Sensitivity and specificity of portable transient otoacoustic emission (TEOAE) in newborn hearing screening.

Early identification and management of hearing impairment is very valuable. The goal standard measurement of hearing loss is by brainstem evoked response (BSER). This prospective study was conducted in Hospital University Kebangsaan Malaysia (HUKM) to determine the sensitivity and specificity of transient evoked otoacoustic emission (TEOAE) as a screening tool for hearing impairment from February 1999 to February 2000. One hundred and thirty-three newborns from postnatal ward and seventy-eight newborns from neonatal intensive care unit (NICU) were screened for possible hearing loss using portable TEOAE. This study showed that TEOAE is a very sensitive but moderately specific screening tool.

A comparison of EEG determinants of near-awakening from isoflurane and fentanyl anesthesia. Spectral edge, median power frequency, and delta ratio.

To evaluate the efficacy of the computer-processed electro-encephalogram (EEG) for determining near-awakening from anesthesia, 14 patients were monitored during emergence from either isoflurane or fentanyl anesthesia at the termination of major surgical procedures. The raw EEG was obtained using bilateral frontomastoid electrodes. The compressed spectral array was digitized and recorded on disk in 4-s epochs using a Tractor Northern "Nomad" processor. The EEG information was displayed in four formats: 1) the frequency-power spectrum from 1-20 Hz, 2) the 95% power frequency, 3) the 50% power frequency, and 4) the ratio of power in the 8-20 Hz frequency range to the power in the 1-4 Hz frequency range (delta ratio). During emergence from isoflurane, there were obvious changes in the EEG frequency-power spectrum that occurred several minutes before patients opened their eyes in response to verbal stimuli. Although no one descriptor of EEG activity could be shown to be superior in anticipating when patients would respond by opening their eyes, awakening was always presaged by an abrupt decrease in power in the 1-4 Hz frequency range; this resulted in a marked increase in the delta ratio value. During emergence from fentanyl anesthesia, however, there was no obvious change in the overall EEG frequency-power spectrum. However, the same numeric EEG descriptors that were predictive of awakening from isoflurane also occurred during emergence from fentanyl, even though they usually occurred within 1 min of awakening. It is concluded that EEG criteria for identifying when patients will awaken from anesthesia are more reliable with isoflurane than with fentanyl.

Induction of endogenous xenotropic retrovirus expression by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner (0.0016 to 4.0 microM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly. TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of induction may be different from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity to study the modulation of retrovirus gene expression by TPA.

Amino acid analogs inhibit murine xenotropic retrovirus expression and cell cycle progression.

Certain functional analogs of amino acids were examined for their capacity to inhibit chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus transformed BALB/c (K-BALB) mouse cells. Partially synchronized cells cultured with aminoethylcysteine (AEC), parafluorophenylalanine (PFA), or valinol, and subsequently induced with either 5-iododeoxyuridine (IUdR), cycloheximide, or histidinol, showed inhibition of virus activation. Inhibition was concentration- and time-dependent (1 to 4 h) and not a consequence of cytotoxicity. Inhibition was competed out by the analogous amino acid and was specific to the induction process. After a 4 h analog treatment, heteronuclear RNA synthesis was reduced 24, 38, and 35% by PFA, AEC, and valinol, respectively, whereas cycloheximide or actinomycin D reduced synthesis by 60 and 90%, respectively; therefore, the analogs did not seem to inhibit induction through a general transcriptional block. Culture of cells in the presence of the analogs resulted in an abrupt reduction (70 to 90%) in DNA synthesis. Using synchronized cells, it was found that 0.1 mM AEC added in G1 phase and followed by IUdR induction almost totally inhibited virus expression. No inhibition was observed when AEC was added during S phase concomitantly with the inducer. AEC added to synchronous cells in G1 phase inhibited the progression of cells into S phase and the onset of DNA synthesis. The results show that K-BALB cells have an AEC-sensitive restriction point in G1 phase that might relate to the effects amino acid analogs have on cell replication and S phase dependent gene expression, as well as subsequent differentiation.

Enhancement of endogenous xenotropic murine retrovirus expression by tuftsin.

The exposure of a high-passage clone of Kirsten sarcoma virus transformed Balb/c (K-Balb) mouse cells to tuftsin (Thr-Lys-Pro-Arg) enhanced the expression of endogenous xenotropic retrovirus. The tetrapeptide increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion (0.001-1000 micrograms/ml). Increased virus expression could be achieved during short-term incubations (3-4 hr), with maximum enhancement occurring over longer time periods (16-18 hr). The enhancement of virus expression by tuftsin was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp 70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. Tuftsin stimulated DNA, RNA, and protein synthesis in K-Balb cells during 16-hr incubations. Increased cellular proliferation was also seen at various time periods. The effects observed using K-Balb cells offer an opportunity to study the modulation of gene expression by tuftsin in a fibroblast culture system.

Juvenile hormones inhibit murine cell cycle progression and expression of type C viruses.

Juvenile hormones (JH), congeners of retinoic acid, were examined for their capacity to inhibit cell cycle progression and chemically induced expression of endogenous xenotropic retrovirus in Kirsten sarcoma virus-transformed BALB (K-BALB) mouse cells. JHI, II, and III were found to inhibit induction of virus by 5-iododeoxyuridine (IUdR) and histidinol (Hdl) in a concentration-dependent fashion. Some inhibition of macromolecular synthesis was observed upon culture of the cells with JH; the most affected was RNA synthesis, which was reduced 27 to 40% within 4 h by the juvenoids. Epoxide hydrase (EH) activity, as determined by high-pressure liquid chromatography (HPLC), was present in amounts sufficient for the cells to convert the hormones metabolically to an ultimate form. A contact-inhibited K-BALB variant was synchronized by mitotic arrest and the cell cycle-specific effect of JHIII on virus induction during S phase was studied. JHIII added during G1 phase, and followed by induction, inhibited virus expression 95 and 76% by IUdR and Hdl, respectively. Induction was inhibited only 35% when JHIII was added during S phase concomitantly with the inducers and no inhibition was observed when JHIII was added during G2 phase followed by the inducers. JHIII added to synchronous cells in G1 phase inhibited progression of cells into S phase and the onset of DNA synthesis. The results indicate that mouse fibroblasts have a juvenile hormone-sensitive restriction point in G1 phase that might relate to the effects these hormones have on cell replication and differentiation.

Cell cycle-specific inhibition by retinoic acid of xenotropic murine retrovirus expression.

Several retinoids were examined for their capacity to block chemically induced expression of endogenous xenotropic retrovirus from Kirsten sarcoma virus-transformed BALB/c mouse cells. Retinoic acid (RA) was found to inhibit induction of virus by 5-iododeoxyuridine, cycloheximide, and histidinol; inhibition was concentration (10(-4) to 10(-6) M) and time dependent (1 to 7 hr) and not a consequence of cytotoxicity. Following a 6-hr treatment with 10(-4) M RA, [3H]thymidine and [3H]uridine incorporation into total cellular DNA and RNA was reduced 37 and 63%, respectively. Heteronuclear RNA synthesis was reduced 36 and 7% within 4 hr by 10(-4) and 10(-5) M RA, respectively, indicating that inhibition was not the result of a general transcriptional block. Using synchronized cells, it was found that 5 X 10(-5) M RA added in G1 phase and followed by cycloheximide or 5-iododeoxyuridine induction inhibited virus expression 60 and 84%, respectively. Little or no inhibition was observed when RA was added during S phase with the inducers or during G2 phase followed by inducers. Cells synchronized by mitotic arrest showed a RA-mediated restriction point in early-to-mid-G1 phase as indicated by a delay in the onset of DNA synthesis and an inhibition of virus induction during S phase. The results show the presence in Kirsten sarcoma virus-transformed BALB/c cells of a RA-sensitive G1 restriction point for cell progression and suggest that inhibition of retrovirus activation may be related to an extended G1 phase.

Increased expression of endogenous xenotropic murine retrovirus by treatment with the tetrapeptides, tuftsin and kentsin.

Enhancement of endogenous xenotropic virus expression has been found upon treatment with tetrapeptides of a high-passage clone of Balb/c (K-Balb) mouse cells transformed with Kirsten sarcoma virus. Tuftsin (Thr-Lys-Pro-Arg) and kentsin (Thr-Pro-Arg-Lys) increased the expression of virus that was infectious for rat, but not mouse, cells in a concentration-dependent fashion. The enhancement of virus expression by the tetrapeptides was proportional to the spontaneous release of virus. The infectivity of the enhanced virus was neutralized by goat anti-RLV gp70 serum. Actinomycin D inhibited the induction of virus, suggesting that enhanced expression required de novo RNA synthesis. The effects observed using K-Balb cells offer an opportunity to study the many biological effects of these peptides in a fibroblast culture system.

Inhibition of endogenous murine retrovirus expression by L-beta-3,4-dihydroxyphenylalanine (L-dopa) methyl ester.

Induction of endogenous xenotropic type-C virus from Kirsten sarcoma-virus-transformed BALB/c (K-BALB) mouse cells was inhibited by short-term exposure to L-beta-3,4-dihydroxyphenylalanine (L-dopa) methyl ester. Partially synchronized cells cultured for 1-4 h with 0.8-1.6 mM L-dopa methyl ester and subsequently induced with 5-iododeoxyuridine (IdUrd), cycloheximide and histidinol showed inhibition of virus activation. Incorporation of thymidine, uridine and leucine was measured at the end of the drug treatment and during the subsequent induction period. L-dopa methyl ester had a pronounced effect on DNA synthesis, reducing it by more than 85% during a 4-h incubation period, whereas RNA and protein synthesis remained largely unaffected. Removal of the drug and replacement with fresh medium did not reverse DNA synthesis or virus activation during the subsequent induction interval. L-dopa methyl ester was also shown to potentially function as an analogue of tyrosine in this cell system. These results suggest that inhibition of virus induction may be caused by inhibiting the normal progressing of cells through the S phase of the cell cycle.

Cell cycle-specific enhancement of type C virus activation by sodium n-butyrate.

The effect of sodium n-butyrate on chemical induction of xenotropic virus from synchronized Kirsten sarcoma virus-transformed BALB mouse cells was examined. When added during the last part of the G1 phase, n-butyrate produced a large increase in cycloheximide induction during S phase. Under similar conditions, activation by 5-iododeoxyuridine was inhibited. When added with cycloheximide during the S phase, n-butyrate inhibited activation of virus. Studies with synchronized cultures showed that n-butyrate delayed the onset of DNA synthesis, characteristic of the S phase, and inhibited histone deacetylation in log-phase cells. The effects produced by n-butyrate could, therefore, be the result of lengthening the G1 phase of the cell cycle or a modification of histones affecting transcription during virus activation.

Analysis of transcription during type C viral induction using cell permeabilization techniques.

The reversible cell permeabilization procedure of Castellot et al. has been applied to chemical induction of endogenous type C virus from mouse cells. This procedure has provided a direct demonstration of the alpha-amanitin sensitivity of viral transcription in intact cells at concentrations known to inhibit RNA polymerase II.

C3H/HeN mammary tumor-bearing mice develop type-specific neutralizing antibodies and group-specific precipitating antibodies for the mouse mammary tumor virus.

The development of mouse mammary tumor virus (MMTV)-neutralizing antibodies in various strains of mice was measured by their ability to neutralize the focus-forming capacity of a Kirsten sarcoma virus (C3H MMTV) pseudotype containing the MMTV envelope glycoprotein gp52. C3H/HeN, but not GR/N and RIII, mammary tumor-bearing mice were found to develop neutralizing antibodies to this pseudotype. In addition, non-tumor-bearing C3H/HeN, GR/N, RIII, NIH Swiss, C57BL/6, and BALB/c mice and 13 feral mice were also negative for neutralizing antibodies. The neutralization was immunoglobulin G mediated, and the antibodies of C3H/HeN mammary tumor-bearing mice were type specific and capable of distinguishing C3H and GR/N MMTVs from RIII and C3H/HeNf MMTVs. Precipitating antibodies were detected in sera from RIII and GR/N tumor-bearing mice, GR/N non-tumor-bearing mice, and six of the feral mice, although these same sera did not neutralize the Kirsten sarcoma virus (C3H MMTV) pseudotype. The results of this study and of a previous study demonstrate that C3H/HeN mammary tumor-bearing mice develop three functionally distinct antibody populations: (i) group-specific virus-precipitating antibodies; (ii) type-specific virus-neutralizing antibodies; and (iii) type-specific cytotoxic antibodies.

Mice with spontaneous mammary tumors develop type-specific neutralizing and cytotoxic antibodies against the mouse mammary tumor virus envelope protein gp52.

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.

Effects of protease inhibitors on chemical induction of type C virus.

A role for proteolysis during chemical induction of endogenous xenotropic Type C virus from Kirsten sarcoma virus-transformed mouse cells was examined. Two distinct classes of protease inhibitors, the trypsin inhibitor, alpha-N-tosyl-L-lysine chloromethyl ketone, and two naturally occurring oligopeptide inhibitors, antipain and leupeptin, were found to inhibit induction of virus by cycloheximide and histidinol. Virus activation by 5-iododeoxyuridine was inhibited to a lesser degree. During the time cells were exposed to these compounds, there was little inhibition of [3H]uridine incorporation into total cellular RNA or polyadenylic acid cytoplasmic messenger RNA, suggesting that inhibition of proteolysis, and not RNA transcription, was responsible for blocking virus induction.

Immunological characterization of the low-molecular-weight DNA binding protein of mouse mammary tumor virus.

p14, a low-molecular-weight MMTV protein previously identified as having DNA-binding properties and encoded by the gag region of the MMTV genome, was purified by affinity chromatography on DNA-sepharose. Immunological characterization of the purified protein showed that MMTV p14 shares no cross-reactivity with gp52, gp36 and p10, antigens associated with the MMTV envelope, nor with p27 antigen found in the virion core. Purified MMTV p14 did show cross-reactivity with purified intracytoplasmic A particles, supporting the concept that A particles are morphological precursors to MMTV cores. In addition, shared antigenic determinants between intracytoplasmic A particles and MMTV p27, p20 and p10 were demonstrated. MMTV p14 did not cross-react with the low-molecular-weight DNA-binding proteins of MuLV or of type-C or -D viruses of higher mammals.