Citations

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.

The blood-nerve transfer of human plasma albumin glycated with D-glucose was investigated by measuring the permeability coefficient-surface area product (PS) of the blood-nerve barrier to radioiodinated albumin in normal adult rat sciatic nerve. Human albumin (ALB) from normal individuals, freshly isolated by CM-Affi-Gel Blue affinity chromatography, was glycated in vitro for 1, 3, 10, 19, and 30 weeks. Glycated ALB (gALB) was separated from the nonglycated form by boronate-affinity chromatography. The efficiency of this separation was assessed by chromatography of ALB glycated with [14C]glucose and by rechromatography of isolated ALB and gALB after radioiodination. The gALB was also shown to have a higher molecular weight and be completely separated from ALB after SDS/pore gradient electrophoresis in a Tris borate/EDTA buffer. After 1 week of glycation, the gALB PS was 2.2-fold greater than the ALB PS (0.724 +/- 0.063 x 10(-6) vs. 0.328 +/- 0.053 x 10(-6) ml.g-1.s-1; mean +/- SD; P less than 0.0001) and it increased with the time of glycation reaching a maximum value of 16.2-fold greater at 30 weeks (4.656 +/- 1.117 x 10(-6) vs. 0.288 +/- 0.042 x 10(-6) ml.g-1.s-1; mean +/- SD; P less than 0.0001). No change was observed in the residual endoneurial plasma volume. In addition, the PS of gALB isolated from patients with diabetic polyneuropathy was significantly increased (P less than 0.0001) compared to the PS for ALB isolated from the same patients. It is hypothesized that the increased permeability of gALB and presumably other glycated serum components across the blood-nerve barrier, as well as the observed quantitative increase in ALB, IgG, and IgM in sural nerve biopsies from patients with diabetic polyneuropathy contribute to the development of diabetic polyneuropathy over a prolonged period of time by mechanisms that might involve osmotic changes in the nerve microenvironment, direct toxic effects of glycated macromolecules on cells within the endoneurium, or nerve damage by classical immunological mechanisms due to trapping of glycated immunoglobulins within nerve.

Target structures important for natural killer (NK) cell recognition of virally infected cells are not well defined. Since major histocompatibility complex (MHC) class I molecules bind viral peptides during acute infection, we evaluated whether an interaction between MHC and virus might influence the susceptibility of infected cells to NK cell-mediated lysis. To control for MHC class I expression on target cells, we used either HLA class I-deficient C1R cells or C1R sublines expressing transfected HLA class I gene products. Human NK cells were unable to preferentially lyse class I-deficient C1R cells after infection with herpes simplex virus (HSV). In contrast, HLA class I transfectants were significantly more susceptible to NK cell-mediated cytotoxicity after HSV infection. This occurred for HSV-infected C1R cells expressing any of the three HLA class I gene products tested (i.e., HLA-B27, HLA-A3, or HLA-Aw68), indicating that NK cell recognition in this system does not require "self" MHC and is not unique for a single haplotype. Productive HSV infection is required for the increased killing, since inoculation with UV-inactivated virus did not lead to increased lysis. In addition, since HSV infection of the transfectants did not significantly alter the level of class I expression, the change in susceptibility appears to be due to qualitative changes in the target structures on HSV-infected, HLA class I+ targets. These results demonstrate a role for MHC class I in regulating NK cell-mediated killing of virus-infected cells.

A primer-directed cDNA library was used to obtain cDNA clones corresponding to the 5' end (i.e., the ligand-binding domain) of the avian c-erbB gene. Bacterial c-erbB fusion proteins were synthesized and used to obtain polyclonal antisera specific for the ligand-binding domain of the avian receptor. These antisera and antisera specific for the carboxyl terminal domain of the chicken c-erbB gene product have been used to study the native protein products of the c-erbB locus in primary cell cultures by in vivo labeling and immunoprecipitation. Our studies reveal that three c-erbB gene products of Mr 300,000, Mr 170,000, and Mr 95,000 are synthesized in uninfected chicken embryo fibroblasts. Only the Mr 300,000 and Mr 170,000 species can be precipitated by using antisera specific for the cytoplasmic domain of the c-erbB product. The 95,000 species is not recognized by the antiserum directed against the carboxyl-terminal domain of c-erbB and is specifically released into the culture medium. Northern transfer studies reveal a lower molecular weight transcript of approximately 2.6 kilobases that selectively hybridizes to the ligand-binding domain of the avian c-erbB gene product but does not hybridize with probes specific for the cytoplasmic kinase domain of c-erbB. An additional cDNA clone corresponding to this transcript has been isolated, and its sequence suggests it may arise via alternative processing. Together, these data suggest that a truncated form of this growth factor receptor--i.e., a Mr 95,000 species--is synthesized from a low molecular weight c-erbB transcript that exclusively encodes the ligand-binding domain of the receptor. Secretion of truncated growth factor receptors has been reported recently in several systems, and our results are discussed in the light of these findings.

This report describes a rapid regulation of the expression of the c-jun protooncogene by the antiestrogen tamoxifen (Tam). The c-jun protooncogene codes for an important component of the AP-1 transcription factor complex, which regulates the expression of many unlinked genes. Repeated experiments have shown that Tam rapidly increases the steady-state c-jun mRNA levels in the avian oviduct but decreases the levels in the liver. The Tam effects are time- and dose-dependent. These results are supported by other studies that have demonstrated that 17 beta-estradiol decreases steady-state levels of c-jun protooncogene mRNA in oviducts of animals fully withdrawn from estradiol. The effect of Tam in the avian oviduct is in contrast to the reported effects of Tam on the expression of practically all other genes in the avian oviduct and other animal tissues. Transcription analyses using nuclear runoff experiments with oviduct nuclei demonstrate a decrease in the c-jun gene transcription within minutes after Tam treatment with a return to 75% of control values by 4 hr. The fact that Tam transiently decreases the transcription of the c-jun gene but increases the steady-state c-jun mRNA levels suggests that Tam must alter both transcriptional and post-transcriptional events. The results support a role of the c-jun protooncogene as a regulatory gene in the cascade model for steroid action whereby steroids rapidly regulate the regulatory genes, which in turn regulate many other structural genes.

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone alpha subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 125I-labeled bovine TSH to porcine thyroid membranes. Removal either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys31, Cys32, Phe33, Arg35, Arg42, Lys44, and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly30 and Ser34, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg35 (leaving the alpha-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the alpha-subunit 26-46 sequence. The first lies between residues Gly30 and Arg35 and includes Cys31, Cys32, and Phe33 as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys44 as an important residue and Ser43 as a less important component.

Although in vivo effects of 17 beta-estradiol (estrogen) on bone turnover have been shown to occur mainly through influences on osteoclast-mediated bone resorption, the mechanism by which estrogen reduces bone resorption is unclear. To approach this question, we have examined authentic osteoclasts for evidence of a direct osteoclast response to estrogen in vitro. Highly purified (greater than (90%) viable avian osteoclasts from birds maintained on a low calcium diet were obtained using an osteoclast-specific monoclonal antibody coupled to magnetic beads. Isolated cells were either analyzed directly for estrogen receptor (ER) levels or cultured to assess the biological effects of estrogen. Northern blot analysis revealed a 5.2-kilobase mRNA that hybridized with a cDNA to human ER mRNA in the osteoclasts. An anti-human ER antibody recognized proteins of 66 kDa and 140 kDa in osteoclast extracts by Western blot analysis. The 66-kDa size is in close agreement with the reported size of the human ER. Nuclear binding of estrogen to intact viable osteoclasts was steroid-specific and saturable, with 5662 +/- 1420 molecules bound per nucleus (mean +/- SEM). In vitro estrogen responses in osteoclasts included a dose-dependent decrease in resorption as well as an increase in nuclear protooncogene mRNA levels. These observations indicate that osteoclasts are capable of directly responding to estrogen in vivo.

Natural killer (NK) cells are a unique subpopulation of lymphocytes with the capability to kill malignant cells via one of two alternative mechanisms: (i) Fc receptor-dependent cytotoxicity against antibody-coated targets or (ii) direct cell-mediated cytotoxicity. However, the molecular mechanisms that trigger and subsequently regulate NK cell cytotoxicity are incompletely understood. We have therefore investigated the role of protein tyrosine phosphorylation in the transmembrane signaling initiated after Fc receptor stimulation or direct tumor cell contact in clonal CD16+/CD3- human NK cells. We report that stimulation of the Fc receptor rapidly induced the tyrosine phosphorylation of a number of NK cell proteins. These effects occurred within 2 min, were maximal at 10 min, and declined toward baseline after 60 min. In addition, Fc receptor ligation increased the in vitro protein kinase activity of NK cell phosphotyrosyl proteins. We have also demonstrated that direct contact of NK cells with K562 tumor cells induced the rapid phosphorylation of distinct NK cell phosphotyrosyl proteins. Furthermore, the protein-tyrosine kinase inhibitor herbimycin A blocked NK cell cytotoxic function in a concentration-dependent manner. Our results suggest that protein tyrosine phosphorylation is an obligatory early proximal signal in activating the cytotoxic function of NK cells.

Whether compression nerve injury is due to ischemia, direct mechanical injury, or both remains unsettled. To assess structural changes of nerve during compression, peroneal nerves of rats were compressed at various pressures for different times, and the structural alterations were stopped by simultaneous in situ and perfusion fixation. The structural changes observed during a few minutes of compression cannot be explained by ischemic injury because the pathologic alterations characteristic of ischemia take many hours to develop and in any case are different from the ones found here. The pressure- and time-related structural changes observed in the present study under the cuff were (i) decrease in fascicular area and increase in fiber density due to expression of endoneurial fluid; (ii) compression and expression of axoplasm, sometimes to the point of fiber transection; (iii) lengthening of internodes; and (iv) obscuration of nodes of Ranvier due to cleavage and displacement of myelin and overlapping of nodes by displaced loops of myelin. At the edges of the cuff the changes were (i) increase of fascicular area probably from expressed endoneurial fluid; (ii) widening of nodal gaps, perhaps mainly from translocated axonal fluid; and (iii) disordered structure of axoplasm. We suggest that the process of paranodal demyelination and axonal transection are linked, occur during the act of compression, and are due to shear forces. The initial event is expression of endoneurial fluid, followed by compression and expression of axoplasm and cleavage and displacement of layers of myelin. Conceivably, with prolonged cuff compression ischemic injury might be found to be superimposed on mechanical injury.