Interaction of the human protein kinase PKR with the mouse PKR homolog occurs via the N-terminal region of PKR and does not inactivate autophosphorylation activity of mouse PKR.

The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes, HIS3 and IacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of the HIS3 and lacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathione S-transferase (GST) fusions in Escherichia coli were purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1-184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR.

The PKR protein kinase--an interferon-inducible regulator of cell growth and differentiation.

Post-translational modifications such as protein phosphorylation provide an important mechanism by which the functional activity of proteins can be controlled and, hence, biological processes regulated. Interferons (IFN) are a multigene family of cytokines that can profoundly affect a wide variety of functions in animal cells including virus replication, cell growth and differentiation, and the immune response. Changes in protein phosphorylation mediated by the IFN-inducible, RNA-dependent protein kinase (PKR) are implicated in the control of cell proliferation mediated by IFNs. Our knowledge of the structure, regulation and function of PKR will be summarized in this brief review, with focus on those aspects of protein phosphorylation and interferon action involving PKR that are central to the roles of the enzyme in the control of cell growth and proliferation.

Cat scratch disease and acquired immunodeficiency disease: diagnosis by transmission electron microscopy.

A 33-year-old, homosexual, cat-owning, African-American man with human immunodeficiency virus infection by positive serologic tests and acquired immunodeficiency syndrome by CD4 lymphocyte count alone (39 cells/mL) presented with a one-year history of intermittent fever, weight loss, and generalized lymphadenopathy. A malignant lymphoma was suspected clinically. Light microscopic study of a left inguinal lymph node biopsy specimen revealed effacement of the lymph node architecture by a diffuse infiltrate of large, atypical reticulum cells, loose, patchy granulomatous inflammation, diffuse hyaline fibrosis, diffusely proliferated blood vessels, and multifocal degeneration and necrosis. Lymph follicles were absent and lymphocytes were moderately depleted. Microorganisms were not seen in lymph node sections stained with special histochemical stains (including the Warthin-Starry stain). These light microscopic changes were considered suggestive of a malignant lymphoma, especially Hodgkin's disease. The diagnosis of cat scratch disease (CSD) became apparent only after transmission electron microscopic study of the lymph node revealed clusters of small, pleomorphic bacteria in degenerated collagenous tissue and in blood vessel walls. This case illustrates the value of transmission electron microscopy in making the diagnosis of CSD, especially when light microscopic changes are superimposed on those of late human immunodeficiency virus infection of the lymph node.

Trichomoniasis complicating esophageal intramural pseudodiverticulosis: diagnosis by transmission electron microscopy.

A 58-year-old African-American man presented with a progressive esophageal stricture of unknown etiology complicated by esophageal candidiasis, broncho-esophageal fistula, four episodes of aspiration pneumonia, and a 40-lb weight loss. He ultimately required an esophagectomy. Pathologic examination showed marked thickening of the esophageal wall by submucosal pseudodiverticula typical of esophageal intramural pseudodiverticulosis (EIPD) and extensive mucosal and submucosal chronic inflammation and fibrosis. Small, oval cells with ill-defined nuclei were present in lumens of some pseudodiverticula, light microscopically. Their exact nature could not be determined by light microscopy. The diagnosis of trichomoniasis became apparent only after transmission electron microscopic study of these cells demonstrated characteristic features of trichomonad protozoa. These included four anteriorly placed flagella with kinetosomes, a recurrent flagellum associated with an undulating membrane, a costa, a peltar-axostylar complex, and a small Golgi body with parabasal filaments. This case of EIPD is unusual in that the associated broncho-esophageal fistula and trichomoniasis have not been previously reported as complications of EIPD.

Mechanism of interferon action. Biochemical and genetic evidence for the intermolecular association of the RNA-dependent protein kinase PKR from human cells.

The interferon-inducible protein kinase (PKR) is activated by an RNA-dependent autophosphorylation. Structure-function studies of the 551 amino acid PKR kinase from human cells have revealed that catalytic-deficient PKR mutants such as PKR(1-551)K296R display a dominant negative behavior when expressed in transfected cells. The potential for PKR to form protein multimers has therefore been examined. Three types of studies, including both genetic and biochemical analyses, demonstrated that PKR from human cells undergoes an intermolecular association that is not dependent upon RNA. First, the intermolecular association of PKR in vitro was demonstrated in the context of an enzyme-substrate interaction. Purified recombinant histidine-tagged PKR(1-551)K296R mutant protein was phosphorylated by purified wild-type PKR; this intermolecular phosphorylation of PKR was dependent on double-stranded RNA. At a fixed RNA concentration, high concentrations of the HIS-PKR(1-551)K296R mutant both impaired the autophosphorylation of wild-type PKR and blocked the trans-phosphorylation of itself. Second, the yeast two-hybrid system was used to probe the intermolecular association of PKR in vivo. Coexpression of the full-length catalytic-deficient phosphotransfer mutant PKR(1-551)K296R as a fusion protein with the Gal4 activation domain and the Gal4 DNA binding domain resulted in the expression of two Gal4-responsive reporter genes, HIS3 and lacZ. The full-length RNA-binding deficient PKR(1-551)K64E/K296R double mutant also interacted with PKR(1-551)K296R sufficiently to activate Gal4-responsive reporter genes; however, other PKR mutants including PKR(1-280)wt and PKR(281-551)K296R as well as p53, RAS, and BCL2 did not. Third, both PKR(1-551)K296R and PKR(1-551)K64E/K296R enhanced the expression of the reovirus S1 gene and S1/S4 chimeric gene in cotransfected COS cells. By contrast, the expression of the reovirus S4 gene was not enhanced by cotransfection with either PKR(1-551)K296R or PKR(1-551)K64E/K296R. These results indicate that PKR interacts with itself in an intermolecular manner both in vivo and in vitro, and that RNA binding is neither necessary nor sufficient for PKR multimerization.

Herpetic bronchitis with a broncho-oesophageal fistula.

Tracheobronchitis and oesophagitis due to herpes simplex virus (HSV) are rare. Tracheo-oesophageal fistula due to HSV oesophagitis has been described in the immunocompromised host. A case is reported of a broncho-oesophageal fistula which developed secondary to herpetic bronchitis in an apparently immunocompetent patient.

Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity.

The interferon-induced P1/eIF-2 alpha protein kinase cDNA, designated PKR, was expressed both in Escherichia coli and in transfected monkey COS cells. TrpE-PKR fusion proteins and PKR nonfusion proteins were examined for their RNA-binding activity by Northwestern blot analysis. PKR is a RNA-binding protein that possesses two copies of a highly conserved motif, RI and RII, within the N-terminal region of the protein. Amino acid residues between 11 and 243 of PKR, which includes both copies of the R motif, displayed RNA-binding activity comparable to that of the full-length 551-amino-acid PKR protein. Analysis of substitution and deletion mutant PKR proteins revealed that motif RI was both necessary and sufficient for RNA-binding activity, whereas motif RII was not. Substitution of the highly conserved lysine at position 64 within the RI motif abolished RNA-binding activity, both of full-length PKR and the N-terminal 243-amino-acid truncated PKR. Finally, PKR substitution and deletion mutant cDNAs deficient for kinase function were expressed to much higher levels in transfected monkey cells than was the full-length wild-type PKR cDNA.

Clonal rearrangement of the T-cell receptor beta-chain gene in hyperplastic lymphadenopathy associated with lung cancer.

A patient presenting with marked inflammatory lymphadenitis and Jaccoud's arthritis was found to have a rearranged gene for the beta-chain of the T-cell receptor (TCR-beta) antigen in the lymph node DNA digests and normal germ line DNA in the peripheral blood lymphocytes. Four months later, the patient was diagnosed to have poorly differentiated adenocarcinoma of the lung with small foci of metastatic tumor in lymph nodes that contained the same extensive lymphocytic and inflammatory cell infiltrates noted earlier. Rearranged TCR-beta chain genes were detected in both lymph node and peripheral blood lymphocyte DNA at this time. The most likely explanation for the florid lymph node reaction and the unusual arthropathy appears to be a paraneoplastic immune response. The rearranged TCR-beta genes indicate a clonal T-cell expansion that most likely resulted from the aberrant immunologic response to the lung cancer.

Thrombosis: the major Hickman catheter complication in patients with solid tumor.

Major complications of Hickman catheter placement (thrombosis and infection) were determined in 168 patients with solid tumor (lung, 79; head and neck, 56; esophagus, 24; and miscellaneous, 9). Catheter-related thrombosis was clinically detected in 22 individuals and was detected at autopsy in six (total 17 percent). The 17 percent figure underestimates the true incidence of thrombosis since only 25 percent of study patients had autopsies. Patients with adenocarcinoma of the lung constituted a high risk group. Nine of 20 (45 percent) of these patients had thrombosis compared to 25, 9, and 16 percent of patients with squamous cell cancers of lung, head and neck and esophagus, respectively (p less than 0.002). Three patients with thrombosis had pulmonary emboli and two died. Thrombosis occurred despite daily heparin catheter flushing. INfections occurred in 11 patients. One had suspected endocarditis, one had a subcutaneous tunnel infection, and nine had exit site infections. All responded to local or systemic antibiotics. Better methods to prevent thrombosis are needed.

Necrolytic migratory erythema, presenting as candidiasis, due to a pancreatic glucagonoma.

A 54-year-old male with diabetes, weight loss, glossitis and Candidiasis presented with the typical cutaneous eruption of necrolytic migratory erythema. The suspicion of pancreatic glucagonoma was confirmed by an elevated plasma glucagon level. Surgical removal of the pancreatic alpha cell tumor resulted in a complete disappearance of all symptoms. The importance of the recognition of the skin eruption of necrolytic migratory erythema as a clue to the presence of pancreatic glucagonoma is emphasized.