Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor. Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.


The role of the mature domain of proOmpA in the translocation ATPase reaction. The export of proOmpA, the precursor of outer membrane protein A from Escherichia coli, requires preprotein translocase, which is comprised of SecA, SecY/E, and acidic phospholipids. Previous studies of proOmpA translocation intermediates (Schiebel, E., Driessen, A. J. M., Hartl, F.-U., and Wickner, W. (1991) Cell 64, 927-939) suggested that the "slippage" of the translocating polypeptide chain and the high level of ATP hydrolysis, characteristic of the "translocation ATPase," were part of a futile cycle. To examine the role of the mature domain of proOmpA in its translocation-dependent ATP hydrolysis, we used chemical cleavage to generate NH2-terminal fragments of this preprotein. Each fragment contained the 21-residue leader region and either 53 or 228 residues of the mature domain (preproteins P74 and P249, respectively). As observed with full-length proOmpA, the translocation of each fragment requires ATP and both the SecA and SecY/E domains of translocase and is stimulated by the transmembrane proton electrochemical gradient. The apparent maximal velocities of P74 and proOmpA translocation are similar. While the translocation of P74 and of proOmpA show the same apparent Km for ATP, far less ATP is hydrolyzed during the translocation of P74. Thus, the mature carboxyl-terminal domain of proOmpA has a major role in supporting the translocation ATPase.


Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase. The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.


The primary structure of rat ribosomal protein S5. A ribosomal protein present in the rat genome in a single copy. The amino acid sequence of the rat 40 S ribosomal subunit protein S5 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed by the determination, directly from the protein, of 17 residues near the NH2 terminus. S5 has 204 amino acids; the molecular weight is 22,863. The protein designated S5a has the same amino acid sequence as S5 except that it lacks the NH2-terminal 5 residues. It is not known whether the conversion of a portion of S5 to S5a is physiological or fortuitous. The mRNA for S5 has about 820 nucleotides. Hybridization of the S5 cDNA to digests of nuclear DNA indicates that the rat genome has only a single copy of the gene; this is in distinction to the mouse and human genomes which have three to six copies of the S5 gene. Rat ribosomal protein S5 is related to the eubacteria, the arachaebacteria, and the chloroplast family of S7 ribosomal proteins. There is a peptide of 16 residues at the carboxyl terminus of S5 that is highly conserved in 18 species spanning the three kingdoms and chloroplasts.


A Saccharomyces cerevisiae DNA helicase associated with replication factor C. A novel DNA helicase has been isolated from Saccharomyces cerevisiae. This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC. Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl. The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP. It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM. The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP. The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.


Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity. De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.


Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants. Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.


Probing the phosphocholine-binding site of human C-reactive protein by site-directed mutagenesis. Human C-reactive protein (CRP) can activate the classical pathway of complement and function as an opsonin only when it is complexed to an appropriate ligand. Most known CRP ligands bind to the phosphocholine (PCh)-binding site of the protein. In the present study, we used oligonucleotide-directed site-specific mutagenesis to investigate structural determinants of the PCh-binding site of CRP. Eight mutant recombinant (r) CRP, Y40F; E42Q; Y40F, E42Q; K57Q; R58G; K57Q, R58G; W67K; and K57Q, R58G, W67K were constructed and expressed in COS cells. Wild-type and all mutant rCRP except for the W67K mutants bound to solid-phase PCh-substituted bovine serum albumin (PCh-BSA) with similar apparent avidities. However, W67K rCRP had decreased avidity for PCh-BSA and the triple mutant, K57Q, R58G, W67K, failed to bind PCh-BSA. Inhibition experiments using PCh and dAMP as inhibitors indicated that both Lys-57 and Arg-58 contribute to PCh binding. They also indicated that Trp-67 provides interactions with the choline group. The Y40F and E42Q mutants were found to have increased avidity for fibronectin compared to wild-type rCRP. We conclude that the residues Lys-57, Arg-58, and Trp-67 contribute to the structure of the PCh-binding site of human CRP. Residues Tyr-40 and Glu-42 do not appear to participate in the formation of the PCh-binding site of CRP, however, they may be located in the vicinity of the fibronectin-binding site of CRP.


Purification and characterization of the rat spermatid basic nuclear protein TP4. Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.


Differential regulation of biglycan and decorin by retinoic acid in bovine chondrocytes. The small, leucine-rich proteoglycans, decorin and biglycan, are prominent components of many extracellular matrices and are differentially regulated in various tissues. We have examined the effects of retinoic acid (RA) on the expression of biglycan and decorin at the protein and mRNA levels in cultured bovine articular chondrocytes. Biglycan protein expression is rapidly turned off after 1-2 days of treatment with RA. In contrast, decorin protein expression is increased 12-18-fold following 3 days of RA treatment. The level of biglycan mRNA was also rapidly reduced upon RA treatment, mirroring the protein expression. The reduction was apparent by 6 h, and, by 4 days, the levels were nearly undetectable. In contrast, decorin mRNA was induced upon treatment with RA. The increase in decorin message levels was first apparent by 24 h, reaching maximum by 2 days, and remained constant through 4 days. The repression of biglycan mRNA displayed equal sensitivity to RA concentrations from 10(-5) to 10(-9) M. Decorin mRNA was induced in a dose-dependent fashion by RA. Retinoic acid at a concentration of 10(-5) M, the highest dose examined, resulted in maximal induction of the message, and control levels were obtained with 10(-8) M. The protein synthesis inhibitor cycloheximide inhibited the induction of decorin mRNA, indicating that the induction by RA was a secondary event. In contrast, the repression of biglycan by RA was not significantly altered by cycloheximide, showing that the repression was a direct effect. Actinomycin D inhibited the induction of decorin mRNA, indicating that transcription was required for the induction. Nuclear run-on assays confirmed that RA was regulating biglycan mRNA expression at the transcription level. A 24-h RA treatment decreased the level of transcription of the biglycan gene 5-fold. In contrast, no increase in transcription from the decorin gene could be detected by nuclear run-on assays. Therefore, the elevation in decorin mRNA levels observed after RA treatment was the result of a post-transcriptional event, most likely the consequence of stabilization of the message. This study demonstrates that the genes for these two similar proteoglycans are under very different forms of regulation by RA in chondrocytes. The pattern of differential expression of biglycan and decorin could serve as an additional marker for indicating changes of the cartilage phenotype.


Identification of an upstream regulatory region essential for cell type-specific transcription of the pro-alpha 2(V) collagen gene (COL5A2). The transcriptional features of the human alpha 2(V) collagen gene (COL5A2) were examined by transfection experiments coupled to various DNA binding assays. This approach identified an upstream region essential for the cell type-specific expression of the COL5A2 promoter. Within this region are two nuclear factor-binding sites, FP-A and FP-B, responsible for the formation of distinct DNA-protein complexes. Mutations introduced across each of the two binding sites eliminated the formation of the cognate complex and decreased promoter activity by about 3-fold (FP-A) and 40-fold (FP-B) in transfection experiments. Competition experiments using recognition sequences for known transcription factors exhibiting some similarity to the FP-A- and FP-B-binding sites failed to inhibit COL5A2/protein interactions. Thus, COL5A2 expression appears to be under the positive control of a short regulatory sequence likely to harbor two novel nuclear factor-binding sites.


Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol. Pseudomonas exotoxin (PE) is a three-domain toxin which is cleaved by a cellular protease within cells and then reduced to generate two prominent fragments (Ogata, M., Chaudhary, V. K., Pastan, I., and FitzGerald, D. J. (1990) J. Biol. Chem. 265, 20678-20685). The N-terminal fragment is 28 kDa in size and contains the binding domain. The 37-kDa C-terminal fragment, which translocates to the cytosol, contains the translocation domain and the ADP-ribosylation domain. Cleavage followed by reduction is essential for toxicity since mutant forms of the toxin that cannot be cleaved by cells are nontoxic. Previous results with these mutants suggest that cleavage occurred in an arginine-rich (arginine residues are at positions 274, 276, and 279) disulfide loop near the beginning of the translocation domain, but the exact site of cleavage was not determined. Since very few molecules of the 37-kDa fragment are generated within cells it was not possible to determine the site of cleavage by performing a conventional N-terminal sequence analysis of the 37-kDa fragment. Two experimental approaches were used to overcome this limitation. First, existing amino acids near the cleavage sites were replaced with methionine residues; this was followed by the addition of [35S]methionine-labeled versions of these toxins to cells. The pattern of radioactive toxin fragments recovered from the cells indicated that the toxin was cleaved either just before or just after Arg279. Second, [3H]leucine-labeled toxin was produced and added to the cells. Sequential Edman degradations were performed on the small amount of radioactive 37-kDa fragment that could be recovered from toxin-treated cells. A peak of radioactivity in the fifth fraction indicated that leucine was the 5th amino acid on the C-terminal side of the cleavage site. This result confirmed that cleavage was between Arg279 and Gly280.


Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica. A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.


Interleukin 2-induced activation of Ras requires two domains of interleukin 2 receptor beta subunit, the essential region for growth stimulation and Lck-binding domain. Interleukin 2 (IL-2) can stimulate the proliferation of various kinds of T-cell lines. The receptor for IL-2 is composed of at least two subunits (alpha and beta), of which beta subunit plays the major role in transducing growth signals into the cells. A nonreceptor-type tyrosine kinase, Lck, is associated with IL-2 receptor beta subunit, and the binding of IL-2 to its receptor induces the activation of Lck. On the other hand, it has been shown that stimulation of T-cells with IL-2 causes rapid activation of Ras protein. In this paper, we describe that both of the two regions in IL-2 receptor beta subunit, the indispensable region for the induction of cell growth (serine-rich region) and the binding region of Lck protein (acidic region), are required for the activation of Ras. These two regions are also required for tyrosine phosphorylation of an 85-kDa cellular protein (p85) and the accumulation of fos and jun mRNAs. This observation suggests also that the activation of a receptor-associated tyrosine kinase in response to IL-2-stimulation is primarily responsible for subsequent activation of the pathway through Ras to Fos and Jun.


The Na-K-Cl cotransport protein of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization. The Na-K-Cl cotransporter mediates the coupled transport of Na, K, and Cl across the plasma membrane of many animal cell membranes. It is inhibited by loop diuretics such as furosemide, bumetanide, and benzmetanide. We have developed a panel of monoclonal antibodies directed against the 195-kDa shark rectal gland Na-K-Cl cotransport protein. Four representative antibodies (J3, J4, J7, and J25), each of which recognizes a discrete structural domain, were selected for detailed characterization. When a radiolabeled loop diuretic is bound to the cotransporter prior to solubilization, each antibody immunoprecipitates the same diuretic-protein complex. Of the four antibodies, J4 favors the native protein over the denatured one and does not bind well to proteolytic fragments; in contrast, J7 recognizes the cotransporter only after it has been solubilized. J3, J7, and J25 each recognize a unique ensemble of proteolytic fragments of the 195-kDa protein; analysis of the patterns of recognition has yielded a tentative assignment of the approximate location of the epitopes within the peptide. When the cotransport protein is treated with N-glycanase to remove N-linked oligosaccharides, its apparent mass decreases to approximately 135 kDa. The deglycosylated form is recognized by each of the antibodies except J25; this suggests that the J25 epitope is within the oligosaccharide component or in a peptide domain whose folding is disturbed by carbohydrate removal. An immunoaffinity matrix constructed with the J4 antibody permits single-step purification of the 195-kDa protein; other proteins copurify with the large glycoprotein, but none of these appear to be subunits of a stoichiometric complex. The amino acid sequence of four fragments of the 195-kDa cotransport protein is reported. Immunofluorescence and immunoelectron microscopy demonstrates, in agreement with physiological evidence, that the 195-kDa protein is distributed along the basolateral membrane and excluded from the apical membrane of the rectal gland secretory cell.


Beta gamma-subunit activation of G-protein-regulated phospholipase C. The availability of purified G alpha 11 and the G-protein-regulated phospholipase C from turkey erythrocytes has allowed an examination of the direct effects of G-protein beta gamma-subunit on the components of the inositol lipid signaling system. Reconstitution of purified turkey erythrocyte or bovine brain beta gamma-subunit into phospholipid vesicles containing G alpha 11 inhibited AlF4- induced activation of phospholipase C. However, beta gamma-subunit at higher concentrations increased phospholipase C activity. This stimulatory effect of beta gamma-subunit on phospholipase C did not require the presence of the alpha-subunit. G alpha o had no effect on the catalytic activity of phospholipase C. However, coreconstitution of G alpha o and beta gamma-subunit shifted to the right the concentration-effect curve for beta gamma-subunit-promoted activation of phospholipase C. As was observed with G alpha 11, the increase in activity observed in the presence of beta gamma-subunit occurred as an increase in the maximal activity and with no change in the apparent affinity for Ca2+ for phospholipase C activation. The concentration dependence of G alpha 11 for activation of turkey erythrocyte phospholipase C and bovine brain phospholipase C-beta, as well as the concentration dependence of the two enzymes for activation by G alpha 11, were very similar. In contrast, beta gamma-subunit was a much less effective activator of bovine brain phospholipase C-beta than the turkey erythrocyte enzyme. The observation of direct effects of free beta gamma-subunit on phospholipase C extend the possibilities for receptor-mediated regulation of this signaling pathway.


Mutational analysis of the adenovirus 2 IVa2 initiator and downstream elements. The initiator element of the adenovirus type 2 IVa2 promoter is sufficient to direct accurate initiation by RNA polymerase II. Analysis of the effects of substitution of specific base pairs on initiator activity in in vitro transcription systems indicated that specific sequences between positions -4 and +5 were essential for initiator activity. Mutations that impaired or eliminated initiator activity altered both base pairs that are conserved in sequence-related initiators and nonconserved sequences. Neither the downstream TA-rich sequence of the IVa2 promoter, nor the adenovirus 2 major late TATA element placed at the same downstream site could overcome the severe inhibitory effects of initiator mutations, indicating that the initiator is the primary determinant of the specificity and direction of IVa2 transcription. By contrast, when the ML TATA element was placed 31 nucleotides upstream of the IVa2 initiator, the precise specificity, but neither the efficiency nor direction of transcription, depended on the presence of a functional initiator. Activity of the IVa2 promoter was relatively insensitive to changes in the orientation or nature of the TA-rich sequence. Furthermore, only a promoter containing the ML TA-TAAAA sequence downstream of the IVa2 initiator was competent to direct both IVa2 transcription and transcription from the opposite strand. The implications of this functional difference for recognition of the downstream element are discussed.


High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation. The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.


Polypyrimidine tract binding protein interacts with sequences involved in alternative splicing of beta-tropomyosin pre-mRNA. Previous studies of alternative splicing of the rat beta-tropomyosin gene have shown that nonmuscle cells contain factors that block the use of the skeletal muscle exon 7 (Guo, W., Mulligan, G. J., Wormsley, S., and Helfman, D. M. (1991) Genes & Dev. 5, 2095-2106). Using an RNA mobility-shift assay we have identified factors in HeLa cell nuclear extracts that specifically interact with sequences responsible for exon blockage. Here we present the purification to apparent homogeneity of a protein that exhibits these sequence specific RNA binding properties. This protein is identical to the polypyrimidine tract binding protein (PTB) which other studies have suggested is involved in the recognition and efficient use of 3'-splice sites. PTB binds to two distinct functional elements within intron 6 of the beta-tropomyosin pre-mRNA: 1) the polypyrimidine tract sequences required for the use of branch points associated with the splicing of exon 7, and 2) the intron regulatory element that is involved in the repression of exon 7. Our results demonstrate that the sequence requirements for PTB binding are different than previously reported and shows that PTB binding cannot be predicted solely on the basis of pyrimidine content. In addition, PTB fails to bind stably to sequences within intron 5 and intron 7 of beta-TM pre-mRNA, yet forms a stable complex with sequences in intron 6, which is not normally spliced in HeLa cells in vitro and in vivo. The nature of the interactions of PTB within this regulated intron reveals several new details about the binding specificity of PTB and suggests that PTB does not function exclusively in a positive manner in the recognition and use of 3'-splice sites.


Genomic organization of the mouse granzyme A gene. Two mRNAs encode the same mature granzyme A with different leader peptides. Granzyme A is a serine protease that, together with the other granular components of cytotoxic T lymphocyte (CTL) cells, has been implicated in the cytolysis process. We report here two different messages and the genomic organization of the mouse granzyme A gene. The granzyme A gene is composed of six exons spanning 7 kilobases. Alternative splicing of the second exon results in the two transcripts. The two mRNA species encode the same mature granzyme A protein but with different leader sequences. The first (HF1) encodes a typical leader signal sequence similar to other granzymes, but the second (HF2) putative leader sequence is different and less hydrophobic. Both messages are present in cultured CTL cell lines and in normal lymphoid tissues. They are both induced when CTL cells are activated in vitro or in vivo. Both messages can be translated in vitro, although the HF1 message appears to be much more efficient as a template. The putative 5' promoter region of the HF gene sequenced (500 base pairs of upstream sequences) contains no well defined promoter sequences aside from the TATA box. The results suggest that (a) granzyme A may be produced with putative different leader sequences from two different mRNAs; (b) this may provide a model system for studying alternate splicing and the evolution of a complex enzymatic system in an organelle; and (c) the genomic DNA reported will be useful for studying transcription regulations involved in controlling the specific expression pattern of this gene.


Impact of O-glycosylation on the function of human intestinal lactase-phlorizin hydrolase. Characterization of glycoforms varying in enzyme activity and localization of O-glycoside addition. Lactase-phlorizin hydrolase (LPH) is an integral intestinal brush border membrane glycoprotein responsible for the hydrolysis of lactose, the primary carbohydrate in mammalian milk. To assess the role of N- and O-glycosylation on the function of LPH, lectin-binding experiments combined with enzymatic and chemical deglycosylation of purified LPH molecules were performed. These investigations provided evidence for the existence of two forms of brush border LPH, an N-glycosylated molecule (LPHN) and an N- and O-glycosylated molecule (LPHN/O). These two forms could be discriminated on the basis of (i) their binding capacity to Helix pomatia lectin, which has high specificity toward O-linked oligosaccharides, and (ii) their deglycosylation patterns with endo-beta-N-acetylglucosaminidase F/GF, O-glycanase, and trifluoromethanesulfonic acid. Interestingly, both forms have identical Km values (approximately 14 mM) when assayed with lactose, but hydrolyze this substrate at different rates. Thus, the N- and O-glycosylated form exhibits almost a 4-fold higher Vmax than that of the N-glycosylated enzyme (3.28 nM/min versus 0.90 nM/min) and is therefore enzymatically more active than the latter. Sequential affinity chromatography of glycopeptides derived from [3H]mannose-labeled LPHN and LPHN/O on lectin columns revealed similar patterns of N-linked glycosylation of both forms indicating that the presence of O-linked oligosaccharides did not affect or alter the processing of N-linked oligosaccharides. O-Linked glycosylation of LPH appears to occur in the Golgi apparatus, since the earliest detectable forms of LPH, the mannose-rich precursor (pro-LPH) is not O-glycosylated. In view of the fact that differentiation of intestinal crypt cells to mature epithelial cells is accompanied by significant phenotypical, morphological, and structural alterations, including changes in the levels of several Golgi glycosyl, -sialyl, galactosyl-, and N-acetylgalactosaminyltransferases, and since O-glycosylation is a Golgi event, we suggest that the generation of LPHN and LPHN/O is strongly linked to differentiation of intestinal cells. Finally, the variations in the enzymatic activity of the two forms propose a role for O-glycosylation in posttranslational regulation of LPH activity.


Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.


Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule. Type I procollagen was purified from the medium of dermal fibroblasts cultured from four individuals with osteogenesis imperfecta (OI) type II who had mutations in the COL1A1 gene of type I procollagen. The procollagens were mixtures of normal molecules and molecules that contained substitutions of aspartate for glycine 97, arginine for glycine 550, cysteine for glycine 718, and aspartate for glycine 883 in one or both of the alpha 1 (I) chains of the molecule. The procollagens were cleaved more slowly than control type I procollagen by procollagen N-proteinase. Double-reciprocal plots of initial relative velocities and initial substrate concentrations indicated that the OI procollagens were all cleaved slowly by N-proteinase because of decreased Vmax, rather than increased Km. This suggested that slow cleavage of the OI procollagens by N-proteinase was the result of slow conversion of the N-proteinase-procollagen complex. Further experiments showed that the vertebrate collagenase A fragment of the aspartate for glycine alpha 1(I) 883 OI procollagen that contained the N-proteinase cleavage site but not the site of the substitution was also cleaved more slowly by N-proteinase than the normal vertebrate collagenase A fragments in the samples. These data show, for the first time, that an altered triple-helical structure is propagated from the site of a substitution of a bulky residue for glycine to the amino-terminal end of the procollagen molecule and disrupts the conformation of the N-proteinase cleavage site. Rotary shadowing electron microscopy of molecules in the preparation of cysteine for glycine alpha 1(I)-718 showed the presence of a kink in approximately 5% of a population of molecules in which 60% were abnormal and 20% contained a disulfide bond. In contrast, procollagens containing aspartate and arginine for glycine were indistinguishable by rotary shadowing electron microscopy from those in control samples. The results here confirm previous suggestions that substitution of cysteine for glycine in the alpha 1(I) chain of type I collagen can introduce a kink near the site of the substitution. However, the presence of a kink is not a prerequisite for delayed cleavage of abnormal procollagens by N-proteinase.


A tripeptide deletion in the triple-helical domain of the pro alpha 1(I) chain of type I procollagen in a patient with lethal osteogenesis imperfecta does not alter cleavage of the molecule by N-proteinase. Dermal fibroblasts from a fetus with perinatal lethal osteogenesis imperfecta synthesized normal and abnormal type I procollagen molecules. The abnormal molecules contained one or two pro alpha 1(I) chains in which glycine, alanine, and hydroxyproline at positions 874, 875, and 876 in the triple-helical region were deleted as the result of a 9-base pair genomic deletion. Molecules that contained abnormal chains were overmodified from the site of the deletion toward the amino-terminal region of the molecule. Secretion of the overmodified molecules was impaired. The thermal stability of molecules containing abnormal chains was lower than that of normally modified molecules. After cleavage of molecules with vertebrate collagenase, the temperature of thermal denaturation of the overmodified A fragments was greater than that of the fragments from the normal molecules. The rates of cleavage of the normal and the abnormal molecules by N-proteinase were indistinguishable. Our findings suggest that the tripeptide deletion introduces a shift in the phase of the chains in the triple helix. This structural change is propagated from the site of the deletion toward the amino terminus of the molecule, but the subsequent alteration in the structure of the N-proteinase cleavage site is not sufficient to cause a decrease in the rate of cleavage by the enzyme.


Positive regulation of the skeletal alpha-actin gene by Fos and Jun in cardiac myocytes. Transcription of the skeletal alpha-actin gene is selectively activated in rat myocardiocytes undergoing hypertrophy both in vivo and in vitro. In most of these models, transient expression of certain proto-oncogene transcription factors precedes hypertrophy and sarcomeric gene induction. Using expression vectors encoding Fos and Jun, the main constituents of transcriptional activator protein AP-1, we analyzed the role of these oncoproteins in mediating the transcriptional induction of skeletal alpha-actin by adrenergic stimulation. Both c-fos and c-jun were induced early after beta-adrenergic stimulation, with peak mRNA levels preceding skeletal alpha-actin induction by several hours. A second peak of c-jun mRNA coincided with skeletal alpha-actin induction. Co-transfection assays in cardiac myocytes and P19 teratocarcinoma cells demonstrated that over-expression of c-jun, or c-fos plus c-jun, transactivated the skeletal alpha-actin promoter by about 5-fold. Comparable activation was not seen for alpha-myosin heavy chain or cardiac alpha-actin promoters. Skeletal alpha-actin promoter sequences between -153 and -36 were required for maximal transactivation by c-fos/c-jun, and purified Fos and Jun were bound specifically within this region. A direct physiological role is suggested for the AP-1 transcription factor complex in regulating skeletal alpha-actin gene expression and alpha-actin isoform switching during the onset of signal-mediated cardiac myocyte hypertrophy.


Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli. Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).


Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).


The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase. X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.


In situ imaging of agonist-sensitive calcium pools in AR4-2J pancreatoma cells. Evidence for an agonist- and inositol 1,4,5-trisphosphate-sensitive calcium pool in or closely associated with the nuclear envelope. The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.


Protein LG: a hybrid molecule with unique immunoglobulin binding properties. Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.


Human interferon consensus sequence binding protein is a negative regulator of enhancer elements common to interferon-inducible genes. The promoter regions of many interferon-inducible genes share a short DNA sequence motif, termed the interferon consensus sequence (ICS) to which several regulatory proteins bind. A murine cDNA which encodes an ICS binding protein has been reported (M-ICSBP). The cloning of the human homologue of ICSBP (H-ICSBP) is described. H-ICSBP shares high sequence homology with its murine cognate. The derived sequence of H-ICSBP reveals restricted homology within the first 120 amino acids to three other interferon regulatory factors, IRF-1, IRF-2, and ISGF3 gamma. Truncated ICSBP lacking the first 33 amino-terminal amino acids fails to bind to the ICS, indicating that at least part of the DNA binding domain is located within the well conserved amino terminus. H-ICSBP is expressed exclusively in cell lines of hematopoietic origin. The results of transient transfection assays carried out either in hematopoietic or nonhematopoietic cells suggest that ICSBP acts as a negative regulatory factor on ICS-containing promoters. Furthermore, either interferon-gamma (IFN-gamma) or IFN-beta can alleviate the repression mediated by ICSBP. Therefore, ICSBP may be involved in maintaining submaximal transcriptional activity of IFN-inducible genes in hematopoietic cells. IFN treatment would then alleviate repression allowing maximal transcriptional activity of these genes.


DNA binds and activates complement via residues 14-26 of the human C1q A chain. The mechanism by which DNA activates the classical complement pathway was investigated, with emphasis upon the C1q binding sites involved. DNA bound to both the collagen-like and globular regions of C1q. Binding reactivity with DNA was retained after reduction/alkylation and sodium dodecyl sulfate treatment of C1q. DNA bound preferentially to the A chain of C1q. Binding sites for DNA were localized by using synthetic C1q A chain peptides to two cationic regions within residues 14-26 and 76-92, respectively. Peptides 14-26 and 76-92 avidly bound DNA in enzyme-linked immunosorbent and gel shift assays. Peptide 14-26 also precipitated with DNA and blocked its ability to bind C1q and activate C. Replacement of the two prolines with alanines or scrambling the order of the amino acids resulted in loss of ability of peptide 14-26 to inhibit C1q binding and complement activation by DNA; similar investigations showed a sequence specificity for peptide 76-92 as well. These experiments identify C1q A chain residues 14-26 as the major site, and residues 76-92 as a secondary site, through which DNA binds C1q and activates the classical complement pathway, and demonstrate that a peptide identical to residues 14-26 can modulate C1q binding and complement activation by DNA.


Sequence and transcriptional analysis of the Escherichia coli rnt gene encoding RNase T. The Escherichia coli rnt gene encoding the enzyme RNase T, which is responsible for the end-turnover of tRNA, was cloned on a 1.5-kilobase DNA fragment. When placed in pUC18 and pUC19 vectors this fragment led to approximately a 40-fold overexpression of RNase T activity. The cloned fragment was sequenced and was found to contain an open reading frame sufficient to encode a protein of 215 amino acids with a molecular weight of 23,521, which is close to the subunit molecular weight of RNase T; the fragment also contains a second incomplete open reading frame with some sequence similarity to RNA helicases. The derived sequence of RNase T showed no similarity to any of the other E. coli exoribonucleases sequenced to date. Primer extension analysis and deletion of part of the upstream region were used to identify the transcription start point and the promoter of the rnt gene. Northern and primer extension analysis revealed that the rnt message also included the second open reading frame, suggesting that rnt is part of an operon.


Functional analysis of the human platelet-derived growth factor A-chain promoter region. The platelet-derived growth factor (PDGF) A-chain gene is a developmentally regulated gene that is expressed in high levels in a limited number of normal and transformed cell lines and in cells stimulated by cytokines, including PDGF itself. We have now analyzed potential regulatory elements in 3.6 kilobase pairs (kb) of the 5'-flanking sequences of the human PDGF A-chain gene using reporter gene constructs and transient transfection analyses. The region between base pairs (bp) -618 and +392 (relative to the transcription initiation site) is sufficient for optimal promoter activity. A highly G + C region containing three contiguous Sp1 binding sites between bp -150 and -33 contributes over 80% of promotor activity. DNase I footprinting analyses indicates that Sp1 binds to and protects over 57 bp of this G + C region. A functional serum response element is located within bp -477 and -468 and positively regulates induction of PDGF A by PDGF. A negative regulatory (silencer) element is located from -1.9 to -0.9 kb. The results suggest that the major constitutive expression of the PDGF A-chain gene requires a highly G + C-rich region containing three Sp1 binding sites and that induction of the PDGF A-chain gene by PDGF is mediated by a SRE located at bp -477 to -468.


Wrist anatomy.

Wrist anatomy. The multiple joints within the wrist form a complex structure capable of transmitting significant loads to the upper extremity while providing a mobile base for the precision movements of the hand. This duality is accomplished through a fine interplay between the carpal bones and an arrangement of highly specific ligamentous structures, a system loaded by balanced muscle forces.


Ligamentous injuries to the wrist. Imaging techniques. The variety of imaging techniques available to the clinician facilitates the diagnosis of acute and chronic wrist disorders. In most cases, conventional radiographs provide sufficient information. Computed and trispiral tomography, arthrography, video-fluoroscopy, and magnetic resonance imaging are additional imaging techniques that are useful in making the correct diagnosis and deciding on a course of treatment.


Wrist arthroscopy.

Wrist arthroscopy. Wrist arthroscopy has become a routine method of evaluating and treating wrist disorders. Technical advances and improved techniques have resulted in therapeutic modalities with a much lower morbidity rate. Specific indications for wrist arthroscopy are described, as well as techniques and precautions.


The distal radioulnar joint.

The distal radioulnar joint. A phylogenic review shows the DRUJ to be a highly evolved and specialized structure. The diarthrodial joint, in combination with the TFCC, ligament complex, and oblique fibers of the distal interosseous membrane, permit rotation while preserving stability and transmitting the load borne by the radiocarpal joint to both bones of the forearm. Our improved, yet imperfect, understanding of the anatomy, biomechanics, and pathophysiology of the region, abetted by new imaging modalities and wrist arthroscopy, permit more precise diagnosis and thus more rational treatment. The persistent inconsistency of our results belies our imperfect comprehension. Thus humbled, consider Goethe's admonition as a call to further scrutiny and investigation, "Theory and experience are opposed to each other in constant conflict. Only action can reconcile them."


Treatment of scapholunate dissociation. Rotatory subluxation of the scaphoid. The treatment of scapholunate dissociation remains controversial. Although good to excellent results have been reported using a variety of surgical techniques, they have often not been confirmed by other authors. Limited wrist mobility and the likelihood that degenerative changes will progress appear to be inherent in most approaches. The ideal treatment is to restore the normal anatomy and, with it, the function of the wrist. Destroying a normal joint by even a limited fusion as a means to provide stability to an abnormal joint is counter to our usual approach to disease. It is for that reason that more reliable soft-tissue reconstructive procedures are necessary. Those interested in arthroscopy are pursuing endoscopic methods of repair, but, at present, little progress is apparent. The physician who solves this problem with an uncomplicated and reliable surgical solution will have indebted us all.


Lunate-triquetral and midcarpal joint instability. Instability of the ulnar side of carpus centers around the triquetrum, which is suspended by the ulnar triquetral ligaments and supported proximally by the TFCC. The triquetrum guides the lunate by an interosseous membrane and stout palmar ligaments that provide a relatively rigid connection between the two bones. Disruption of the LT ligament is frequently associated with pathology in the ulnar carpal area and may progress to triquetral instability, VISI, and finally, degenerative arthritic changes on the ulnar side of the carpus. The diagnosis of LT injuries is made by stress radiographs, arthrography, video-fluoroscopy, and arthroscopy. Treatment is initially nonoperative, but if symptoms persist, surgery is warranted. Arthroscopic debridement and pinning the LT joint, ligament repair or reconstruction, and intercarpal arthrodesis have all been reported as successful treatments. For the chronic problem confined to the LT joint, a limited intercarpal arthrodesis of the joint is the most predictable procedure for relieving pain without causing any significant restrictions in wrist motions. When there is a dissociation pattern in addition to LT instability, a more extensive intercarpal arthrodesis is required. Midcarpal instability occurs at the triquetral-hamate joint and is characterized by a dynamic subluxation of the joint. During ulnar deviation, the joint undergoes an exaggerated shift from volar flexion to dorsiflexion. Supportive care is generally successful; although in chronic cases, a midcarpal joint arthrodesis is often required.


Rehabilitation techniques for ligament injuries of the wrist. The goal of treatment after any wrist injury is a pain-free, stable joint with sufficient strength and mobility to carry out the daily recreational, and occupational tasks required by the individual. Treatment varies considerably depending on the age of the patient, the severity of the initial injury, the operative procedure performed, and the specific guidelines requested by the referring physician.


Anatomy of the joints of the thumb.

Anatomy of the joints of the thumb. The three joints of the thumb differ markedly with respect to anatomy and function. Each contributes its own anatomic personality, and when functioning together, they allow the thumb to move with remarkable versatility and grace yet with the stability necessary to perform a wide variety of tasks. The carpometacarpal and interphalangeal joints allow for thumb mobility while the metacarpophalangeal joint provides stability. Thumb rotation primarily takes place at the CMC joint, although the MP and IP joints each contribute a small amount of pronation with flexion. Although much is known about the static structural anatomy of the thumb, its dynamic anatomy requires further investigation. The fluid, elegant and powerful movements of the thumb are complex, and defining the contributions of the various individual structures is difficult. Hopefully, future investigators using newer techniques will unravel the complexities of the functional anatomy of this remarkable digit.


Anatomy of the joints of the fingers.

Anatomy of the joints of the fingers. The anatomy and function of the three joints within each finger are mutually complementary; none functions independently of the other two. A complex, yet precise, intrinsic and extrinsic soft-tissue support links these three joints and provides a precisely articulated balance for the mobility of the finger. This complex interrelationship requires both strength and stability, which are inherent in the design of the bony architecture and their surrounding soft tissues. Understanding this interrelationship is essential for the accurate diagnosis and treatment of the numerous problems with which these joints are sometimes afflicted.


Trapeziometacarpal joint injuries.

Trapeziometacarpal joint injuries. This article begins with discussions of anatomy and thumb movement. Palmar (anterior) ligament reconstruction is then discussed. Peritrapezial arthritis is also discussed.


Metacarpophalangeal joint injuries of the thumb. Dorsal capsular injuries, volar plate injuries, ulnar collateral ligament injuries, and radial collateral ligament injuries are all discussed in this article. Treatment is both discussed and illustrated for ease of comprehension.


Carpometacarpal joint injuries of the fingers. Injuries to the carpometacarpal joints are uncommon and are sometimes overlooked on initial radiographs. Their recognition depends on a careful physical and radiographic examination that may require trispiral tomograms and CT scans. Most injuries can be treated successfully by closed reduction and percutaneous fixation of the joint(s) using Kirschner wires. Chronic joint injuries can also be effectively treated, and if surgery is required, an arthrodesis will restore stability and eliminate discomfort.


Metacarpophalangeal joint injuries in fingers. Although uncommon, injuries to the MP joints of fingers must not be ignored. A loss of function in even a single MP joint can seriously impair overall hand function. The potential dire consequences of these injuries can be avoided by an accurate diagnosis at the time of injury and the institution of appropriate treatment.


Capsular injuries of the proximal interphalangeal joint. Although capsular injuries of the PIP joints are common, their management is frequently complicated. Successful treatment must begin with a detailed history because reviewing the mechanism of injury may provide information relevant to the pathomechanics of the capsular disruption and facilitate making an accurate diagnosis. Grades I and II volar plate and collateral ligament sprains represent the vast majority of PIP joint injuries. They are best treated with a short period of dorsal splinting followed by supervised mobilization. Although splinting is also applicable for grade II sprains associated with instability and most grade III sprains, the initial period of immobilization should be longer. The prognosis for recovery is generally good, although some residual tenderness or joint stiffness are common complications. Dorsal capsular injuries, if unrecognized, result in deformity rather than instability. The majority of these injuries can also be treated by closed means, but they require more prolonged immobilization and more commonly result in reduced mobility than volar plate and collateral ligament injuries. Capsular injuries that are compound, irreducible, or associated with a large intraarticular fracture can result in serious problems. Frequently, these injuries require primary surgical treatment, particularly in the case of the irreducible dislocation, which always requires surgery. An exception to the generally poor prognosis of these injuries is the irreducible volar dislocation because the central tendon remains intact permitting early postoperative joint mobilization. A chronic dislocation or late instability are fortunately not common sequela of capsular injuries; however, when they do occur, surgery is required.


Distal joint injuries of the thumb and fingers. The DIP joints of fingers and the interphalangeal joints of thumbs provide important stability and contribute to the flexion and extension arc of the digit. A variety of injuries can affect these joints, involving their ligaments, bones, and tendons. Instituting proper and effective treatment is predicated on making an accurate diagnosis of the injury and understanding the function of each anatomic structure.


Contractures of the proximal interphalangeal joint. Posttraumatic stiffness of proximal interphalangeal joints remains a difficult problem that severely compromises hand function. The emphasis of treatment must be on preventing the problem from developing, which begins within hours of the injury. Effective treatment involves a careful evaluation of the injury and then instituting a rational course of therapy. When surgery is necessary, the operative technique must be precise, and it requires a thorough understanding of the anatomic and pathomechanical factors that cause the contracture. Effective postoperative therapy and rehabilitation are integral parts of any surgical procedure, and they usually continue for many months.


The boutonniere deformity.

The boutonniere deformity. Understanding the pathophysiology of the boutonniere deformity requires a complete understanding of the anatomy of the dorsal tendon apparatus. This unique tendon mechanism often becomes unbalanced, requiring correction of its components. Splinting is the cornerstone of treatment for the boutonniere deformity. In the acute stage, splinting ensures that the continuity of the central tendon to its insertion into the middle phalanx is maintained, and in the chronic stage, its function is to correct the flexion contracture of the PIP joint and stretch the retinacular ligaments. Splinting is also important postoperatively because it permits healing of the central tendon and lateral bands in their correct anatomic positions. Without proper splinting, the patient with the boutonniere deformity could not be successfully treated. Frequently, surgery is necessary, and the choice of procedure depends on the stage of the condition and the extent of the defect in the extensor tendon mechanism. The procedure also depends on the success of the splinting program and stretching of the tight retinacular structures. If passive joint mobility can be restored and if tendon imbalance and retinacular tightness persist, rebalancing is necessary. This rebalancing can be accomplished by a tenotomy of the terminal extensor tendon, a lysis or release of the retinacular structures, or release of the insertion of the extensor tendon at the base of the proximal phalanx. Reconstituting the defect in the central tendon over the PIP joint is accomplished by using a variety of procedures, including mobilization and advancement of the more proximal portion of the central tendon, shifting the lateral bands, or a tendon graft.


Rehabilitation techniques for ligament injuries of the hand. The contributions of the hand therapist to the treatment of ligament injuries in the hand requires a combination of skills. A thorough knowledge of the pathomechanics of these injuries is a necessary prerequisite in the initial evaluation and treatment planning stage. Focus should never be entirely on the injury; it is also important to understand the emotional effect that the injury has had on the patient's work and leisure activities. Therapy programs usually require edema control and reduction and a variety of static and dynamic splints. The ultimate goal is returning the patient to fully functional activities.


High-performance liquid chromatographic separation of purine deoxyribonucleoside monophosphate-benzo[a]pyrene adducts. Chromatographic methods that allow the separation of adducts of purine nucleoside 3'-phosphates with the pure enantiomers of the anti-dihydrodiol epoxide of benzo[a]pyrene are developed. The optimization procedure includes evaluation of the effect of buffer molarity, the pH of the buffer, and the role of organic modifiers. The method can be utilized to prepare standards with known absolute configuration that can be further used in the Randerath 32P-postlabeling procedure.


Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems. The tiny injection volumes that are usually necessary to maintain the high efficiency of capillary zone electrophoresis present a major problem if only limited sample amounts are available. To increase the sample load, discontinuous buffer systems were developed that allow the on-column concentration of dilute samples. Injection volumes can be increased in this way by at least a factor of 30. These stacking systems were applied to the analysis of tryptic peptides, to the purity checking of high-performance liquid chromatographic fractions and for the micropreparative separation of peptides with subsequent amino acid sequence analysis.


Effects of 2,3-butanedione monoxime on the crossbridge kinetics in frog single muscle fibres. The effects of 2,3-butanedione monoxime (BDM) on contraction characteristics were studied at 5 degrees C in single intact fibres isolated from the tibialis anterior muscle of the frog. The force-velocity relation was determined using the controlled-velocity method in either whole fibres or short fibre segments in which sarcomere shortening was measured by a laser light diffraction method. It is shown that 3 mM BDM decreases the speed of rise and the amount of tetanus tension, reduces the maximum velocity of shortening and increases the curvature of the force-velocity relation, as well as the value for the stiffness to tension ratio. BDM also slowed down the redevelopment of tetanus tension after a period of unloaded shortening both in fixed-end and in length-clamp conditions. In normal and in BDM-treated fibres length-clamping increased the speed of the initial rise of tetanus tension but not that of the recovery after shortening. The observed force-velocity data points were fitted by the Huxley (1957) equation. It was found that BDM produces a conspicuous decrease of the rate constant for crossbridge attachment. This effect, and also a reduction of the force per crossbridge, are responsible for the depression of the contractile characteristics produced by BDM.


Two different acto-S1 complexes.

Two different acto-S1 complexes. Based on change in anisotropy of fluorescently labelled S1 and on increase in turbidity of acto-S1 complex when S1 bound to F-actin, we reported previously that depending on the molar ratio of S1 to actin two different complexes of actin monomer (A) and myosin subfragment 1 (S1) could be formed: A1*S1 (one actin with one S1) and A2*S1 (two actins with one S1). Here we extend these findings to F-actin labelled with pyrene and cross-linked to S1 with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC). The fluorescence of pyrene F-actin decreased with increase in S1 concentration and reached saturation at a molar ratio of S1 to actin of either 0.5 or 1.0, depending on whether S1 was added slowly (5 min) or quickly (10-20 s between additions). Incubation of A2*S1 complex in excess of S1 for > 1 h caused a shift in equilibrium towards the A1*S1 complex. The A2*S1 complexes were not formed at high S1 to actin ratios (> 1.0) owing to competition between heads. Crosslinking experiments showed that the formation of EDC crosslinked products, 175-185 kDa doublet and 265 kDa band, depended on the ratio S1 to actin. To assess the relative ratio of S1 and actin in crosslinked products, we labelled S1 and F-actin with different fluorescent probes (5-IAF and IATR). The S1 to actin ratio was proportional to the ratio of intensities of fluorescence of labelled S1 and actin. The S1 to actin ratio in 265 kDa product was two times smaller than in 175-185 kDa doublet (which is believed to be A1*S1 complex) and therefore 265 kDa band corresponded to A2*S1. Transition between two types of binding may be important to understanding how muscle contracts.


Early stages of myogenesis in a large mammal: formation of successive generations of myotubes in sheep tibialis cranialis muscle. The generation of myotubes was studied in the tibialis cranialis muscle in the sheep hindlimb from the earliest stage of primary myotube formation until a stage shortly before muscle fascicles began to segregate. Primary myotubes were first seen on embryonic day 32 (E32) and reached their maximum number by E38. Small numbers of secondary myotubes were first identified at E38, and secondary myotube numbers continued to increase during the period of study. The ratio of adult muscle fibre to primary myotube numbers was approximately 70:1, making it seem unlikely that every later generation myotube used a primary myotube as scaffold for its formation, as described in small mammals. By E62, some secondary myotubes were supporting the formation of a third generation of myotubes. Experiments with diffusible dye markers showed that primary myotubes extended from tendon to tendon of the muscle, whereas most adult fibres ran for only part of the muscle length, terminating with myo-myonal attachments to other muscle fibres in a series arrangement. Acetylcholinesterase (AChE) and acetylcholine receptor (AChR) aggregations appeared in multiple bands across the muscle shortly after formation of the primary generation of myotubes was complete. The number of bands and their pattern of distribution across the muscle as they were first formed was the same as in the adult. Primary myotubes teased from early muscles had multiple focal AChE and AChR deposits regularly spaced along their lengths. We suggest that the secondary generation of myotubes forms at endplate sites in a series arrangement along the length of single primary myotubes, and that tertiary and possibly later generations of myotubes in their turn use the earlier generation myofibres as a scaffold. Although the fundamental cellular mechanisms appear to be similar, the process of muscle fibre generation in large mammalian muscles is more complex than that described from previous studies in small laboratory rodents.


Myosin heavy chain composition of single fibres and their origins and distribution in developing fascicles of sheep tibialis cranialis muscles. The myosin heavy chain (MHC) composition of single muscle fibres in developing sheep tibialis cranialis muscles was examined immunohistochemically with monoclonal antibodies to MHC isozymes. Data were collected with conventional microscopy and computerized image analysis from embryonic day (E) 76 to postnatal day (PN) 20, and from adult animals. At E76, 23% of the young myofibres stained for slow-twitch MHC. The number of these fibres considerably exceeded the number of primary and secondary myotubes. By E100, smaller fibres, negative for slow-twitch MHC, encircled each fibre from the initial population to form rosettes. A second population of small fibres appeared in the unoccupied spaces between rosettes. Small fibres, whether belonging to rosettes or not, did not initially express slow-twitch MHC, expressing mainly neonatal myosin instead. These small fibres then diverged into three separate groups. In the first group most fibres transiently expressed adult fast myosin (maximal at E110-E120), but in the adult expressed slow myosin. This transformation to the slow MHC phenotype commenced at E110, was nearing completion by 20 postnatal days, and was responsible for approximately 60% of the adult slow twitch fibre population. In the other two groups expression of adult fast MHC was maintained, and in the adult they accounted for 14% (IIa MHC) and 17% (IIb MHC) of the total fibre numbers. We conclude that muscle fibre formation in this large muscle involves at least three generations of myotube. Secondary myotubes are generated on a framework of primary myotubes and both populations differentiate into the young myofibres which we observed at E76 to form rosettes. Tertiary myotubes, in turn, appear in the spaces between rosettes and along the borders of fascicles, using the outer fibres of rosettes as scaffolds.


Tetragonal deformation of the hexagonal myofilament matrix in single skinned skeletal muscle fibres owing to change in sarcomere length. Single skinned skeletal muscle fibres were immersed in solutions containing two different levels of activator calcium (pCa: 4.4; 6.0). Sarcomere length was varied from 1.6 to 3.5 microns and recorded by laser diffraction. Slack length was 2.0 microns. Small-angle equatorial X-ray diffraction patterns of relaxed and activated fibres at different sarcomere lengths were recorded using synchrotron radiation. The position and amplitude of the diffraction peaks were calculated from the spectra based on the hexagonal arrangement of the myofilament matrix, relating the position of the (1.0)- and (1.1)-diffraction peaks in this model by square root of 3. The diffraction peaks were fitted by five Gaussian functions (1.0, 1.1, 2.0, 2.1 and Z-line) and residual background was corrected by means of a hyperbola. The coupling of the position of the (1.0)- and (1.1)-peak was expressed as a factor: FAC = [d(1.0)/d(1.1)]/square root 3. In the relaxed state this coupling factor decreased at increasing sarcomere length (0.9880 +/- 0.002 at 2.0 microns; 0.900 +/- 0.01 at 3.5 microns). The coupling factor tends toward the one that will be obtained from the squared structure of actin filaments near the Z-discs. At shorter sarcomere lengths a decrease of the coupling factor has also been seen (0.9600 +/- 0.005 at 1.6 microns), giving rise to an increased uniform deformation of the hexagonal matrix, when sarcomere length is changed from slack length. From these experiments we conclude that a change in sarcomere length (from slack length) increases the deformation of the actin-myosin matrix to a tetragonal lattice.


Galactose to ceramide linkage is essential for the binding of a polyclonal antibody to galactosyl ceramide. Characterization of a polyclonal antibody to galactosyl ceramide (Gal-Cer) which inhibits the internalization and infection of HIV-1 in neural cell lines was carried out. Polyclonal antibody to Gal-Cer was produced by injecting rabbits with Gal-Cer liposomes. The specificity of anti-Gal-Cer binding was studied by high performance thin layer chromatography (HPTLC)-based immunoassay. Using natural and semisynthetic lipids, the specificity of anti-Gal-Cer interaction was studied. The antibody bound to Gal-Cer and its derivatives. The antibody did not bind to glucosyl ceramide or lactosyl ceramide. Glucosyl ceramide differs from Gal-Cer by a hydroxyl group at the fourth carbon and in lactosyl ceramide galactose is linked to ceramide by an intervening glucose molecule. This indicates that D-galactose linked to ceramide is essential for binding. Removal of fatty acid from Gal-Cer, as seen with N-palmitoyl- and N-oleoyl Gal-Cer, had no effect on the binding. It appears that the third carbon of Gal-Cer is not involved in the binding. This is supported by the binding of anti-Gal-Cer to sulfatide or GM4 in which sulfate or sialic acid are added at the third carbon of Gal-Cer, respectively.


Immunochemical characterization of a monoclonal antibody specific for Alzheimer's disease associated protein. In this study, the monoclonal antibody PHF-1 which recognizes epitopes unique to Alzheimer's disease associated proteins (ADAP) has been characterized. Crossed affinity immunoelectrophoresis was used to estimate the binding constant for the interaction of PHF-1 with ADAP and to estimate the fraction of PHF-1 reactive protein. The binding constant of PHF-1 was determined to be 1.3 x 10(-8) M. Furthermore, the effect of dephosphorylation on the electrophoretic pattern of the PHF-1 reactive protein and the ensuing changes in its immunoreactivity were demonstrated.


Presynaptic inhibitory effect of TNF-alpha on the release of noradrenaline in isolated median eminence. The effect of tumor necrosis factor-alpha (TNF-alpha) on the stimulation-evoked release of noradrenaline (NA) from isolated rat median eminence (ME) was investigated, using a low-volume perfusion system. Median eminence, loaded with [3H]noradrenaline, was superfused with Krebs solution and stimulated electrically (2 Hz, 120 shocks). The effect of TNF-alpha was studied on the S2/S1 ratio. It was found that stimulation-evoked release of NA from noradrenergic axon terminals in the isolated rat ME was inhibited by TNF-alpha and this effect was concentration-dependent. In contrast, TNF-alpha had no effect on the release of [3H]NA from the spleen. Since NA released in the ME might be involved in the modulation of corticotropin-releasing factor (CRF) production, it is suggested that TNF-alpha, through presynaptic modulation of NA release from noradrenergic nerve terminals in the ME, might regulate CRF and other neurohormone release in this hypothalamic structure.


Human glioblastoma cell line 86HG39 activates T cells in an antigen specific major histocompatibility complex class II-dependent manner. The capacity of three different human glioblastoma cell lines to activate human T cells was analysed by measuring major histocompatibility complex (MHC) antigen expression, monokine secretion and lectin, mAb OKT3 and antigen-driven T cell proliferation. All glioblastoma cells tested were able to induce PHA and concanavalin A (ConA)-driven T cell proliferation in a dose-dependent fashion, while all failed to induce T cell activation with mAb OKT3. In addition, the glioblastoma cell line 86HG39 was able to induce tetanus toxoid and toxoplasma lysate antigen-specific T cell proliferation. The responding T cell lines originated from only one out of five different donors. This foreign antigen-specific T cell proliferation induced by 86HG39 cells could be inhibited with mAb L243 directed against HLA-DR molecules. The study of monokine secretion by 86HG39 cells showed a strong interleukin (IL)-6 secretion after lipopolysaccharide (LPS) treatment, whilst no IL-1 secretion was observed. Furthermore, only 86HG39 cells were positive for HLA-DR molecules, whereas interferon (IFN) gamma treatment of 87HG28 and 87HG31 cells was necessary for the induction of class II antigen expression. Thus, cell line 86HG39 shows many features of an antigen presenting cell and the interaction of these cells with MHC compatible human T cells might be a useful model to study cellular immune reactions within the central nervous system.


Expression of major histocompatibility complex (MHC) class I genes in astrocytes correlates with the presence of nuclear factors that bind to constitutive and inducible enhancers. The molecular basis of constitutive and inducible major histocompatibility complex (MHC) class I gene expression was studied in murine astrocytes in primary culture. Astrocytes constitutively expressed MHC class I molecules and treatment of these cells with interferon-gamma (IFN-gamma) further induced expression. The conserved region containing the upstream MHC class I regulatory element (MHC-CRE) and juxtaposed interferon consensus sequence (ICS) enhanced constitutive MHC class I promoter activity. As seen with cell surface expression of MHC molecules, treatment of astrocytes with IFN-gamma increased MHC class I promoter activity. Inducible expression required the presence of the MHC-CRE/ICS enhancer region. Nuclear factors that bind to the MHC-CRE and ICS were constitutively expressed in cultured astrocytes and IFN-gamma treatment further induced binding activity both to the MHC-CRE and ICS and correlated with induction of MHC class I gene expression. This study identifies the MHC-CRE and ICS as the major cis elements in controlling MHC class I promoter activity and suggests that the expression of nuclear factor binding activities to these enhancer elements is a basic transactivating mechanism for the expression of MHC class I genes in astrocytes.


Type I interleukin-1 receptors in the mouse brain-endocrine-immune axis labelled with [125I]recombinant human interleukin-1 receptor antagonist. Iodine-125-labelled recombinant human interleukin-1 (IL-1) receptor antagonist ([125I]IL-1ra) was utilized to further determine the characteristics of IL-1 receptors in the brain-endocrine-immune axis. The binding of [125I]IL-1ra in homogenates of mouse hippocampus, spleen and testis was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity (KD, 20-30 pM). In competition studies, IL-1ra, recombinant human IL-1 alpha, IL-1 beta and a weak IL-1 beta analog inhibited [125I]IL-1ra binding to mouse tissues in parallel with their biological activities. In autoradiographic studies, [125I]IL-1ra and [125I]IL-1 alpha binding showed comparable distribution patterns with highest densities of binding sites present in the dentate gyrus of the hippocampus, choroid plexus, anterior pituitary, marginal zones and red pulp regions of the spleen, epididymis and interstitial area of the testis. The binding characteristics and distribution of [125I]IL-1ra are comparable to those of previously characterized Type I IL-1 receptors. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immune responses to physiological and pharmacological stimuli.


Specific immunotherapeutic strategy for myasthenia gravis: targeted antigen-presenting cells. The pathogenesis of myasthenia gravis (MG) involves a T cell-dependent antibody-mediated autoimmune response directed against acetylcholine receptors (AChR). Inactivation of AChR-specific T cells should interrupt the immune response, resulting in therapeutic benefit. Since each individual's repertoire of T cells responds to a heterogeneous and unique spectrum of AChR epitopes presented in association with self-major histocompatibility complex (MHC) class II, an individualized approach is required to target all relevant AChR-specific T cells. The individual's own antigen-presenting cells (APC) can be used for this purpose, since they process and present the antigen appropriately, and express the correct MHC class II. A novel method of binding AChR to surface immunoglobulin with a heterobifunctional antibody conjugate allows us to use all B cells as APC. Conjugate-plus-AChR-treated B cells (AChR-APC) effectively targeted AChR-specific T cells, stimulating vigorous proliferative responses in a rat cell culture system. If APCs are 'fixed' with cross-linking reagents, they induce long-lasting or permanent 'anergy' of the specific T cells. We prepared AChR-APC, allowed them to process AChR in vitro, and fixed them with paraformaldehyde. Pre-culture of these fixed AChR-APC with AChR-specific T cells induced anergy: when restimulated with fresh AChR-APC, the T cells exhibited markedly reduced proliferative responses and IL-2 production, compared with responses of T cells pre-cultured with control fixed B cells. Implications for the design of antigen-specific therapeutic strategies for MG and other immune disorders will be discussed.


Identification of a 58-kDa antigen with increased immunoreactivity in the cerebella of multiple sclerosis patients. An increase in immunoreactivity associated with a 58-kDa antigen was found in a majority of MS cerebellar homogenates examined by Western blot analysis using antisera obtained by selective immunization of rabbits with autopsy cerebella. Two-dimensional immunoblotting demonstrated that the majority of the increased immunoreactivity observed in MS cerebella was associated with the highest apparent pI of three immunoreactive species at 58 kDa. Immuno-crossreaction with rat cerebellar homogenates demonstrated that the 58-kDa antigen was developmentally regulated, showing the greatest immunoreactivity at embryonic day 15. The 58-kDa cerebellar antigen may represent a membrane protein which is re-expressed as part of the onset of MS.


Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-alpha. Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-alpha had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-alpha production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-alpha production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.


Evidence of a neurogenic component during IgE-mediated inflammation in mouse skin. IgE-mediated inflammation was measured in mouse footpads that lacked sciatic innervation. Mice were passively sensitized with a monoclonal antibody, IgE anti-dinitrophenol, or were immunized for specific IgE production. Antigen-induced swelling in the denervated footpads was reduced 23-39% when compared to sham or untreated controls. Reduced IgE-mediated swelling responses were attributed to the loss of a mast cell-nerve interaction and not to blood vessel sensitivity to vasoamines. Furthermore, electrical stimulation of the distal segment of the sciatic nerve completely restored IgE-mediated inflammation. These data provide in vivo evidence that peripheral nerves participate in cutaneous IgE-mediated swelling reactions with the net effect of increasing inflammation.


Assessing treatment acceptance: the Abbreviated Acceptability Rating Profile. As part of a broad focus on service accountability, increased attention has been devoted to the assessment of consumer judgments of treatment acceptability. Current treatment acceptability measures are limited by item complexity and time-intensiveness. These limitations were addressed with the modification of an existing instrument (Intervention Rating Profile; Witt & Elliott, Journal of Abnormal Child Psychology, 13, 59-67, 1985). Psychometric support was found for the revised measure (Abbreviated Acceptability Rating Profile; AARP) in an initial sample of parents (N = 60) who rated child treatments. Results were replicated in a cross-validation study of a second independent sample of parents (N = 80). A third sample of subjects was used to assess the time-intensiveness and readability of the AARP. The usefulness of the modified instrument is discussed.


Some relevant components of adherence behavior. Adherence to medical or psychological prescriptions is analyzed as a type of rule and self-rule following behavior. Variables that increase the likelihood of compliance are: (1) the specific motivational condition, usually aversive when treatment or preventive prescription is sought, (2) the medium of contact and context used when receiving the prescription, (3) attentive responses under the control of the prescription, (4) understanding the complexity of the prescription, (5) the specific content of the prescription, (6) verbalization on the willingness to follow the prescription, (7) appropriate abilities for producing the behaviors described in the prescription, (8) physical facilities and appropriate biological conditions for producing the behaviors, (9) verbalization describing the relationship between the adherence response and the immediate and long-term consequences, functioning as a discriminative stimulus and as a reinforcing event, (10) compatibility between daily activities and the behaviors described in the prescription: response cost and self-control behavior when several behaviors are possible and probable. Strategies which facilitate self-control as well as incompatible behavior are suggested.


The case of a newspaper phobia in a 9-year-old child. The case of E, a 9-year-old, male, with a newspaper phobia, is presented. The phobia developed over a 4-year period and gradually spread until it included many significant areas of E's life. Treatment took place over a 4-month period. It involved use of a token economy administered by E's parents. Cognitively, the treatment involved the concepts of the acquisition of perceived self-control as a consequence of the token economy agreement. This included volition, attention, and self-control by E as well as parental reinforcement.


Extinction and DRO in the treatment of aggression in a boy with severe mental retardation. A novel use of extinction was applied in combination with differential reinforcement of other behavior to reduce dangerous levels of aggression displayed by an 11-year-old boy with severe mental retardation and cerebral palsy. Data taken during actual treatment sessions and in the client's "natural" environment (classroom and dormitory) suggest that the intervention was immediately effective and that the improvements were maintained and generalized.


B. F. Skinner and the cognitive revolution. The behaviorism that cognitive scientists attack is a caricature, drawn primarily from the more polemical writings of J. B. Watson and B. F. Skinner. In this brief commentary, I discuss the fact that these writings, and especially Skinner's, offered the neocognitivists such a polar difference from their own position, that it was easier to ignore the distinction between behaviorism and neobehaviorism than to recognize it. I point out that there are factors in Skinner's intellectual history that may account for the emergence of his more radical behaviorism out of what was essentially his own theoretical, neobehavioristic approach to the study of conditioning and learning.


Listening to our clinical partners: informing researchers about children's fears and phobias. We surveyed a national sample of clinical psychologists to expand on Graziano and De Giovanni's (Behaviour Research and Therapy, 1979, 17, 161-162) findings that 6.8% of clinically referred children present with problems with fears and/or phobias. The survey was also conducted to assess how clinicians could inform researchers about current needs for investigations in this area. Questions involved epidemiological, demographical, and treatment characteristics of cases recently seen by therapists. The percentage of referred cases for fears/phobias in children was determined to be similar over time. In addition, types of fears, length of history of fear, intensity of fear, length of treatment, types of treatments used, and degree of parental involvement were measured and reported. The results were found to suggest a substantial degree of heterogeneity among types of fears and treatment methods for this population, as well as important lines of future research. They also suggest the need for controlled studies to assess the efficacy of a number of widely used treatments that lack an empirical base.


Dietary restraint anxiety and its relationship to human eating behavior. Ninety-five female university students completed Stunkard and Messick's (Journal of Research Psychosomatic Research, 29, 71-83, 1985) Three-Factor Eating Questionnaire, measuring three dimensions of human eating behavior: cognitive restraint, disinhibition, and hunger susceptibility. They were led through the visualization of a neutral or pleasant scene and a food scene, but were instructed that they would not be able to eat the food. Only hunger susceptibility was found to be significantly correlated with ratings of subjective anxiety (SUDS) and urge to eat in the not eating situation after any variability associated with the neutral scene was removed. That is, internal (hunger) and external (incentive) cues when linked to not eating are sources of anxiety. Treatment programmes for obesity might well include desensitization of these cues.


The non-standardization of the Fear Survey Schedule. The Fear Survey Schedule-III was administered to 860 college students. The results are presented in the form of frequencies, in deliberate contrast to previous work which present results in the form of z-scores. The argument is made that these z-scores do not normalize the original item distributions and thus provide no opportunity for normative comparisons. Data are provided allowing the researcher to assess the characteristics of FSS-III item distributions and make limited normative comparisons.


Improving parent-child interactions for families of children with developmental disabilities. Child Management Training (CMT) involves compliance training with a focus on consistent use of antecedents and consequences. Planned Activities Training (PAT) focuses on teaching parents to plan for and engage in activities with their children. A multiple probe design counterbalancing PAT and CMT showed that PAT and CMT were about equally effective in improving mother-child interactions in four families with children with developmental disabilities. Responses to a social validation questionnaire indicated that parents were satisfied with the services received, and that PAT was the slightly preferred treatment. Prior research demonstrated that PAT enhanced the results of CMT. The practical advantages of PAT over CMT are discussed.