial wall but not to the host-derived extrahaustorial membrane.Abbreviations ConA Concanavalin A - LCA Lens culinaris agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - DAPI 4,6-diamidinophenylindol×2 HCl