Studies of the secondary structures of amelogenin from bovine tooth enamel

Circular dichroism and Fourier transform infrared spectroscopic studies of the major amelogenin protein of developing bovine tooth enamel in solution and in the solid state suggest a unique secondary structure containing beta-sheet and repetitive beta-turn structures. The repetitive beta-turn structure at the C-terminal end results from the unique primary structure of amelogenin.     

Immunosuppression in viral oncogenesis. III. Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness

Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus. We studied the effects of MV infection and MV-associated tumor-induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2. Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E. coli endotoxin. Neither live virus nor TISF alters the production or the responsiveness to IL 1. However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient. Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4. TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2. Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants. Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2. Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed. We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally. Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2.     

Localization of the ganglioside-binding site of fibronectin

It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule.     

The effect of calcium supplements on plasma alkaline phosphatase and urinary hydroxyproline in postmenopausal women

Although calcium supplements are widely used in the treatment of osteoporosis, their beneficial effect is not conclusively established. We now report some effects of a calcium supplement (1 g/day) given for 6 to 12 weeks to 15 postmenopausal osteoporotic women. The mean fasting urinary hydroxyproline/creatinine ratio decreased from 0.021 +/- 0.002 to 0.015 +/- 0.001 (P less than 0.0025), indicating a significant reduction in bone resorption. The mean plasma alkaline phosphatase fell from 123 +/- 5 U/l to 104 +/- 3.1 U/l (P less than 0.01), probably representing some secondary reduction in bone formation following the inhibition of bone resorption. These results support the concept that calcium supplementation is useful in the treatment of postmenopausal osteoporosis.     

Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs.

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions.     

Reassembly of lipid-protein complexes of pulmonary surfactant. Proposed mechanism of interaction

We studied the interaction at 37 degrees C between a major apolipoprotein of pulmonary surfactant and 11 mixtures of lipids. The experiments were carried out in the presence of either 3 mM Ca2+ or 10 mM EDTA. The amount of apolipoprotein associated with lipid was independent of Ca2+. However the binding was sensitive to the percentage of gel-state lipid in the vesicles, and the amount of apolipoprotein in the recombinant lipoprotein complex decreased as the percentage of fully saturated phospholipid was reduced. Maximum association of the apolipoprotein occurred with lipid vesicles containing 85% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 15% 1,2-dipalmitoyl-sn-glycero-3-phospho-1-glycerol or 1,2-dipalmitoyl-sn-glycerol. Fluorescence measurements on the apolipoprotein indicated that the tryptophan side chains were in a relatively hydrophobic environment, and that the wavelength of maximum fluorescence emission was not changed upon the binding of lipid. The results suggest that the principal mode of interaction between the apolipoprotein and lipids of surfactant is hydrophobic bonding. The most extensive binding occurs with lamellar lipids in a gel that would be expected to have inhomogeneities in packing density due to the presence of acidic phospholipids or other glycerolipids. The role of Ca2+ in this interaction has not been fully determined. Although it is not needed to effect the binding of the lipids and the apolipoprotein, it does influence the physical state of the complex, and possibly the stoichiometry of lipid to protein. Some of the processes mediated by Ca2+ in this interaction may be analogous to those observed in membrane fusion. Thus, Ca2+ probably causes segregation of the lamellar phospholipids into domains, inducing vesicular disruption and fusion. This lipid aggregates about hydrophobic sites on the protein, thereby forming high molecular weight reassembly complexes.     

Heat stress and thermal dehydration: lactacidemia and plasma volume regulation.

This investigation was undertaken to study heat stress and dehydration effects on 1) plasma lactic acid (LA) concentration and 2) plasma LA effect on plasma volume conservation during thermal dehydration. Experiments were performed on conscious nonacclimated and heat-acclimated laboratory rats subjected to various levels of heat stress and/or dehydration (37-42 degrees C with and without drinking water). During the exposures, rectal temperature (Tre), plasma LA pyruvic acids, and hematocrit were measured. From these data, excess LA, indicative of anaerobic metabolism, was calculated. In separate experiments, transvascular protein efflux (half time of Evans blue-labeled albumin) was measured before and after plasma LA elevation, either by LA infusion or thermal dehydration. The results show that elevation of plasma LA was associated with a rise in Tre, with accelerated elevation within a Tre range of 41-42 degrees C. LA concentrations were similar for the same Tre in all experimental groups. In nonacclimated rats, this rise was accompanied by a significant rise in excess LA. In acclimated rats, only a minor rise in excess LA was observed. A positive correlation was found between plasma LA elevation and the increase in plasma protein efflux. It is concluded that there is a temperature threshold for the rise in plasma LA. In nonacclimated rats, local hypoxia may contribute to this rise. The data also suggest that, in nonacclimated rats, lactacidemia accelerates plasma protein and fluid loss, leading to circulatory failure during acute thermal dehydration.     

The Location of a Disease-Associated Polymorphism and Genomic Structure of the Human 52-kDa Ro/SSA Locus (SSA1)

Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exons were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race.     

HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.