Endothelium-derived nitric oxide and cyclooxygenase products modulate corpus cavernosum smooth muscle tone.

Relaxation of penile corpus cavernosum smooth muscle is controlled by nerve and endothelium derived substances. In this study, endothelium-dependent relaxation of corporal smooth muscle was characterized and the role of arachidonic acid products of cyclooxygenase in endothelium-dependent relaxation was examined. Endothelium removal from rabbit corpora was performed by infusion with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and was confirmed by transmission electron microscopy. Strips of human and rabbit corporal tissues were studied in the organ chambers for isometric tension measurement. The accumulation of cyclic guanosine monophosphate (cGMP) and the release of eicosanoids from corporal tissue was measured by radioimmunoassay and correlated to smooth muscle relaxation. Our study showed that relaxation of corpus cavernosum tissue to acetylcholine, bradykinin and substance P was endothelium-dependent; potentiated by indomethacin; and inhibited by NG-monomethyl-L-arginine, methylene blue or LY83583. Relaxation to papaverine and sodium nitroprusside was endothelium-independent, and unaffected by NG-monomethyl-L-arginine. Relaxation to vasoactive intestinal polypeptide was partially endothelium-dependent; potentiated by indomethacin; attenuated by NG-monomethyl-L-arginine or methylene blue. The tissue level of cGMP was enhanced by acetylcholine and nitric oxide. Methylene blue inhibited both basal and drug-stimulated levels of cGMP. The release of eicosanoids was enhanced by acetylcholine and blocked by indomethacin. In conclusion, nitric oxide or a closely related substance accounts for the activity of endothelium-derived relaxing factor in the corporal tissue. Inhibition of the release of eicosanoids potentiates the relaxing effect of nitric oxide. Nitric oxide increases tissue cGMP which appears to modulate corporal smooth muscle relaxation.


Immunoreactive prostatic specific antigen in male periurethral glands.

Prostatic specific antigen (PSA) is considered an antigen unique to benign and malignant prostatic tissue. Recent evidence in the literature has raised serious doubts about the specificity of this antigen. In this study twenty male urethral specimens were evaluated for PSA and prostatic acid phosphatase (PAP) from patients without evidence of prostatic cancer. Eight of these 20 urethral specimens exhibited strong immunostaining for both PSA and PAP, localized in the periurethral glands. Five of the 17 urethral biopsies were positive for both antigens, while all three of the whole mount autopsy specimens stained positive for PSA and PAP. Within the autopsy series, there was heterogenous staining of the periurethral glands within the same specimen. This evidence disproves the fact that PSA and PAP are organ specific as previously described. More than likely any tissue of cloacal origin has potential for staining positive for prostatic specific antigen and prostatic acid phosphatase.


Bladder cancer cells express functional receptors for granulocyte-colony stimulating factor.

The effects of human recombinant granulocyte-colony stimulating factor (G-CSF) on the growth of cultured human bladder cancer cells and G-CSF receptor on these cells were investigated. Human bladder cancer cell lines, KU-1 or NBT-2, were incubated with and without G-CSF. At 48 hours, 3H-thymidine uptake of both cells was significantly higher with G-CSF (one ng./ml., 10 ng./ml.) than that of control without G-CSF (p less than 0.05). The binding of 125I-labeled KW-2228, a muteins of G-CSF, to KU-1 and NBT-2 was inhibited by unlabeled KW-2228 in a dose-dependent manner. These results demonstrated that G-CSF stimulates the clonal growth of human bladder cancer cells by binding to its specific receptors.


Long-term followup of 150 patients with testicular cancer treated at a single institution.

Between 1977 and 1988, 150 patients with disseminated primary testicular germ cell tumors were treated with cisplatin, vinblastine and bleomycin. Of the 150 patients 90 (60%) achieved a complete response to chemotherapy. An additional 33 patients achieved a complete response after removal of residual masses following chemotherapy. Thus, 123 of 150 patients (82%) achieved a disease-free status following chemotherapy with or without an operation. After a median followup of 49 months the estimated long-term probability of remaining without failure and of surviving is 77%. With this data base a multivariate analysis of prognostic factors determined the Indiana University staging system to be highly predictive. Other staging systems proved to be less useful. The subset of patients with minimal and moderate disease by Indiana staging containing mature teratoma in the orchiectomy specimen has a particularly excellent prognosis (99% actuarial survival) with chemotherapy.


Enhancement of HIV-1 infection by the capsular polysaccharide of Cryptococcus neoformans.

Patients with AIDS who become infected with Cryptococcus neoformans have a poor prognosis. We speculated that the presence of cryptococcal capsular polysaccharide may enhance HIV-1 infection. In an in-vitro study, the presence of cryptococcal polysaccharide significantly increased (p less than 0.05) production of p24 antigen after infection of H9 cells with HIV-1-infected H9 cells. We also found similar results when lymphocytes from an HIV-1-infected patient were co-cultured with mononuclear cells from an uninfected individual. Our findings suggest a new pathogenic role for the capsular polysaccharide--namely, the capacity to enhance HIV-1 infectivity.


Carbon monoxide poisoning in children riding in the back of pickup trucks.

OBJECTIVE - To describe the case characteristics of a series of children poisoned with carbon monoxide while traveling in the back of pickup trucks. DESIGN - Pediatric cases referred for treatment of carbon monoxide poisoning with hyperbaric oxygen between 1986 and 1991 were reviewed. Those cases that occurred during travel in the back of pickup trucks were selected. Clinical follow-up by telephone interview ranged from 2 to 55 months. SETTING - A private, urban, tertiary care center in Seattle, Wash. PATIENTS - Twenty children ranging from 4 to 16 years of age. INTERVENTION - All patients were treated with hyperbaric oxygen. MAIN OUTCOME MEASURES - Characteristics of the poisoning incident and clinical patient outcome. RESULTS - Of 68 pediatric patients treated for accidental carbon monoxide poisoning, 20 cases occurred as children rode in the back of pickup trucks. In 17 of these, the children were riding under a rigid closed canopy on the rear of the truck, while three episodes occurred as children rode beneath a tarpaulin. Average carboxyhemoglobin level on emergency department presentation was 18.2% +/- 2.4% (mean +/- SEM; range, 1.6% to 37.0%). Loss of consciousness occurred in 15 of the 20 children. One child died of cerebral edema, one had permanent neurologic deficits, and 18 had no recognizable sequelae related to the episode. In all cases, the truck exhaust system had a previously known leak or a tail pipe that exited at the rear rather than at the side of the pickup truck. CONCLUSIONS - Carbon monoxide poisoning is a significant hazard for children who ride in the back of pickup trucks. If possible, this practice should be avoided.


Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor.

Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.


An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12.

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.


Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure,


Rapid-time-course miniature and evoked excitatory currents at cerebellar synapses in situ.

Neurotransmission from mossy fibre terminals onto cerebellar granule cells is almost certainly mediated by L-glutamate. By taking advantage of the small soma size, limited number of processes and short dendrite length of granule cells, we have obtained high-resolution recordings of spontaneous miniature excitatory postsynaptic currents (m.e.p.s.cs) and evoked currents in thin cerebellar slices. Miniature currents have a similar time-course and pharmacology to evoked currents and consist of an exceptionally fast non-NMDA (N-methyl-D-aspartate) component (measured rise-time, 200 microseconds; estimated pre-filtered rise-time less than 100 microseconds; decay time constant, tau = 1.0 ms), followed by 50 pS NMDA channel openings that are directly resolvable. We could find no evidence for the recent proposal that miniature currents in granule cells are mediated solely by NMDA channels with a novel time course. The non-NMDA receptor component of m.e.p.s.cs has a skewed amplitude distribution, which suggests potential complications for quantal analysis. The difference in time course between the m.e.p.s.cs reported here and other synaptic currents in the brain could reflect differences in synaptic function or electrotonic filtering; the relative contribution of these possibilities has yet to be established.


Three-dimensional heteronuclear NMR studies of RNA.

Multidimensional heteronuclear NMR has revolutionized solution structure determinations of proteins. But this technique has not been applied to nucleic acids because of difficulties in the synthesis of isotopically (13C and/or 15N) labelled molecules. Here we report the application of three-dimensional heteronuclear NMR to the study of a uniformly 13C/15N or 15N-labelled RNA duplex of defined sequence. These experiments simplify resonance assignment and the analysis of proton-proton nuclear Overhauser effects (and therefore distance information) in the molecule. Our results show that it is now possible to determine the structures of larger and more complex RNAs using multidimensional heteronuclear NMR.


Tyrosine phosphorylation is required for ligand-induced internalization of the antigen receptor on B lymphocytes.

The membrane immunoglobulin (mIg) receptor for antigen mediates signal transduction in B lymphocytes. Multivalent ligand induces several early activation events including an increase in intracellular calcium concentration, hydrolysis of phosphatidylinositol, and activation of protein kinase C. Most recently, it has been demonstrated that anti-immunoglobulin antibodies induce the rapid accumulation of tyrosine phosphorylated proteins and anti-phosphotyrosine immune complex-associated kinase activity, both of which require receptor crosslinking. Multivalent ligand binding of mIg also results in its association with detergent-insoluble cytoskeletal components and with a slight lag period, in a characteristic pattern of patching, followed by polar capping and finally internalization of the receptors. In this report, we demonstrate that two specific inhibitors of tyrosine phosphorylation, a tyrphostin and genistein, retard the modulation of mIg on the cell surface and inhibit ligand-induced receptor internalization. We conclude that in B cells, tyrosine phosphorylation occurs as the result of crosslinking mIg and is required for subsequent internalization of mIg-ligand complexes. This suggests that tyrosine phosphorylation may be important for B cells to function as specific antigen presenting cells.


Synthetic copolymer 1 inhibits human T-cell lines specific for myelin basic protein.

Copolymer 1 (Cop 1) is a synthetic basic random copolymer of amino acids that has been shown to be effective in suppression of experimental allergic encephalomyelitis and has been proposed as a candidate drug for multiple sclerosis. Cop 1 is immunologically cross reactive with myelin basic protein (BP) and was shown to inhibit murine BP-specific T-cell lines of various H-2 restrictions. In the present study these findings were extended to include human T-cell lines. Cop 1 competitively inhibited the proliferative responses and interleukin 2 secretion of six BP-specific T-cell lines and 13 clones with several DR restrictions and epitope specificities. Conversely, BP inhibited--albeit to a lesser extent--the response of all the Cop 1-specific T-cell lines and clones, irrespective of their DR restrictions. Another random copolymer of tyrosine, glutamic acid, and alanine, denoted TGA, had no effect on these lines. Neither Cop 1 nor BP inhibited the response of lines and clones specific for purified protein derivative. Cop 1 and BP exerted their cross-inhibitory effects only in the presence of antigen-presenting cells. These results suggest that Cop 1 can compete with BP for the binding to human major histocompatibility complex molecules. In view of recent studies implicating BP reactivity in multiple sclerosis, these findings suggest a possible mechanism for the beneficial effect of Cop 1 in this disease.


Infectious enveloped RNA virus antigenic chimeras.

Random insertion mutagenesis has been used to construct infectious Sindbis virus structural protein chimeras containing a neutralization epitope from a heterologous virus, Rift Valley fever virus. Insertion sites, permissive for recovery of chimeric viruses with growth properties similar to the parental virus, were found in the virion E2 glycoprotein and the secreted E3 glycoprotein. For the E2 chimeras, the epitope was expressed on the virion surface and stimulated a partially protective immune response to Rift Valley fever virus infection in vivo. Besides providing a possible approach for developing live attenuated vaccine viruses, insertion of peptide ligands into virion surface proteins may ultimately allow targeting of virus infection to specific cell types.


Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.


Sulfhydryl oxidation down-regulates T-cell signaling and inhibits tyrosine phosphorylation of phospholipase C gamma 1.

Early events in both T-cell receptor (CD3)- and CD4-induced signal transduction pathways include tyrosine phosphorylation of protein substrates, the generation of phosphatidylinositol-phosphate breakdown products, and the mobilization of intracellular Ca2+. Oxidative stress in T cells mediated by sulfhydryl-reactive nonpolar maleimides was shown previously to down-regulate both receptor-mediated Ca2+ mobilization and interleukin 2 production. Here we show that N-ethylmaleimide suppresses both CD3- and CD4-induced Ca2+ responses in human T cells correlating with a reduction in the level of phospholipase C gamma 1 (PLC gamma 1) tyrosine phosphorylation. The inhibition of tyrosine phosphorylation of PLC gamma 1 and additional protein substrates was observed at concentrations of N-ethylmaleimide above 20 microM, whereas lower concentrations of oxidant appeared to increase tyrosine kinase activity following cell stimulation. Sulfhydryl oxidation did not directly affect the catalytic activity of PLC gamma 1, since immunopurified enzyme from N-ethylmaleimide-treated T cells was fully active. Although N-ethylmaleimide treatment of T cells did not cause a direct effect on total pp56lck kinase activity measured in vitro, the interaction between CD4 and pp56lck was oxidation-sensitive in vivo. However, CD3-induced signaling was inhibited at N-ethylmaleimide concentrations lower than that required for CD4/pp56lck dissociation, suggesting that CD3-associated tyrosine kinase activity involves acutely sensitive regulatory thiols. In addition to chemically induced sulfhydryl oxidation, naturally regulated cellular redox states appear to dictate the potential for T-cell responsiveness, since degranulating human peripheral blood neutrophils inhibited CD3-induced Ca2+ mobilization in T lymphocytes. These data indicate that signal transduction in T cells involves the activation of PLC gamma 1 by tyrosine phosphorylation through an oxidation-sensitive intermediate between surface receptors and tyrosine kinases, perhaps including the interaction between CD4 and pp56lck.


Single amino acid change in the helicase domain of the putative RNA replicase of turnip crinkle virus alters symptom intensification by virulent satellites.

The virulent satellite [satellite C (sat C)] of turnip crinkle virus (TCV) is a small pathogenic RNA that intensifies symptoms in TCV-infected turnip plants (Brassica campestris). The virulence of sat C is determined by properties of the satellite itself and is influenced by the helper virus. Symptoms produced in infections with sat C differ in severity depending on the helper virus. The TCV-JI helper virus produces more severe symptoms than the TCV-B helper virus when inoculated with sat C. To find determinants in the TCV helper virus genome that affect satellite virulence, the TCV-JI genome was cloned and the sequence compared to the TCV-B genome. The genomes were found to differ by only five base changes, and only one of the base changes, at nucleotide position 1025, produced an amino acid change, an aspartic acid----glycine in the putative viral replicase. A chimeric TCV genome (TCV-B/JI) containing four of the five base changes (including the base change at position 1025) and a mutant TCV-B genome (TCV-B1025G) containing a single base substitution at position 1025 converted the TCV-B genome into a form that produces severe symptoms with sat C. The base change a position 1025 is located in the helicase of the putative viral replicase, and symptom intensification appears to result from differences in the rate of replication of the satellite supported by the two helper viruses.


Molecular basis of recognition by the glycoprotein hormone-specific N-acetylgalactosamine-transferase.

Lutropin (LH) bears asparagine-linked oligosaccharides terminating with the unique sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2Man alpha, whereas follitropin (FSH) bears oligosaccharides terminating predominantly with the sequence Sia alpha-Gal beta 1-4GlcNAc beta 1-2Man alpha, where Sia is sialic acid. We previously identified a glycoprotein-hormone-specific N-acetylgalactosamine-transferase (GalNAc-transferase) that recognizes a peptide-recognition marker(s) present on the common glycoprotein hormone alpha subunit and beta subunits of human chorionic gonadotropin and LH but not on the beta subunit of FSH. We have now identified an amino acid sequence motif, Pro-Leu-Arg, that is essential for recognition by the GalNAc-transferase. This tripeptide sequence is found 6-9 residues on the amino-terminal side of a glycosylated asparagine on the alpha subunit and beta subunits of LH and human chorionic gonadotropin but is not present on the beta subunit of FSH. The presence of this motif accounts for the differences in LH and FSH oligosaccharide structures. Additional proteins containing this recognition motif have been identified and were determined to bear sulfated oligosaccharides with the same structures as those on the glycoprotein hormones, indicating that these structures are not restricted to the glycoprotein hormones.


CFTR protein expression in primary and cultured epithelia.

The gene responsible for the lethal disorder cystic fibrosis encodes a 1480-amino acid glycoprotein, CFTR. Using polyclonal antibodies directed against separate phosphorylation sites in the pre-nucleotide-binding fold (exon 9) and the R domain (exon 13), we have identified a 165-kDa protein in Xenopus laevis oocytes injected with recombinant CFTR cRNA transcribed from the full-length CFTR plasmid pBQ4.7. A protein of the same mobility was also detected with Western blotting techniques in whole cell extracts of cells that express CFTR mRNA (T84, FHTE, HT-29), including biopsied human nasal and bronchial tissue. Immunodetectable 165-kDa protein was concentrated in the apical membrane fraction of ileal villus tissue. We also report that the 165-kDa protein levels can be modulated pharmacologically, and these levels are appropriately correlated with second-messenger-regulated Cl- efflux. Thus, native or recombinant CFTR can be recognized by these anti-CFTR peptide polyclonal antibodies.


Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells.

One approach to identify postreceptor molecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G0/G1-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical G0/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G0/G1-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor beta, reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon- or IL-6-induced G0/G1-specific arrest requires at least these two receptor-generated events.


Tolerance to a self-protein involves its immunodominant but does not involve its subdominant determinants.

We have produced transgenic mice expression hen egg-white lysozyme (HEL) under the control of a ubiquitous promoter, so that in transgenic animals, HEL is presumably present in the serum and thymus throughout the period of establishment of the T-cell repertoire. We show that HEL transgenic H-2d mice with HEL blood levels greater than 10 ng/ml are tolerant to HEL as well as to the immunodominant peptide 108-116. Thus, their T lymphocytes do not proliferate in response to the immunodominant peptide 108-116 after in vivo immunization with HEL or peptide 108-116. In contrast, in transgenic mice tolerant to HEL, the state of tolerance to subdominant peptides 1-18 and 74-96 appears variable and highly depended on HEL blood levels. Complete unresponsiveness is seen when HEL serum levels are high, and this unresponsiveness is reached at a lower HEL concentration for peptide 1-18 than for peptide 74-96. Thus, a hierarchy exists among the three peptides (108-116 much greater than 1-18 greater than 74-96) for induction of a response to HEL and for HEL tolerance induction in T cells specific for these peptides. Persistence in the periphery of autoreactive T cells recognizing subdominant peptides of self-proteins, as shown in this transgenic model, indicates that self-tolerance is limited to a subset of dominant self-peptides and suggests a role for T lymphocytes specific for subdominant determinants in autoimmunity.


Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor beta after antigen-specific triggering.

Oral administration of myelin basic protein (MBP) is an effective way of suppressing experimental autoimmune encephalomyelitis (EAE). We have previously shown that such suppression is mediated by CD8+ T cells, which adoptively transfer protection and suppress immune responses in vitro. In the present study we have found that modulator cells from animals orally tolerized to MBP produce a suppressor factor upon stimulation with MBP in vitro that is specifically inhibited by anti-transforming growth factor beta (TGF-beta) neutralizing antibodies. No effect was observed with antibodies to gamma interferon, tumor necrosis factor alpha/beta, or indomethacin. In addition, the active form of the type 1 isoform of TGF-beta 1 (TGF-beta 1) can be directly demonstrated in the supernatants of cells from animals orally tolerized to MBP or ovalbumin after antigen stimulation in vitro. Antiserum specific for TGF-beta 1 administered in vivo abrogated the protective effect of oral tolerization to MBP in EAE. Furthermore, injection of anti-TGF-beta 1 serum to nontolerized EAE animals resulted in an increase in severity and duration of disease. These results suggest that immunomodulation of EAE induced by oral tolerization to MBP and natural recovery mechanisms use a common immunoregulatory pathway that is dependent on TGF-beta 1. Implications of such an association are of therapeutic relevance to human autoimmune diseases and may help to explain one of the mechanisms involved in the mediation of active suppression by T cells.


Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes.

Leishmania are obligatory intracellular parasites in mammalian macrophages that gain entry by receptor-mediated phagocytosis. Their major cell surface glycoconjugate, lipophosphoglycan (LPG), has been implicated in this process. A monoclonal antibody specific for Leishmania major LPG (WIC 79.3), which has been shown to block promastigote attachment to macrophages, was used to identify a macrophage-binding determinant of LPG. WIC 79.3 bound exclusively to the phosphorylated repeats of LPG and not to the saccharide core or lipid anchor. Furthermore, the epitope recognized by WIC 79.3 mapped to the phosphorylated oligosaccharide P5b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-, which is unique to the LPG of promastigotes of L.major. Phosphorylated oligosaccharides P3, PO4-6[Gal(beta 1-3)[Gal(beta 1-4) Man(alpha 1-, and P4b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)] Gal(beta 1-4)Man(alpha 1-, were also recognized by WIC 79.3 but with considerably lower (approximately 100-fold) affinities. The phosphorylated oligosaccharide P5b inhibited attachment of promastigotes of L. major to the macrophage cell line J774 to the same degree as phosphoglycan (derived from LPG) and Fab fragments of WIC 79.3, suggesting that P5b is a site of L. major LPG that is recognized by macrophage receptor(s) and is an important determinant in the attachment of promastigotes to host macrophages and initiation of infection.


Monoclonal IgM rheumatoid factors bind IgG at a discontinuous epitope comprised of amino acid loops from heavy-chain constant-region domains 2 and 3.

A combination of site-directed mutagenesis and exon exchange has been used to further define the structure on IgG recognized by monoclonal IgM rheumatoid factors (RFs) from patients with Waldenstrom macroglobulinemia. Most of these RFs bound IgG1, -2, and -4 but not IgG3. For these RFs, His-435 is a critical residue for binding and replacing it with arginine, the residue present in IgG3, destroys or reduces RF binding. However, additional polymorphic sequences in both the heavy-chain constant-region domains (CH) 2 and 3 are important for RF binding. Among the important residues in CH2 are amino acids 252-254 and 309-311, which are conserved among IgG isotypes and comprise two loops of amino acids on the surface of the domain. Therefore, at least three regions, two from CH2 and one from CH3, contribute significantly to the epitope recognized by the RFs. Although this epitope contains many of the same residues as the staphylococcal protein A binding site on IgG, the binding specificities of staphylococcal protein A and monoclonal RFs are not identical. Sera from patients with rheumatoid arthritis contain antibodies directed not only at this epitope but also at other sites on IgG.


Characteristics of anal canal motility after ileoanal anastomosis.

This study attempted to determine the mechanism of anal canal motility after ileoanal anastomosis with the use of alpha-, beta- and muscarinic receptor agents. Forty-five patients, 19 +/- 2 months after operation (mean plus or minus S.E.M.) and 48 control volunteers were studied. Anal manometry indicated no difference in maximum resting pressure and squeeze pressure between the patients and the normal controls. Greater amplitude and less frequent anal canal slow waves were a particular characteristic during the postoperative period. Phentolamine (alpha-blocker) exhibited a stimulatory effect on anal resting tone in the normal rectum, while propranolol (beta-blocker) and atropine (muscarinic blocker) had no effect. However, alpha-, beta- and muscarinic receptors all had stimulatory effects on postoperative anal tone. The muscarinic receptor had dominant effects on the reduction in anal canal slow wave frequency after ileoanal anastomosis. Our results indicated that neorectum after ileoanal anastomosis had hybrid characteristics of both rectum and ileum. Through analysis of receptor-related motor function, we may be able to improve our understanding of dyscontinence after ileoanal anastomosis.


Cadmium's action on NRK-49F cells to produce responses induced also by TGF beta is not due to cadmium induced TGF beta production or activation.

Transforming growth factor beta (TGF beta) is a multifunctional regulator of cell growth that has either a stimulatory or inhibitory effect on cell proliferation, depending on TGF beta concentration and on cell type, history and culture conditions. Cadmium mimics some of the effects of TGF beta in cultured cells. In this study the effects of Cd2+ and TGF beta on EGF-induced DNA synthesis in a clonal subpopulation (N1) of NRK-49F cells were compared. It was found that TGF beta 1 and cadmium both inhibit EGF-induced DNA synthesis and cell proliferation in a dose-dependent fashion, but that neither inhibits EGF-induced myc oncogene accumulation. TGF beta 1 and cadmium added at the same time as EGF or several hours after EGF addition showed similar inhibitory effects on EGF-induced [3H]Tdr incorporation, indicating that the inhibitory effect of TGF beta 1 and cadmium on EGF-induced DNA synthesis does not involve early G1 events. Rather, they occur in late G1, at the G1/S boundary or during S phase. Because of the similarities in nature and timing of the Cd2+ and TGF beta responses, the possibility that Cd2+ acts through stimulation of TGF beta production and/or activation was explored. It is shown in this paper however that TGF beta neutralizing antibody blocks the effects of TGF beta 1, but not the cadmium effects, on EGF-induced DNA synthesis, suggesting that cadmium is not functioning through activation or preinduction of TGF beta.


Effect of 2-acetylaminofluorene on intracellular free Ca2+ in isolated rat hepatocytes.

The effect of 2-acetylaminofluorene (2-AAF) on the intracellular free Ca2+ ([Ca2+]i) and viability of isolated rat hepatocytes has been investigated using the fluorescent probes quin 2 and propidium iodide respectively. At the highest concentration tested (224 microM), 2-AAF produces an elevation of [Ca2+]i which shows a biphasic profile. A small initial increase is observed during the first 5 min; this is followed by a considerable rise which reaches up to 2.5 times the control value at 15 min. These changes in intracellular calcium are not accompanied by detectable alterations in cell viability. In order to determine the mechanisms by which this effect of 2-AAF takes place, three calcium antagonists, namely verapamil, TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) and ruthenium red (RuR), have been used. The results suggest that the first phase is dependent upon internal Ca2+ store mobilization, while the second phase seems to be related to Ca2+ entry from the extracellular space. The data obtained with RuR further indicate that mitochondria may be involved in the perturbation of calcium homeostasis caused by 2-AAF. In addition, in the experiments involving antagonists, no consistent pattern emerges that suggests a close relationship between intracellular Ca2+ levels and cell viability. The present study provides further information on the mechanisms by which these well-known hepatotoxin 2-AAF may interact with liver cells. It also shows that when these cells are exposed to a toxin, short-term changes in [Ca2+]i may not be accompanied by loss of cell viability, and conversely, that changes in cell viability may occur without alterations in [Ca2+]i.


MR imaging of joints: analytic optimization of GRE techniques at 1.5 T.

To clarify the choice of imaging parameters for optimal gradient-recalled echo MR scanning of joints, we analyzed the behavior of contrast-to-noise and signal-to-noise ratios for spoiled (i.e., fast low-angle shot [FLASH] or spoiled GRASS) and steady-state (i.e., gradient-recalled acquisition in the steady state [GRASS] or fast imaging with steady precession) techniques at 1.5 T. The analysis is based on tissue characteristics derived from spin-echo measurements of hyaline cartilage and synovial fluid signal in the patellofemoral joints of 11 volunteers. Separate analysis of contrast-to-noise and signal-to-noise ratios for multiplanar (long TR) acquisitions shows that these parameters are each improved compared with single-slice methods. At TRs greater than 250 msec, there is no significant difference in the contrast behavior of FLASH and GRASS. For optimal contrast-to-noise ratio (synovial fluid-cartilage), the best multiplanar sequence (for TE less than 23 msec) is with a short TE and a large flip angle (e.g., 400/9/73 degrees [TR/TE/flip angle]). If a single-scan or three-dimensional technique is desired, than a GRASS sequence at minimal TR and TE and intermediate flip angle (18/9/32 degrees) is best. For optimal signal-to-noise ratio (for both synovial fluid and hyaline cartilage), the best multiplanar sequence uses a short TE and an intermediate flip angle (e.g., 400/9/30 degrees). If a short TR, high signal-to-noise technique is desired, then GRASS (18/9/13 degrees) is superior to FLASH.


Ventricular dysrhythmias in patients undergoing coronary artery bypass graft surgery: incidence, characteristics, and prognostic importance. Study of Perioperative Ischemia (SPI) Research Group.

To determine the incidence and characteristics of ventricular dysrhythmias (premature ventricular contractions greater than 30/min, ventricular tachycardia greater than or equal to 3 beats, and ventricular fibrillation) and whether a relationship exists between ventricular tachycardia and myocardial ischemia in patients undergoing coronary artery bypass graft surgery, we continuously monitored 50 patients for 10 perioperative days using two-lead electrocardiography. Electrocardiographic changes consistent with ischemia were defined as a reversible ST depression greater than or equal to 1.0 mm, or ST elevation greater than or equal to 2.0 mm from baseline, lasting at least 1 minute. Ventricular dysrhythmias developed in 10% of patients preoperatively and in 16% intraoperatively before bypass surgery. The highest incidence occurred postoperatively, with ventricular dysrhythmias developing in 66% of patients (22% to 44% of patients on any postoperative day 0 to 7). Premature ventricular contractions were greater than 30/hr in 6% of patients preoperatively, in 8% intraoperatively before bypass, and in 34% postoperatively (6% to 23% of patients on any postoperative day). Twenty-nine patients (58%) developed 76 verified episodes of greater than or equal to 3 beats of ventricular tachycardia. Ventricular tachycardia occurred in 6% of patients preoperatively (four episodes), in 8% of patients intraoperatively prior to bypass (four episodes), and 54% of patients postoperatively (5% to 21% on any postoperative day). No patient developed ventricular fibrillation. All postoperative ventricular tachycardia episodes (after tracheal extubation) were asymptomatic. Postoperatively, 48% of patients developed ischemia, compared with 12% preoperatively and 10% intraoperatively before bypass surgery. Only 5 of 68 (7%) postoperative ventricular tachycardia episodes occurred within 3 hours of an ischemia episode.(ABSTRACT TRUNCATED AT 250 WORDS)


Two amino acid substitutions in apolipoprotein B are in complete allelic association with the antigen group (x/y) polymorphism: evidence for little recombination in the 3' end of the human gene.

We report the identification of an A-to-G base change, in exon 29 of the apolipoprotein B (apo B) gene, that results in the substitution of serine for asparagine at residue 4311 of mature apo B100. In a recent publication, Huang et al. have reported a C-to-T base change in exon 26 that causes the substitution of leucine for proline at residue 2712 of apo B. We have found complete linkage disequilibrium between the alleles at both these sites and an immunochemical polymorphism of LDL designated antigen group (x/y) (Ag(x/y)) in a sample of 118 Finnish individuals. This implies that either one of these substitutions--or both of them combined--could be the molecular basis of the Ag(x/y) antigenic determinants, with the allele encoding serine4311 plus leucine2712 representing the Ag(x) epitope, and that encoding asparagine4311 plus proline2712 the Ag(y) epitope. In a sample of 90 healthy Swedish individuals the Leu2712/Ser4311 allele is associated both with reduced serum levels of LDL-cholesterol and apo B and with raised levels of HDL. However, these differences are of smaller effect than those associated with the XbaI RFLP of the apo B gene in this sample. We have also genotyped 523 individuals from European, Asian, Chinese, and Afro-Caribbean populations and have found complete association between the sites encoding residues 2712 and 4311 in all of these samples, although there are large allele frequency differences between these populations. In addition, there is strong linkage disequilibrium with allelic association between the alleles of these sites and those of the XbaI RFLP in all the populations examined. Taken together, these data suggest that, since the divergence of the major ethnic groups, there has been little or no recombination in the 3' end of the human apo B gene.


Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease.

Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation (delta F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cystic fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57 (60%) are of Ashkenazi origin. Together with the delta F508 (23% in this group), G542X, N1303K, and 1717-1G----A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the delta F508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease.


[A case of verrucous carcinoma showing a good partial response by C.P.E. (CDDP, PEP, etoposide) chemotherapy].

A 60-year-old woman was admitted to our hospital with a complaint of rough feeling on the oral mucosa and diagnosed as verrucous carcinoma with histopathological examination. She was treated with CPE chemotherapy, and showed a good response and improvement of clinical symptoms. Toxicities were leukopenia, alopecia and anorexia. However, these were slight side effects. The patient is currently healthy with no recurrence after two years and 3 months. CPE chemotherapy is considered to be effective for a patient with verrucous carcinoma.


Antibody-dependent enhancement of hantavirus infection in macrophage cell lines.

Antibody-dependent enhancement (ADE) of hantavirus infections (strains Hantaan 76-118 and SR-11) was studied using macrophage-like cell lines (J774.1, P388D1, and U937). Significantly higher virus titers (1,000 to 4,000 FFU/ml) were obtained by pretreatment of the virus with immune serum as compared to normal serum (less than 20 FFU/ml). Monoclonal antibodies (MAbs) to strain Hantaan 76-118 were employed to determine the antigenic determinants responsible for the ADE activity. ADE of the infection occurred with MAbs to both G1 and G2 envelope glycoproteins, but not with MAbs to nucleocapsid protein. Antigenic determinants related to haemagglutination or virus neutralization were found to cause ADE of the infection.


Effect of macrophage source and activation on susceptibility in an age-dependent model of murine hepatitis caused by a phlebovirus, Punta Toro.

The Adames strain of a bunyavirus, Punta Toro virus (PTV), is an hepatotrophic virus that has been described to produce an age-dependent lethal hepatic necrosis in 3-4 week old C57BL/6 mice, but 8 week old mice survive with minimal necrosis. The course of PTV infection in vitro in macrophages derived from these mice served as a model to study the pathogenesis of phlebovirus infection. Peripheral blood monocytes, resident or elicited peritoneal macrophages, and Kupffer cell liver macrophages, as well as hepatocytes, were able to support replication of PTV in vitro to a variable extent. Kupffer cells were the only population of macrophages, however, that expressed an age-related ability to affect viral infection and replication in vitro, suggesting that liver macrophages may have a unique modulatory effect on the occurrence and severity of PTV-induced hepatitis in mice. Whereas PTV showed minimal replication in resident peritoneal macrophages, the virus could replicate effectively in peritoneal macrophages elicited by thioglycolate. Activation of peritoneal macrophages with endotoxin resulted in a significant inhibition of intrinsic PTV replication (p less than 0.001), and a modest extrinsic inhibitory effect on PTV replication in cocultured hepatocytes. Both effects persisted in the presence of anti-interferon. These results indicate that the source and state of activation of macrophage/monocyte populations can influence the course of infection in vitro by the phlebovirus, Punta Toro, and can modulate infection in cocultured target cells.


Molecular cloning and characterization of the fusaric acid-resistance gene from Pseudomonas cepacia.

Fusaric acid-resistance genes (fus) were isolated from Pseudomonas cepacia. The nucleotides of the 5437 base pairs containing the fus genes were sequenced.


Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors.

Two properties were found to distinguish neuronal from muscle nicotinic acetylcholine receptors (nAChRs). First, neuronal nAChRs have a greater Ca2+ permeability. The high Ca2+ flux through neuronal nAChRs activates a Ca(2+)-dependent Cl- conductance, and the Ca2+ to Cs+ permeability ratio (PCa/PCs) is 7 times greater for neuronal than for muscle nAChRs. A second difference between the receptor types is that neuronal nAChRs are potently modulated by physiological levels of external Ca2+. Neuronal nAChR currents are enhanced by external Ca2+ in a dose-dependent manner. The results indicate that changes in extracellular Ca2+ modulate neuronal nAChRs and may modulate cholinergic synapses in the CNS. Also, activation of neuronal nAChRs produces a significant influx of Ca2+ that could be an important intracellular signal.


Slow voltage-dependent changes in channel open-state probability underlie hysteresis of NMDA responses in Mg(2+)-free solutions.

Many single-channel studies rely on the assumption that the channels are functioning under steady-state conditions. In examining the basis for nonlinear whole-cell current-voltage curves in Mg(2+)-free solutions we discovered that N-methyl-D-aspartate (NMDA) channels in excised patches reversibly shifted their open-state probability (Po) in a voltage-dependent way, exhibiting approximately 3- to 4-fold greater Po at positive potentials than at rest. Changes in Po were mainly attributable to shifts in frequency of channel opening. Po changed remarkably slowly (2-15 min), explaining the hysteresis of whole-cell current-voltage curves obtained in nonequilibrium conditions. The slow increase in Po provides a mechanism by which NMDA channels can substantially increase Ca2+ influx in cells depolarized for prolonged periods of time and may play a role in excitotoxicity.


Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.

Functionally diverse GluR channels of the AMPA subtype are generated by the assembly of GluR-A, -B, -C, and -D subunits into homo- and heteromeric channels. The GluR-B subunit is dominant in determining functional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and unedited forms, GluR-B(R) and GluR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GluR-B(R) channels expressed in 293 cells display a low divalent permeability, whereas homomeric GluR-B(Q) and GluR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine- and glutamine-containing subunits generates cells with varying divalent permeabilities depending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GluR-B(R) subunit determines low divalent permeability in heteromeric GluR channels and that changes in GluR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell.


Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.

We have cloned a functional cDNA encoding the cyclic nucleotide-gated channel selectively expressed in catfish olfactory sensory neurons. The cyclic nucleotide-gated channels share sequence and structural features with the family of voltage-gated ion channels. This homology is most evident in transmembrane region S4, the putative voltage sensor domain, and the H5 domain, thought to form the channel pore. We have characterized the single-channel properties of the cloned catfish channel and compared these properties with the channel in native catfish olfactory sensory neurons. The channel is activated equally well by cAMP and cGMP, shows only a slight voltage dependence of gating, and exhibits a pH- and voltage-dependent subconductance state similar to that observed for the voltage-gated L-type calcium channel.


Sexual differentiation and regulation of cytochrome P-450 CYP2C7.

The multigene family of proteins known as the cytochrome P-450-dependent monooxygenases play a central role in the metabolism of hormones and foreign compounds. As part of our studies into the function and regulation of these proteins we have isolated a little studied constitutively expressed isozyme CYP2C7 and have investigated its substrate specificity and mode of regulation. Interestingly the haem of this enzyme in its isolated form is almost 100% in the high spin state. The enzyme was active in the metabolism of a range of model resorufin substrates, but exhibits highest activity towards benzyloxyresorufin. Indeed, this isozyme appears to play a significant role in the metabolism of this substrate in microsomal samples from untreated male rats. Tissue distribution studies indicated that CYP2C7 was expressed in liver, kidney and possibly muscle tissue. Cytochrome P-450 CYP2C7 could not be significantly induced by any of a wide range of known modulators of cytochrome P-450 expression at the mRNA level, however some significant changes in protein expression were observed. Some of the agents used (e.g., diethylnitrosamine and carbon tetrachloride) caused a significant reduction in the expression of this protein. In agreement with other reports where mRNA levels were measured we found that the level of CYP2C7 protein expression was sexually differentiated. Female rats express two to three times the level found in males, the sex difference being reversible by hypophysectomy.


Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro.

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10(-9) M. At concentration 10(-8) M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.


Electron image analysis of ribosomal subunits from Thermus aquaticus.

Electron micrographs of ribosomal subunits from the thermophilic bacterium Thermus aquaticus were analysed using multivariate statistical analysis and characteristic views constructed to reproducible spatial resolutions ranging from 1.9 to 3.6 nm. These views were comparable to morphological classes of Escherichia coli ribosomal subunits, albeit with differences in fine features also found in archaebacterial ribosomes.


The 16S rRNA gene of Streptomyces lividans TK64 contains internal promoters.

A 632-bp Sau3AI fragment of Streptomyces lividans TK64 genome was found to confer promoter activity in Streptomyces and Escherichia coli. This fragment showed almost identical sequence (97.8%) to the S. coelicolor 16S rRNA segment encompassing from nucleotide 706 to 1337 region. The transcription start points of this fragment were identified by the primer extension method. Analysis of the nucleotide sequence upstream the transcription start points revealed two putative E. coli-like promoters resided within this fragment. The occurrence of internal promoters active in Streptomyces and E. coli was also confirmed in the 16S rRNA gene of rrnE operon from TK64.


Nucleotide sequence of the complementary DNA for human Pit-1/GHF-1.

Human cDNA clones encoding Pit-1/GHF-1, a pituitary-specific DNA binding factor, were obtained by PCR following reverse transcription of human pituitary RNA. It is approx. 1.3 kb in size with 0.1 kb 5' non-coding region, 0.9 kb protein-coding region and 0.3 kb 3' non-coding region. The predicted human Pit-1/GHF-1 peptide structure has 291 amino acids and is highly conserved among mouse, rat and bovine. In addition, the 5' non-coding region is highly conserved with rat pit-1/GHF-1 sequence to the transcription start site.


Organization and nucleotide sequence of carp gonadotropin alpha subunit genes.

We have used PCR to amplify and align the sequence of two genes encoding cGTH alpha. Both genes comprise four exons and three introns. The organization of cGTH alpha genes is very similar to that of mammalian GTH alpha genes. However, the cGTH alpha genes only span a region of 1.2 kb which is much smaller than those mammalian GTH alpha genes.


Abnormal collagen fibril structure as studied by electron microscopy.

Transmission electron microscopy has emerged as an ideal tool for the study and diagnosis of various disorders that involve collagen, since the information obtained by this technique is at the ultrastructural level. Structural alterations of collagen fibrils brought about by these disorders are discussed. The positive staining pattern of such fibrils is also investigated. In addition, this review describes how quantitative studies of electron-optical images from abnormal collagen fibrils can lead to information about the changes produced by collagen defects which relate to molecular or fibril architecture.


Interleukin-11 enhances human megakaryocytopoiesis in vitro.

We investigated the effect of recombinant human interleukin-11 (rhIL-11) on human megakaryocytopoiesis. Nonadherent and T-cell-depleted human bone marrow (BM) mononuclear cells were cultured in a serum-free agar culture system. rhIL-11 alone did not stimulate the growth of human megakaryocyte colonies. However, when rhIL-11 was combined with optimal or suboptimal doses of rhIL-3, the number and size of the megakaryocyte colonies increased. The same results were obtained when highly purified BM CD34-positive cells were used as target cells. Next, we investigated the effect of rhIL-11 on the ploidy of megakaryocytes. The ploidy distribution of individual cells in megakaryocyte colonies obtained by rhIL-11 in combination with rhIL-3 was significantly shifted towards higher values. Furthermore, when highly purified CD41-positive BM cells were cultured in the presence of rhIL-11, the ploidy distribution was shifted towards higher values. This effect was not suppressed by anti-IL-6 antibody. These results suggest that rhIL-11 acts directly as a megakaryocyte potentiator and may play a role in regulating human megakaryocytopoiesis.


Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro.

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.


Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor.

Stem cell factor (SCF) stimulates the growth of burst-forming unit-erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) by binding to a specific cell surface receptor. The receptor for SCF is encoded by the protooncogene c-kit. After immunizing mice with the human erythroleukemia cell line OCIM1, we obtained a monoclonal antibody (MoAb) that recognizes the human c-kit receptor. This MoAb, designated SR-1, blocks binding of 125I-human SCF to the c-kit receptor, and neutralizes the biologic effects of SCF in hematopoietic colony assays. With few exceptions, c-kit expression was identified on all hematopoietic and lymphoid cell lines tested by indirect immunofluorescent analysis using SR-1 and by binding studies with 125I-SCF. SR-1 recognizes a small fraction of normal bone marrow mononuclear cells, and these cells have the morphologic appearance of blasts. Colony assays show that BFU-E and CFU-GM display the c-kit receptor. SR-1 does not cross-react with murine c-kit protein, indicating that the binding epitopes of the human and murine c-kit receptors are antigenically distinct. This MoAb may be useful to characterize the spectrum of cells that display the c-kit receptor and to further define the role of SCF in hematopoiesis.


Characterization of glycoprotein IIb/IIIa-positive cells in human umbilical cord blood: their potential usefulness as megakaryocyte progenitors.

A need for hematopoietic stem cells, particularly cells destined to enter the megakaryocyte (MK) series, prompted phenotypic analysis of mononuclear leukocytes in human cord blood. To this end, immunohistochemical, flow cytometric, and ultrastructural techniques were used. The immunogold silver enhancement method (IGS) for the detection of the MK-specific glycoprotein (GP) IIb/IIIa epitopes combined with a monocyte-specific stain for alpha-naphthyl butyrate esterase proved to be superior to flow cytometry (FACS) for precise quantitation of cell types in each sample. Immunoelectron microscopy afforded a description of distinctions between precursors bearing GPIIb/IIIa epitopes and other stem cells of the myeloid series. The number of presumed MK progenitors was surprisingly high, averaging 1.8% +/- 1.3% (range, 0.2% to 4.6%) by IGS and 4.1% +/- 3.0% (range, 0.2% to 9.3%) by FACS analysis. The occurrence of GPIIb/IIIa-positive denuded MK nuclei in cord blood was of interest, but was too small to affect these data. These observations should advocate a greater use of cord blood for restitution of MK/platelet-lineage-depleted patients as well as for experimental studies concerned with MK differentiation.


Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells.

The kit ligand (KL), also termed stem cell factor (SCF), is a recently discovered hematopoietic growth factor that augments response of early progenitor cells to other growth factors and supports proliferation of continuous mast cell lines. Histological studies suggest that the receptor for SCF/KL, the c-kit proto-oncogene product, is present in bone marrow megakaryocytes. We studied the effects of SCF/KL on immortalized human megakaryocytic cell lines (CMK, CMK6, and CMK11-5) and on isolated human marrow megakaryocytes. Human SCF/KL alone or in combination with the hematopoietic growth factors, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, stimulated proliferation of these megakaryocytic cell lines. SCF/KL treatment did not alter expression of gpIb, gpIIb/IIIa, LFA-1, ICAM-1, or GMP-140 in CMK cells. No effect on ploidy was observed. Furthermore, human SCF/KL induced expression of IL-1 alpha, IL-1 beta, IL-2, and IL-6 in CMK cells. In a fibrin clot system, SCF/KL modestly potentiated megakaryocyte colony formation when added alone to cultures containing CD34+, DR+ bone marrow cells. Addition of SCF/KL with IL-3 or GM-CSF to these cultures resulted in a more marked marrow megakaryocytic cells. SCF/KL may directly affect megakaryocytopoiesis, as well as secondarily modulate hematopoiesis through induction of cytokines in target cells.


Regulation of B-cell growth and immunoglobulin gene transcription by interleukin-6.

Interleukin-6 (IL-6) stimulates growth and immunoglobulin (lg) secretion in Epstein-Barr virus (EBV)-infected B cells. In this study, we demonstrate that B-cell activation by IL-6 is associated with an initial induction of c-myc, a gene believed to act as a competence factor for increased RNA transcription and DNA replication, and by increases in DNA, RNA, and protein synthesis, as well as cell number. IL-6 increased the levels of lg mRNA per cell in comparison to a non-cycle-dependent cellular mRNA, tubulin. However, two other cell cycle-dependent cellular mRNAs, c-myc and actin, were also induced by IL-6 comparable to lg mRNAs. Increased levels of lg mRNA were not due to significant changes in RNA turnover, but appeared to reflect increased levels of RNA transcription. Together, these findings support the notion that IL-6 plays an important role as a stimulator of DNA and RNA synthesis in EBV-activated B cells.


Clinical significance of multidrug resistance P-glycoprotein expression on acute nonlymphoblastic leukemia cells at diagnosis.

To evaluate the clinical value of the expression of multidrug resistance P-glycoprotein (P-170) on the surface of acute nonlymphoblastic leukemia (ANLL) cells, we analyzed specimens from 150 newly diagnosed patients for staining with MRK16, a monoclonal antibody (MoAb) that binds to an external epitope of P-170. Other surface markers (CD13, CD14, CD15, and CD34) were studied by the same technique. A marker was considered positive when 20% or more cells were stained. Of 150 samples, 71 were P-170-positive. These cases did not differ from P-170-negative cases with regard to age, sex, initial white blood cell (WBC) counts, or French-American-British (FAB) type (except for M3 ANLL, which were more frequently negative). However, leukemias arising from previous myelodysplastic syndrome (MDS) and therapy-induced leukemias were more frequently P-170-positive. CD34 and P-170 expression were significantly associated. All patients were treated by intensive chemotherapy. Complete remission (CR) rates were significantly lower in P-170-positive (23/71, 32%) than in P-170-negative cases (64/79, 81%) (P less than 10(-5)). CD34 positivity was also associated with a low remission rate (P less than 10(-5)). Survival was shorter for P-170- and CD34-positive patients (P less than 10(-5)). The prognostic value of both markers was confirmed in multivariate analysis. CR duration was also shorter for P-170-positive cases, but the difference is less significant (P = .05). It is concluded that P-170 analysis may be an important tool for predicting the outcome of intensive chemotherapy in ANLL patients.


The gene for B7, a costimulatory signal for T-cell activation, maps to chromosomal region 3q13.3-3q21.

B7 is an activation antigen expressed on activated B cells and gamma-interferon-stimulated monocytes. The B7 antigen is the natural ligand for CD28 on T cells. After engagement of T-cell receptor with antigen in association with major histocompatibility complex class II, a second signal mediated through the binding of B7 to CD28 greatly upregulates the production of multiple lymphokines. We have now mapped the B7 gene to human chromosome 3 using the technique of polymerase chain reaction on a panel of hamster x human somatic cell hybrid DNAs. We have further localized the gene to 3q13.3-3q21 using in situ hybridization on human metaphase chromosomes. Trisomy of chromosome 3 is a recurrent chromosome change seen in various lymphomas and lymphoproliferative diseases, particularly diffuse, mixed, small, and large cell lymphomas, human T-cell lymphotropic virus type I-induced adult T-cell leukemia, and angioimmunoblastic lymphadenopathy. A number of chromosomal defects involving 3q21 have been described in acute myeloid leukemia and also in myelodysplastic and myeloproliferative syndromes. The mapping of B7 may permit further insight into disease states associated with aberrant lymphocyte activation and lymphokine synthesis.


Differential expression of cytokines in human blood monocyte subpopulations.

Cytokine expression was analyzed in CD14++ regular monocytes and in the novel subset of CD14+/CD16+ small monocytes. Biologic activity for tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 in the supernatant of elutriator-enriched, cell sorter-purified small monocytes was about 10-fold lower compared with regular monocytes when stimulated with lipopolysaccharide (LPS) for 12 hours. In CD14++ regular monocytes levels were 1,157 U x 10(-3)/mL, 158 U/mL, and 1,337 U/mL for TNF, IL-1, and IL-6, respectively. By contrast, CD14+/CD16+ small monocytes exhibited 137 U x 10(-3)/mL, 14 U/mL, and 60 U/mL for TNF, IL-1, and IL-6, respectively. Additional treatment with interferon-gamma enhanced production of TNF in both subsets, but CD14+/CD16+ small monocytes still exhibited lower levels. Stimulation of the monocyte subsets by platelet-activating factor gave the same pattern of results. Hybridization with 32P-labeled oligonucleotides specific for the respective cytokine messenger RNAs (mRNAs) showed a 10-fold lower prevalence of transcripts for TNF, IL-1, and IL-6, as well. By contrast, the constitutive expression of Glyceraldehyde-3-phosphate-dehydrogenase mRNA was 1.7-fold higher in the CD14+/CD16+ small monocytes. These data indicate that the novel subset of small monocytes is selectively suppressed in the expression of the cytokines TNF, IL-1, and IL-6, suggesting that these cells may comprise a deactivated type of cell. The expression of class II transcripts in the small monocytes is, however, similar to the regular monocytes, and the cell surface expression of class II protein about threefold increased. Thus, the novel subset of small monocytes appears to be a functionally distinct type of cell.


Use of fibrinogen to enhance the antitumor effect of OK-432. A new approach to immunotherapy for colorectal carcinoma.

OK-432 (5 KE), an immunomodulatory agent prepared from an attenuated strain of Streptococcus pyogenes, was dissolved in 1 ml of aprotinin (1000 KIE) and mixed with 80 mg of fibrinogen containing Factor XIII. A single intratumoral injection of the mixture was performed preoperatively under endoscopy in 20 patients with colorectal carcinoma. Postoperative histopathologic examinations revealed the formation of fibrin fibers at the site of injection and marked infiltration of inflammatory cells into the tumor stroma on the day after injection; the formation of granulomas containing many giant cells after 4 to 7 days; and extensive regression of tumor tissue after 14 days. This study suggests that the high concentration of exogenous fibrinogen gelatinized enough to trap OK-432 in tumor stroma and that OK-432 induced granulomatous hypersensitivity to degenerate tumor stroma, thereby causing regression of the tumors.


Alternating non-cross-resistant chemotherapy for non-Hodgkin's lymphoma of intermediate-grade and high-grade malignancy. A pilot study.

Thirty-two patients with advanced non-Hodgkin's lymphoma (NHL) with aggressive histologic findings were treated with cyclophosphamide, doxorubicin, methotrexate with leucovorin rescue, bleomycin, vincristine, etoposide, ifosfamide, and prednisolone (CAMBO-VIP), in which presumably non-cross-resistant myelosuppressive and nonmyelosuppressive agents were administered during alternate weeks for 12 weeks. To ensure the high-dose intensity of the protocol, dose reduction and delay in treatment were minimized. Three patients were treated inadequately. Twenty-six (89.7%) of 29 evaluable patients had a complete response, and three had a good partial response. Relapse occurred in four patients, with a median follow-up of 29 months. The actuarial overall survival and disease-free survival were estimated to be 87.6% and 75.9%, respectively. The CAMBO-VIP treatment was well tolerated; myelosuppression was severe but transient and caused no serious infections. Side effects that affected dose intensity were oral ulceration, occurring in 28 patients, and blister formation under the thickened skin of palms and/or soles, followed by desquamation (5 patients). Hepatic toxicity was generally mild to moderate; it was severe in one patient. A 12-week regimen of CAMBO-VIP was effective for advanced NHL with aggressive histologic findings.


Choroid plexus tumors in childhood. Response to chemotherapy, and immunophenotypic profile using a panel of monoclonal antibodies.

Clinical and immunophenotypic (IP) data are presented on three children with choroid plexus (CP) tumors. Two children ages 0.2 and 2 years old with histologically proven malignant tumors had subtotal tumor resections and were treated with ten monthly cycles of eight-drugs-in-1-day chemotherapy without radiation therapy (XRT). Both are free of tumor 4 and 7 years later. The literature on survival of children with CP carcinomas after chemotherapy and XRT is reviewed. Monoclonal antibodies to 17 neuroectodermal, neuronal, glial, and leukocytic markers on frozen sections were used to IP the two malignant tumors and a CP papilloma. All tumors expressed two neuroectodermal markers (PI-153/3 and UJ 223.8), cytokeratin 19, and a neural and leukocyte marker (Thy-1). Two of three expressed neurofilament protein (NF-H) and glial fibrillary acidic protein (GFAP) and one expressed NF-M and common leukocyte antigen. None had strong expression for the panneuroectodermal antigen UJ13/A. There was variable expression of the other markers. The most common IP profile for CP tumors (cytokeratin 18+, PI-153/3+, Thy-1+, UJ 223.8+, and GFAP+ and UJ13A-, UJ 127.11-, and NF-L-) is discussed in the context of the current knowledge of the ontogenetic origin of the CP. It was concluded that chemotherapy for malignant CP tumors can be associated with long-term survival in young children and that the unique IP profile of CP tumors with coexpression of three intermediate filaments suggests new and provocative evidence of their cellular complexity and heterogeneity.


Cytoskeletal changes in hepatocytes induced by Microcystis toxins and their relation to hyperphosphorylation of cell proteins.

The heptapeptide toxins produced by the blue-green alga (cyanobacterium) Microcystis aeruginosa are selectively hepatotoxic in mammals. The characteristic post-mortem pathology of the liver is extensive lobular disruption due to sinusoidal breakdown, leakage of blood into the tissue and hepatocyte disintegration. Isolated hepatocytes incubated with toxin show severe structural deformity and surface blebbing. This paper demonstrates the effects of Microcystis toxins on the contraction and aggregation of actin microfilaments, and on the relocation and breakdown of cytokeratin intermediate filaments, in cultured hepatocytes. Earlier work did not show changes in the assembly/disassembly of actin; however, this paper demonstrates the change in cytokeratin from intermediate filaments to distributed granules in the cytoplasm of toxin-affected cells. Acrylamide gel electrophoresis of cytoskeletal fractions from hepatocytes did not show changes in total cytokeratins; however, marked changes in the immunogenicity of cytokeratins at 52 and 58 kDa were seen on toxin exposure of cells. Measurement of 32P-phosphorylation of proteins in toxin-affected cells incubated with [32P]orthophosphate showed a dramatic increase compared to control incubations. This is in agreement with research elsewhere describing phosphatase inhibition in vitro by Microcystis toxins. The data indicate that phosphorylated cytokeratin is a major component of cytoplasmic fraction phosphorylated protein after toxin exposure to hepatocytes. It is concluded that the mechanism of Microcystis toxicity to the hepatocyte is through cytoskeletal damage leading to loss of cell morphology, cell to cell adhesion and finally cellular necrosis. The underlying biochemical lesion is likely to be phosphatase inhibition causing hyperphosphorylation of a number of hepatocyte proteins, including those cytokeratins responsible for microfilament orientation and intermediate filament integrity.


Identification of a CD21 receptor-deficient, non-Ig-secreting peripheral B lymphocyte subset in HIV-seropositive drug abusers.

We have studied 61 HIV-seropositive heroin addicts (18 asymptomatic, 20 ARC, and 23 AIDS cases), 26 HIV-seronegative heroin addicts, and 45 healthy blood donors, matching the groups each other for age and sex. We have focused on the phenotypic characteristics of B subpopulations in the peripheral blood of HIV-seropositive and -seronegative drug abusers, paying particular attention to the consistence of the "CD20+" B cell subset, which poorly expresses the CD21 membrane receptor for the C3d and Epstein-Barr virus (EBV) (referred to as "CD20 + CD21-" subset). In healthy blood donors, the ratio CD20 + CD21-/CD20+ x 100 is extremely low (mean +/- SEM = 8.1 +/- 0.9) and rarely exceeds the value of 20. On the contrary, in HIV seropositives, the values are much more dispersed, with higher mean values (mean +/- SEM = 25.8 +/- 1.8) ranging from 50 to 60. An intermediate situation characterizes the class of HIV-seronegative heroin addicts, whose values are slightly higher and more dispersed than that of normal controls (mean +/- SEM = 11.6 +/- 1.3). The extent of the amplification of the CD20 + CD21- subset in HIV-seropositive individuals does not apparently correlate with the progression of the disease and represents an early event in the clinical course of HIV infection. For each subject of the study group, the number of CD20 + CD21- B lymphocytes is not correlated to other early markers of HIV infection, as the T4 lymphocyte number, or total Ig levels in sera. A functional characterization of the CD20 + CD21- B cell subset indicates that, in HIV-seropositive patients, these cells are unable to produce specific and nonspecific immunoglobulins (Ig's), either spontaneously or after pokeweed mitogen stimulation. Furthermore, this cell subset is characterized by poor expression of surface Ig's. The data reported suggest that this cell subset can be regarded as situated at an early level of B cell lineage differentiation.


Human VEP contrast modulation sensitivity: separation of magno- and parvocellular components.

Human cortical visual evoked potentials (VEPs) were retrieved in real time (without averaging). The stimuli were sinusoidal gratings whose contrast was temporally modulated about some mean value. Electrophysiologically determined contrast modulation thresholds were measured at standing contrast over the range from 2.5% to 50%, defining an increment threshold function. Increment threshold functions were obtained under two different spatio-temporal stimulus conditions identified as "sustained" (4 c/deg grating modulated at 1.5 Hz) and "transient" (1 c/deg grating modulated at 20 Hz). Under each of these conditions, threshold responses were retrieved at both the fundamental and second harmonic of the contrast modulation frequency. Under "sustained" conditions log increment threshold responses and the fundamental the second harmonic of the modulation frequency were similar to those at the fundamental except for a saturation effect (i.e., above a mean contrast of 25% there was little further reduction in modulation sensitivity). There was no contrast gain control under "transient" stimulus conditions. In other words, the same absolute amount of contrast change produced threshold responses for all mean levels up to 25%. This was true at both the fundamental and second harmonic of the modulation frequency. Stimulus differences produce striking differences in the electrophysiologically inferred increment threshold function for grating contrast, but fundamental and second harmonic evoked responses reflect processes with similar increment threshold behavior.


The influence of pattern size on amplitude, latency and wave form of retinal and cortical potentials elicited by checkerboard pattern reversal and stimulus onset-offset.

Transient pattern electroretinograms (PERGs) and visual evoked potentials (VEPs) were recorded with checkerboard pattern reversal and equiluminance stimulus onset-offset, elicited by a high quality moving mirror stimulator. Different sized checkerboard patterns (0.35-4.2 c/deg) were used as stimulus patterns. The wave forms of the equiluminance stimulus onset responses were similar to ERGs evoked with luminance decrease and the stimulus offset PERGs were like ERGs elicited by luminance increase. The PERG c wave and the VEP showed spatial frequency tuning with pattern reversal and stimulus offset. Spatial frequency tuning was not detectable with PERG a and b waves. Pattern reversal and stimulus onset evoked PERGs had no major spectral components above 40 Hz; stimulus offset evoked PERGs contained components up to 55.3 Hz. Retino-cortical time--measured as a latency difference of the PERG b wave to VEP P100--was identical with pattern reversal and stimulus onset and about 12 msec longer with stimulus offset. Our results suggest that the 3 stimulation modes, reversal, onset and offset induce different types of processing at the retinal and cortical levels. PERG a and b waves to our high luminance/contrast stimuli contain no pattern specific information and the c waves are the sum of luminance and pattern specific responses.


Sequence-dependent deterioration in the visual evoked potential in the absence of drowsiness.

When visual evoked potentials (VEPs) are tested patients are often expected to focus on a pattern screen for prolonged periods of time. This may lead to fatigue, failure of concentration and drowsiness, and consequently to a deterioration in the recorded VEP. To determine whether there may be time-dependent changes in the VEP of normal subjects independent of the degree of alertness, attention and altertness were controlled using a reaction time (RT) task in which the subjects were required to re-illuminate the fixation point in the middle of the stimulating screen for the VEP. It was first established that the switching of the fixation point produced little contamination of the background VEP to pattern reversal and that the latency and amplitude of P100 to pattern reversal were identical whether or not the subject was engaged in the RT task. A sequence of 16 averages of the VEP to 256 pattern reversals was recorded, alternately with or without the RT task. The measured RTs decreased during the sequence, presumably due to practice. There was a progressive decrease in the amplitude N70-P100, accompanied by an increase in the variability of the latency of P100. These changes cannot be attributed to lack of alertness, given the improvement in RT. Clinicians should be aware of the possibility of a deterioration in the VEP due to physiological mechanisms when the testing protocol involves multiple averages.


Somatosensory evoked potentials in the term newborn.

Median nerve somatosensory evoked potentials (SEPs) were recorded from surface electrodes in 40 healthy term infants (range 36.5-43 weeks postmenstrual age). Electrical stimulation at 5 Hz was used, averaging the response to several runs of 1024 stimuli to each median nerve, bandpass 10-3000 Hz, sweeptime 100 msec. Identifiable potentials were collected over the cervical cord on all runs in all 40 infants and from the cortex in at least some runs in 39 out of 40 infants. The cervical response showed little variation and consisted of a clear negative wave with up to 3 peaks, mean latency of the largest 10.2 +/- 0.7 msec, followed by a positive deflection. The cortical response was very variable in form and latency between infants and to a lesser degree within infants. Four types of cortical wave form were found, symmetrical, asymmetrical, plateau and M shaped, of increasing complexity. In 11% of trials the response was absent or indistinct but could usually be uncovered by alteration in stimulus frequency or intensity. In the whole group, the mean latency for N1 was 30.0 +/- 6.8 msec and for the central conduction time 19.8 +/- 6.5 msec. Significant differences were found between the 4 cortical wave forms in the main variables measured, which gave support for form S being the most primitive and form M the most mature response.


The late somatosensory evoked potential in premature and term infants. I. Principal component topography.

Very little is known about the topographic distribution of the cortical somatosensory evoked potential in premature infants. Principal component analysis (PCA) was applied to the wave forms generated from right median nerve stimuli over a relatively long sweep (483 msec post stimulus) at 16 electrodes in 53 infants with postconceptual ages from 31 to 40 weeks, subdivided into 5 groups by 2 week increments. Factor scores were averaged across subjects, within groups and displayed as topographical maps. Four factors accounted for 71-76% of the variance in each of the 5 groups and the factors extracted from the PCA performed independently in each group were markedly consistent. The first factor (N1/P1) had a left posterior minimum and a left frontal-central maximum and probably represents a tangential dipole located in the post-central gyrus. The second factor (N2) was characterized by a consistent left central minimum with a systematic developmental change in the maximum that seemed to imply that its neural generator was changing in orientation as the infants matured. A third factor (N3) accounted for the most variance and appeared to represent the first evidence of activity in the ipsilateral cortex. Finally, a very late fourth factor appeared only in the more mature groups, with uncertain localization. The topographic maps of the factor scores for these 4 factors appear to account for independent generators in the SEP of the premature and term infant.


The late somatosensory evoked potential in premature and term infants. II. Topography and latency development.

The maturation of latency and scalp voltage topography of the simultaneously bilateral somatosensory evoked potential was studied in 53 neurologically intact pre-term and term infants, from 31 to 40 weeks post-menstrual age. Four peaks (N1, P1, N2 and P2) were reliably identified in all infants. The latency of each peak decreased as the infants matured. Each peak had a unique voltage scalp topography that remained stable as infants matured, even though the maps changed in amplitude intensity. N2 was large, easily identifiable with a central peak, and extremely stable in topography, suggesting that it might be used to evaluate the functional status of the somatosensory cortex in pre-term and term infants who are at high risk for developing intracranial hemorrhage leading to abnormalities of tone and delays in motor development.


Dermatomal somatosensory evoked potentials: cervical, thoracic, and lumbosacral levels.

Somatosensory evoked potentials were recorded at the scalp to stimulation of the skin at C4, C5, C6, C7, C8, T2, T4, T6, T8, T10, T12, L2, L3, L4, L5, and S1 dermatomes and of the tibial nerve. Stimulation and recording techniques are described. Data were obtained from 41 normal subjects, 25 of which had all 16 dermatomes studied. Wave form descriptions include both typical and atypical presentations. Descriptive statistics for latency, amplitude, left to right comparisons, and level to level comparisons are given. Scalp response latencies for distal extremity dermatomes were well correlated with height but not with vertebral column length, whereas latencies for thoracic dermatomes were not well correlated with either height or vertebral column length. Since scalp response amplitude data were not normally distributed, they were logarithmically transformed and minimum and maximum limits for 1 S.D., 2 S.D., and 3 S.D. derived. Left/right amplitude ratios were similarly treated. Level to level comparisons were achieved with a Z score concordance analysis, which showed that the response values at one level can be used to predict the response values at another level.


Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I.

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thr124, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Grässer, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13-22]. The peptide, A-D-S-Q-H-S-T-P-P, which corresponds to the amino acid sequence 118-125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the -4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the -2 and -3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent Km values around 2 mM and Vmax values up to 500 pmol.min-1.ml-1. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first-order rate constant over 20-fold higher than peptides with acidic amino acid residues at the N-terminus only; the apparent Km value was 0.65 mM with a Vmax of 2900 pmol.min-1.ml-1. The effects of modifying Ser120 to phosphoserine were examined by addition of a recognition sequence for the cAMP-dependent protein kinase prior to Ser120. Prior phosphorylation of the peptide at Ser120 lowered the apparent Km to 0.061 mM and increased the Vmax to 360 pmol.min-1.ml-1, a 50-fold decrease in Km for casein kinase I and a 6-fold increase in Vmax as compared to the non-phosphorylated peptide. This indicates that Ser120, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.


Unmasking of cortical SEP components by changes in stimulus rate: a topographic study.

We performed topographic mapping of somatosensory responses to median nerve stimulation delivered at 2, 5 and 10 Hz. Parietal N20 was significantly attenuated in 10 Hz somatosensory evoked potentials (SEPs), while central P22 diminished between 2 and 5 Hz, remaining stable thereafter. The single component most affected by increasing stimulus rate was N30, which abated by more than 50% in 10 Hz SEPs, as compared with basal responses. N30 attenuation disclosed the existence of an earlier negative component, N24, which appeared as a notch on the N30 ascending slope in 2 Hz SEPs, but became a well-defined peak at higher stimulus rates. The N24 negativity was not significantly modified by stimulus rate; it had a parietal counterpart (P24) with the same peak latency and identical behavior during the experimental procedure. Both P24 and N24 could be differentiated from central P22 on the basis of topographical distribution and response to stimulus frequency. P22 topography could be the result of a radially oriented generator, while P24/N24 appeared as the two poles of a neural source tangential to the scalp. P27 was seen in 40% of the subjects only; it is suggested that P27 is itself a composite potential to which the generator of N30 could contribute in part. We conclude that there is no single "optimal" stimulation rate for SEP recording. On the contrary, combination of different frequencies of stimulation should enhance the diagnostic utility of this technique by allowing a more selective assessment of overlapping activities.


Interleukin 1 beta and tumour necrosis factor alpha induce a macrophage-type of nitric oxide synthase in rat renal mesangial cells.

Treatment of mesangial cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) has been shown to increase cGMP formation, most probably due to induction of nitric oxide synthase. Here we report that maximum stimulation of cGMP formation over a 24-h period required the presence of IL-1 beta or TNF alpha during the first 18 h of induction. N4-monomethyl-L-arginine (L-NMMA) was a potent inhibitor of cytokine-induced cGMP formation while N4-nitro-L-arginine (L-NNA) was less active. Formation of nitric oxide was detected in the cytosol of cytokine-treated mesangial cells by activation of purified soluble guanylate cyclase and was stimulated by tetrahydrobiopterin, but not by calcium calmodulin. Treatment of cells with IL-1 beta or TNF alpha markedly attenuated the contractile response to a subsequent challenge with angiotensin II. Furthermore, conditioned medium from IL-1 beta-treated cells increased cGMP in untreated control cells.


Differences in human evoked potentials related to olfactory or trigeminal chemosensory activation.

The aim of the present study was to determine, whether there are differences in the topographical distribution of chemosensory evoked potentials (CSEPs) due to stimulation with different odorous substances. The odorants used in the study which mainly excited the olfactory nerve were vanillin and acetaldehyde; those which additionally excited the trigeminal nerve were sulphur dioxide and ammonia. Twelve subjects participated in the study. The subjects separately estimated the intensity of the odorous and of the painful/pricking sensation caused by the stimuli, and described the odorous qualities in their own words. CSEPs were recorded from 7 positions. After stimulation with "olfactory" substances maximum CSEP amplitudes were recorded at parieto-central sites, and after stimulation with "trigeminal" substances maximum amplitudes were obtained at the vertex. Following stimulation with ammonia and sulphur dioxide amplitudes were largest contralateral to the stimulated nostril. In contrast, little difference in CSEP amplitudes was observed between hemispheres after stimulation with vanillin or acetaldehyde. Thus, the topographical distribution of CSEP amplitudes may provide information with regard to the sensory system (olfactory or trigeminal) activated by the presentation of an odorous stimulus.


Differential responses to interleukin 2 define functionally distinct subsets of human natural killer cells.

Human natural killer (NK) cells can be subdivided into two populations based on the density of cell surface CD56 antigen. The great majority (approximately 90%) of NK cells express CD56 at low levels (the CD56dim phenotype), whereas a small NK cell subset (approximately 10%) exhibits approximately fivefold greater density of surface CD56. Exposure to exogenous interleukin 2 (IL 2) induces tenfold greater proliferation of CD56bright cells compared to CD56dim lymphocytes, even though both subsets constitutively express similar levels of intermediate affinity IL 2 receptor (IL 2R) p75 chains. Incubation with IL 2 alone or irradiated target cells alone could induce expression of the IL 2R p55 chain by both CD56bright and CD56dim NK cells; a combination of both stimuli was most effective. IL 2R p55 induction was evident after co-culture of NK cells with both NK-sensitive and NK-resistant cell lines or with antibody-coated target cells. Activation of NK cells with IL 2 plus target cells resulted in enhanced proliferation compared to activation with IL 2 alone; target cells alone did not induce significant proliferation. Although both NK cell subsets appeared to express high-affinity IL 2R p75/p55 heterodimers after stimulation with target cells and IL 2, proliferation of CD56dim cells remained minimal after such activation; activated CD56dim cells consistently demonstrated less proliferation to IL 2 than did resting CD56bright cells. In contrast, CD56bright NK cells exhibited even greater proliferation after stimulation with target cells. Almost all CD56dim NK cells expressed CD16 (Fc gamma R III) as well as the NK zeta chain, whereas less than 50% of CD56bright cells express either CD16 or zeta. CD56bright and CD56dim lymphocytes, thus, appear to represent distinct subpopulations of NK cells with different functional activities. Unlike CD56bright cells, CD56dim NK cells do not proliferate optimally to IL 2, even after the latter have been stimulated to express both IL 2R p55 and IL 2R p75. Efficient proliferation of CD56dim NK cells may, thus, require additional or alternative signals.


Human auditory evoked potentials recorded using maximum length sequences.

A maximum length sequence (MLS) is a specially constructed pseudorandom binary sequence that can be used to control the presentation of sensory stimuli. The evoked potentials to such a sequence of stimuli can be analyzed to give the response to one stimulus in the sequence. This procedure allows auditory evoked potentials to be recorded at stimulus rates that would cause a confusing overlap of responses with regular averaging. The MLS technique can be used with auditory evoked potentials at all latencies although it is most effective for the brain-stem and middle-latency responses. By demonstrating different refractory periods for different parts of the response, the technique may help delineate the component structure of the evoked potential. As well, an MLS analysis can disentangle the auditory brain-stem response from overlapping middle-latency responses during evoked potential audiometry.