Molecular typing of human leukocyte antigen and related polymorphisms following whole genome amplification.

Reliable, high-resolution genotyping of human leukocyte antigen (HLA) polymorphisms is often compromised by DNA samples of suboptimal quality or limited quantity. We tested the feasibility of molecular typing for variants at HLA and neighboring loci using whole genome amplification (WGA) strategy facilitated by the Phi29 DNA polymerase. With little (5-100 ng) starting genomic DNA of varying quality and source materials, WGA was deemed successful in 167 of 169 DNA from 47 cell lines, 100 European Americans, and 22 native Africans. The Phi29-processed DNA provided adequate templates for polymerase chain reaction (PCR)-based analyses of several HLA (A, B, C, DRB1, and DQB1) and related loci (HFE, MICA, and 10 microsatellites) in the 6p24.3-6p21.3 region, with PCR amplicons ranging from 92 to 2200 bp. Five different genotyping techniques resolved and confirmed 364 genotypes when both original and Phi29-processed DNA worked in PCRs. General population genetic analyses provided additional evidence that WGA may represent a reliable and simple approach to securing ample genomic DNA for typing HLA, MICA, and related variants.

Surgical treatment of multiple skull abscesses associated with coccidioidomycosis.

This case emphasizes that aggressive neurosurgical management may benefit patients with disseminated coccidioidomycosis and skull abscesses. Disseminated infection due to Coccidioides immitis, the causative agent of coccidioidomycosis, is difficult to treat and often requires prolonged antifungal therapy in addition to surgical debridement. We present a case of a young woman with disseminated coccidioidomycosis who had multiple skull lesions, two of which penetrated the skull and invaded the subgaleal and epidural spaces. Despite prolonged aggressive medical management, these lesions failed to resolve until they were surgically drained.

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.

Oxidative stress and heat shock stimulate RGS2 expression in 1321N1 astrocytoma cells.

RGS2, a regulators of G-protein signaling family member, regulates G-protein signaling and is itself controlled in part by regulated expression. We tested if cell stress regulates RGS2 expression in human astrocytoma 1321N1 cells. Treatment with H2O2 increased RGS2 mRNA levels time- and concentration-dependently, with 200 microM H2O2 causing an approximately eightfold increase after 2 h. Peroxynitrite and heat shock also increased RGS2 mRNA levels. H2O2-induced RGS2 expression was negatively regulated by phosphoinositide-3-kinase and extracellular signal-regulated kinases. H2O2 also concentration-dependently increased RGS2 protein levels, and the RGS2 appeared to be predominantly in the nucleus. These results demonstrate that RGS2 expression is up-regulated by cell stress.

Intrasellar cavernous hemangioma. Case report.

The authors present a rare entity, an intrasellar cavernous hemangioma that on neuroimages mimicked a nonfunctioning pituitary macroadenoma in a patient with a known orbital hemangioma. Such lesions can grow extraaxially within the dural sinuses, particularly the cavernous sinus, and present like tumors. A better understanding of the neuroimaging. clinical, and anatomical features of these lesions may prevent difficulties in management.

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.

p125 focal adhesion kinase promotes malignant astrocytoma cell proliferation in vivo.

p125 focal adhesion kinase (p125FAK) is a cytoplasmic tyrosine kinase that is activated upon engagement of integrin cell adhesion receptors, and initiates several signaling events that modulate cell function in vitro. To determine the biologic role of p125FAK in malignant astrocytic tumor cells, U-251MG human malignant astrocytoma cells were stably transfected with p125FAK cDNA using the TET-ON system, and stable clones isolated that exhibited an estimated 5- or 20-fold increase in p125FAK expression on administration of 0.1 or 2.0 microg/ml doxycycline, respectively. In vitro studies demonstrated that induction of p125FAK resulted in a 2- to 3-fold increase in cell migration, increased p130CAS phosphorylation, localization of exogenous p125FAK to focal adhesions, and a 2-fold increase in soft agar growth. To determine the role of p125FAK in vivo, clones were injected stereotactically into the brains of scid mice. A 4.5-fold estimated increase in p125FAK expression was induced by administration of doxycycline in the drinking water. Analysis of xenograft brains demonstrated that, upon induction of p125FAK, there was a 1.6- to 2.8-fold increase in tumor cell number, and an increase in mAb PCNA-labeling of tumor cells in the absence of a change in the apoptotic index. Compared to normal brain, the expression of p125FAK was elevated in malignant astrocytic tumor biopsies from patient samples. These data demonstrate for the first time that p125FAK promotes tumor cell proliferation in vivo, and that the underlying mechanism is not associated with a reduction in apoptosis.

Hypoxic induction of vascular endothelial growth factor (VEGF) protein in astroglial cultures.

Vascular endothelial growth factor (VEGF) couples hypoxia to angiogenesis in ischemic tissues, including brain. Since hypoxia induces VEGF mRNA in astroglial cultures, we examined its effect on VEGF protein, measured by ELISA, in medium from cultures exposed to 95% N2/5% CO2 for up to 24 h. Levels increased with increasing durations of hypoxia, but not glucose deprivation, reaching approximately 30-fold elevation by 24 h. Co2+ and desferrioxamine also increased VEGF levels, while inhibitors of RNA or protein synthesis blocked induction. These findings support a role for astroglia in the hypoxic induction of VEGF expression and release-and potentially angiogenesis-in the ischemic brain.

Vascular endothelial growth factor expression in transient focal cerebral ischemia in the rat.

Vascular endothelial growth factor (VEGF) has been implicated in hypoxia-induced angiogenesis in tumors and ischemia. We examined VEGF mRNA and protein expression after occlusion of the middle cerebral artery (MCA) in rats. VEGF mRNA expression studied by in situ hybridization was increased in the ischemic border zone 24 h after 30, 60 or 120 min of focal cerebral ischemia. VEGF protein expression measured by Western blots was also increased in this region 24 and 48 h after ischemia, and VEGF immunocytochemistry localized this increased expression to astroglia. Thus, VEGF is induced after focal cerebral ischemia and could have a role in pathophysiology and recovery in the ischemic border zone.

Expression of nitric oxide synthase in the cerebral microvasculature after traumatic brain injury in the rat.

Reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry were performed on sections of brain after moderate traumatic brain injury. There was a pronounced increase in NADPHd reactivity and an induction of the endothelial NOS (eNOS) isoform in microvessels surrounding the cortical contusion by 24 h post-injury. This altered microvascular state may contribute to barrier breakdown and hyperemia which characterize traumatic brain injury.

Manganese superoxide dismutase expression in human central nervous system tumors.

Manganese superoxide dismutase (MnSOD) is a superoxide anion scavenger located in mitochondria. Increased expression of MnSOD can diminish oxygen radical-mediated injuries and the cytotoxic effects of tumor necrosis factor alpha, ionizing radiation, and certain chemotherapeutic agents. We used immunohistochemical staining to analyze 42 specimens of human brain tumors and 3 normal brain controls with a polyclonal antibody recognizing human MnSOD. We measured MnSOD in cerebrospinal fluid (CSF) from 14 patients with brain tumors and 7 control patients using an ELISA. Although MnSOD is not readily detected in normal brain, malignant central nervous system tumors, including tumors metastatic to the brain, displayed marked immunoreactivity to MnSOD intracellularly, in the extracellular matrix and in the tumor endothelial cells. Grade IV astrocytomas (glioblastomas), Grade III astrocytomas, and medulloblastomas were strongly immunoreactive, whereas Grade II astrocytomas had much less immunoreactivity. ELISA analysis of CSF samples from patients with malignant tumors also revealed high levels of MnSOD protein, up to 45-fold greater than the level of control CSF samples.

Expression of nitric oxide synthase in human central nervous system tumors.

The nitric oxide synthases (NOS) are a family of related enzymes which regulate the production of NO, a free radical gas implicated in a wide variety of biological processes. Vasodilation and increased tumor blood flow, increased vascular permeability, modulation of host tumoricidal activity, and free radical injury to tumor cells and adjacent normal tissues are pathophysiological features of malignant tumors that may be mediated by NO. We examined human brain tumors for three NOS isoforms and NADPH diaphrase, a histochemical marker of NOS activity in the brain. We detected increased expression of the brain and endothelial forms of NOS [NOS I and NOS II, respectively (C. Nathan and Q. Xie. Cell, 78: 915-919, 1994)] in astrocytic tumors, and the highest levels of expression was found in higher grade tumors. Each of these two isoforms was found in tumor cells and tumor endothelial cells. The macrophage isoform of NOS (NOS III) was less frequently detected and expressed at a lower level, predominantly in tumor endothelial cells. NADPH diaphorase staining for NOS activity paralleled this pattern of NOS expression. Western blot analysis of tumor tissues for these NOS isoforms confirmed these observations. Our data indicate that malignant central nervous system neoplasms express unexpectedly high levels of NOS and suggest that NO production may be associated with pathophysiological processes important to these tumors.

Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B-lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.

Immobilization of enzymes with polyaziridines.

A novel method of enzyme immobilization using a low molecular weight prepolymer of tri-functional aziridines which can immobilize enzymes both by covalent attachment and entrapment within a gel matrix is described. The enzymes are immobilized on a solid support and exhibit an excellent retention of enzymatic activity. The immobilization procedure is essentially a single step process which can be easily performed at room temperature or 4 degrees C in either aqueous solution or in an inert organic solvent. The polyaziridines used in the immobilization are nontoxic, available in bulk at low cost and completely miscible with water and many organic solvents, thus providing one of the most satisfactory methods of immobilization available.

Studies on the serologic response to jellyfish envenomation.

The case histories of three patients with unusual reactions to jellyfish envenomations or increased amounts of anti-jellyfish serum antibodies are presented. These cases demonstrated the following facts: (1) Allergic reactions may play a significant pathophysiologic role in jellyfish envenomation of humans. (2) Elevated specific anti-jellyfish immunoglobulins may persist for several years. (3) Recurrence of the clinical cutaneous reaction to jellyfish stings may occur within a few weeks without additional contact with the tentacles. (4) It is apparent that serologic cross-reactivity between the sea nettle and the man-of-war occur, as do false-positive enzyme-linked immunosorbent assay (ELISA) serologic tests to either jellyfish venom.

Detection of common antigenic sites in lethal proteins of non-related animal venoms.

Monoclonal antibodies neutralizing specific coelenterate lethal toxins were used to determine the presence of homologous antigenic sites on toxin proteins of a rattlesnake (Crotalus durissus terrificus), a hornet (Vespa orientalis) and the sea wasp (Chironex fleckeri). An anti-Portuguese man-o'war toxin antibody was found useful for isolating a C. d. terrificus toxin.

Immunosorbent chromatography of sea nettle (Chrysaora quinquecirrha) venom and characterization of toxins.

A lethal toxic fraction from nematocysts of the sea nettle (Chrysaora quinquecirrha) fishing tentacle was partially purified by immunochromatography using an immobilized monoclonal antibody column. Elution from the immunosorbent was accomplished under mild conditions which conserved the biological activity of the toxin. The isolated fraction, which contained two purified protein bands with molecular weights of 100,000 and 190,000 daltons on SDS polyacrylamide gels, was both cardiotoxic and neurotoxic and exhibited an intravenous lethal activity (LD50) of 0.37 microgram/g in mice.

Sea nettle (Chrysaora quinquecirrha) lethal factor: effect on black lipid membranes.

A lethal toxin from the fishing tentacles of the sea nettle, Chrysaora quinquecirrha, affected ion permeability in black lipid membranes by producing monovalent cation channels. These channels appeared to measure approximately 31 pS.