The P genes of human parainfluenza virus type 1 clinical isolates are polycistronic and microheterogeneous. The nucleotide sequence of the P gene of human parainfluenza virus type 1 (hPIV1) strain C35 was determined directly from genomic viral RNA and by molecular cloning. The gene contained 1893 nucleotides. Four open reading frames (ORF) capable of encoding a P protein (568 amino acids; M(r) = 64,784), a C' protein (219 amino acids; M(r) = 25,997), a C protein (204 amino acids; M(r) = 24,237), and a Y1 protein (182 amino acids; M(r) = 21,471) were identified. The latter three ORFs are in a +1 reading frame relative to P. The sequencing data are consistent with the hPIV1 C' protein being initiated at a GUG codon (nt 68-70), in contrast to the ACG initiation of the Sendai virus (SV) C' protein. Unlike SV, there is no evidence of a hPIV1 ORF capable of encoding a cysteine-rich V protein. Also, there is no ORF capable of encoding a protein analogous to the SV Y2 protein. In vitro transcription, translation, and immunoprecipitation showed that the hPIV1 P gene is polycistronic. Comparison of the P gene with those of two other distinct clinical isolates confirmed the coding potential of the hPIV1 P gene but also revealed genetic heterogeneity among the isolates. Our results indicate that the hPIV1 P gene uses some coding strategies similar to and others that are different from those of other paramyxovirus P genes.


Defective viral particles in caprine arthritis encephalitis virus infection. Attempts to isolate full-length unintegrated circular forms of the caprine arthritis encephalitis virus (CAEV) genome yielded only a large number of molecules with deletions. The 3' borders of most of these deletions were near the U3 region of the long terminal repeat whereas the 5' edges were found at various upstream sites within pol or env. With one exception, gag sequences were always present. Analysis of molecular clones derived from integrated proviral CAEV genomes from the same infected cells showed a similar spectrum of deletions. The presence of transcriptionally active elements within the U3 domain of the defective genomes, as well as cis-acting elements within the leader sequences known to be required for efficient encapsidation of viral RNA, suggested that the defective viral DNA genomes could be transcribed into defective RNA molecules which could then be packaged into virions. Isopycnic density gradient centrifugation of supernatants of infected cell cultures indicated the presence of particles with densities less than that expected for intact virions (1.16 g/cc). Northern analysis revealed the presence of smaller viral-specific RNAs that lacked env sequences. These data, along with the structures of the molecular clones, suggest that CAEV stocks contain particles with defective genomes. The role of these particles in influencing the course of virus infection remains to be determined.


Primer design for specific diagnosis by PCR of highly variable RNA viruses: typing of foot-and-mouth disease virus. A PCR assay for the specific detection and identification of viral sequences that correlate with established serotypes of foot-and-mouth disease virus (FMDV) has been developed. A new analysis based on homology profiles among reported sequences was used for primer design. RNA replicase (3D) gene regions that showed high homology among FMDVs, and low homology to other picornaviruses, were used for PCR amplification. Specific and highly sensitive detection was achieved for RNA of FMDV types C, A, and O, either purified or extracted from vesicular fluids of infected animals, under reaction conditions permissive for the detection of variants present in the virus population. Similarly, serotype-specific primers were designed to amplify the carboxy-terminal end of VP1 gene of FMDV types either C, A, or O. The results of PCR amplification of 15 different FMDV RNAs using type-specific primers are in agreement with the serological typing of the corresponding viruses and show that the primer-selection procedure developed for FMDV constitutes a reliable method of viral diagnosis.


The herpes simplex virus immediate early protein ICP27 encodes a potential metal binding domain and binds zinc in vitro. The herpes simplex virus type 1 (HSV-1) immediate-early regulatory proteins ICP27 and ICP0 each encode putative zinc-finger metal-binding domains. We utilized the technique of metal chelate affinity chromatography to demonstrate that ICP27 and ICP0 were able to bind to zinc in vitro. This property was further exploited to purify ICP27 from extracts of HSV-1-infected cells. The purification procedure also revealed that ICP27 possessed single-stranded DNA-binding activity. Analysis of ICP27 truncated peptides produced by in vitro translation verified that the zinc-binding region of ICP27 resides in the carboxy terminal 105 amino acids spanning the putative metal binding motif. However, a specific configuration of cysteine and histidine residues in this region was not required for binding to occur as demonstrated by the ability of a frame-shift mutation to bind with an efficiency similar to wild type. The mutated peptide retained four histidine and cysteine residues but in a configuration different from the consensus proposed for zinc-finger motifs. Therefore, while the region spanning the metal binding domain of ICP27 is essential for both the activator and repressor functions, and ICP27 binds zinc in vitro, it is not clear whether zinc binding in vivo is necessary for function.


Maturation of mouse mammary tumor virus envelope protein is blocked by a specific point mutation. Expression of a mouse mammary tumor virus (MMTV) envelope gene, ENV2, in COS-7 cells resulted in the synthesis of a precursor protein Pr74 that underwent complete proteolytic processing to the mature envelope species gp52 and gp33. In contrast, expression of a second MMTV envelope gene, ENV1, in COS-7 cells resulted in the synthesis of a precursor protein Pr74 that failed to undergo proteolytic processing to the mature products. Expression and sequencing of various chimeric ENV1/ENV2 genes determined that a single glycine-to-glutamic acid mutation at position 54 was responsible for the nonprocessable phenotype of ENV1-encoded Pr74. The significance of this point mutation in relation to the requirements for precursor protein folding and maturation are discussed.


Modulation of the frequency of human cytomegalovirus-induced chromosome aberrations by camptothecin. The effects of selected DNA repair inhibitors on the frequency of human cytomegalovirus (HCMV)-induced chromosome aberrations were evaluated in human peripheral blood lymphocytes (PBLs). Treatment of HCMV-infected PBLs with camptothecin (0.05 to 0.3 micrograms/ml), an inhibitor of topoisomerase I, for 30 hr resulted in a significant (P less than 0.01) synergistic enhancement of the frequency of HCMV-induced chromosome damage. On the other hand, a significant increase in the frequency of chromosome damage was not noted for infected PBLs treated with either 3-aminobenzamide (3-AB; 3 to 30 micrograms/ml), an inhibitor of poly(ADP-ribose) polymerase, or novobiocin (3 to 30 micrograms/ml), an inhibitor of topoisomerase II or excision repair processes, for 30 hr. Chromatid-type breaks and exchanges were the predominant type of chromosome aberrations observed in the HCMV-infected cells treated with camptothecin, suggesting that HCMV infection is associated with the induction of single-strand DNA breaks. Furthermore, these findings suggest that HCMV infection does not inflict direct DNA damage which is repaired through 3-AB- or novobiocin-sensitive pathways.


Species differences in the response of mammalian photoreceptor cyclic GMP and PIII to a reduction in calcium. Changes in the content of cyclic GMP (cGMP), induced by exposure of isolated, dark-adapted mouse, cat and dog retinas to media depleted of calcium, have been compared with the amplitude of the trans-retinal PIII. Major differences exist in the time-course and magnitude of effects between the species and, in the cat and dog, changes in PIII (potentially a reflection of free cGMP in photoreceptor outer segments) do not correlate with those occurring in total cGMP. The observations imply species variation, not only in the enzymes maintaining cGMP homeostasis in photoreceptors, but also in phototransduction and allied processes.


Evidence for variable rates of recombination in the MHV genome. Mouse hepatitis virus has been shown to undergo RNA recombination at high frequency during mixed infection. Temperature-sensitive mutants were isolated using 5-fluorouracil and 5-azacytidine as mutagen. Six RNA+ mutants that reside within a single complementation group mapping within the S glycoprotein gene of MHV-A59 were isolated which did not cause syncytium at the restrictive temperature. Using standard genetic techniques, a recombination map was established that indicated that these mutants mapped into two distinct domains designated F1 and F2. These genetic domains may correspond to mutations mapping within the S1 and S2 glycoproteins, respectively, and suggest that both the S1 and S2 domains are important in eliciting the fusogenic activity of the S glycoprotein gene. In addition, assuming that most distal ts alleles map roughly 4.0 kb apart, a recombination frequency of 1% per 575-676 bp was predicted through the S glycoprotein gene. Interestingly, this represents a threefold increase in the recombination frequency as compared to rates predicted through the polymerase region. The increase in the recombination rate was probably not due to recombination events resulting in large deletions or insertions (greater than 50 bp), but rather was probably due to a combination of homologous and nonhomologous recombination. A variety of explanations could account for the increased rates of recombination in the S gene.


Spectrum of clinical manifestations of familial adenomatous polyposis. Familial adenomatous polyposis is an autosomal dominant disease that primarily involves the colon, but may involve other organs such as the central nervous system, ampulla of Vater, liver, soft tissue and the remainder of the gastrointestinal tract. In this report we present the adenocarcinoma of the uterus and adenocarcinoma of the ovary as a new addition to the clinical spectrum of familial adenomatous polyposis. Due to the tendency for development of multiple primary carcinomas in patients with familial adenomatous polyposis, these patients need to be followed very closely for detection of new malignancies. If diagnosed early and treated accordingly, they could have a long-term postoperative survival.


Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract. The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 microliters ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the rationale for active mucosal immunization protocols designed to generate an IgA response.


Expression of the gene coding for the major outer capsid protein of SA-11 rotavirus in a baculovirus system. The gene coding for the major outer capsid protein (VP7) of simian rotavirus SA-11 has been expressed in a baculovirus-insect cell system. The resulting protein is 35 kDa and is primarily associated with the endoplasmic reticulum. Neutralizing SA-11 polyclonal antiserum and VP7 monospecific antiserum reacted specifically with the expressed gene product. Antiserum derived against the recombinant VP7 protein neutralized SA-11 rotavirus infectivity in a fluorescent focus assay.


Sequence conservation among the cognate nonstructural NS3/3A protein genes of six bluetongue viruses. Full-length cDNA copies of segment 10 genes of bluetongue virus serotypes 2, 11, 13 and 17 were synthesized by the Clamp-R method and inserted into the plasmid pUC19. The complete nucleotide sequences of these four cognate genes were sequenced and determined to be 822 nucleotides in length, smallest of the 10 genes in the bluetongue virion. These four cognate gene segments contained two in-phase and overlapping open reading frames capable of coding for two non-structural proteins of 229 and 216 amino acids with net charges of +4.5 and +5.5, respectively, at neutral pH. Comparative analyses of the predicted amino acid sequences of bluetongue virus serotypes 1, 2, 10, 11, 13 and 17 revealed (i) a high degree of sequence homology and conservation, (ii) a single conserved tryptophan located at residue 159, (iii) the presence of two conserved cysteines at residues 137 and 181 and two potential N-linked glycosylation sites at residues 63-65 and 150-152, (iv) a cluster of six prolines within a 15-amino acid region near the amino terminus, and (v) the longest 3' noncoding sequence of 113 bases among the 10 bluetongue viral genes. Phylogenetic analyses indicated that BTV-10 and -11 are very closely related and BTV-2 is the distantly related serotype of the five US bluetongue virus serotypes.


Complete genomic sequences of the GP5 protein gene of bluetongue virus serotype 11 and 17. The complete nucleotide sequences of full-length copies of genomic segment 6 or M3 of US bluetongue virus serotype 11 and 17 consisted of 1638 nucleotides. The plus-strand contained an open reading frame for a protein of 526 amino acids which was equivalent to about 59,000 Da, similar to the molecular weight of GP5 as determined by SDS-PAGE analysis. This long open reading frame was flanked by a 5' non-coding region of 29 nucleotides and a 3' non-coding region of 28 bases. When the predicted amino acid sequences of GP5 of BTV-11 and -17 were aligned and compared with those of BTV-2, -10, -13, -1AU and -1SA, four major highly conserved domains interrupted by several variable regions were detected. The potential significance of these discrete domains is discussed. Evolutionary and phylogenetic characteristics of these US BTV serotypes were consistent with our finding concerning BTV-1AU and -1SA.


Bacterial expression of the capsid antigen domain and identification of native gag proteins in spumavirus-infected cells. A bacterial expression plasmid containing the central part of the gag gene of the human spumaretrovirus (HSRV) was constructed and expressed in E. coli. The expected protein product consisting of the complete region of the HSRV capsid antigen and part of the matrix protein was expressed in relatively large amounts. Polyclonal antisera raised against this recombinant protein were used to identify authentic gag precursors of 78 and 74 kDa and processed gag proteins of 60, 58, and 33 kDa in HSRV-infected human embryonal fibroblast cells by radioimmunoprecipitation. The recombinant antigen will be useful for the detection of antibodies against HSRV gag proteins in human sera.


Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals. A hepatitis C virus (HCV) genome was isolated and sequenced from a single Japanese patient with chronic non-A, non-B hepatitis. The genome (HCV-JT), which was constructed with 23 cDNA clones, consisted of 9436 nucleotides with a long open reading frame which could encode a sequence of 3010 amino acid residues. To study the sequence variation of the HCV genome in an individual, we analyzed another sequence of the HCV genome (HCV-JT') constructed with different cDNA clones derived from the same patient. The nucleotide variation between HCV-JT and -JT' was less than 1%, and was distributed throughout the genome except in the 5' non-coding region, where no variation was observed. The diversity was higher (1.6%) in the putative envelope protein region than in other regions. The nucleotide and deduced amino acid sequences of HCV-JT showed homologies of about 91 and 95%, respectively, with those of other Japanese HCV isolates. The nucleotide diversity was high in the gp 70 region (corresponding to the NS 1 region of flaviviruses) and low in the 5' non-coding and p22 (putative core protein) regions. A similar pattern of distribution of nucleotide changes was observed on comparison of HCV-JT with an American isolate HCV-US, where the homologies in nucleotide and amino acid sequences were about 79 and 85%, respectively. Base transversions contributed about 50% of the total base exchanges between the Japanese and American HCV sequences, but only 20% or less of those among Japanese HCV or among American HCV sequences. Thus, the Japanese and American HCVs are genetically distinguishable, supporting our earlier prediction that these two HCVs could be classified as different subtypes.


Inhibition of bovine leukaemia virus replication by the antisense RNA in cell line CC81. A model system has been developed for quantitative evaluation of bovine leukaemia virus (BLV) replication in a permanent cell line CC81. Transfection of the BLV DNA into these cells evoked typical signs of retroviral infection: formation of syncytia, manifestation of reverse transcriptase activity and appearance of characteristic budding retroviral particles. To inhibit BLV replication, a recombinant plasmid pAGR with an antisense RNA gene targeted at the R-U5 region (147th-342th nt) of the viral genome has been engineered. Cotransfection of CC81 cells with infectious BLV DNA and pAGR led to effective inhibition of BLV replication by the antisense RNA, evidenced by a drop in the number of syncytia and reverse transcriptase activity. Maximal inhibition of BLV replication (95-97%) was observed at a weight ratio of input viral and plasmid DNAs equal to 1:10.


Histopathology of laryngeal tumours.

Histopathology of laryngeal tumours. A description is given of the frequent as well as of the less frequent epithelial and mesenchymal tumours of the larynx. The description is based on the light microscopical and histochemical characteristics of these tumours. The relation to the clinical presentation is briefly mentioned. The correlation between the subtypes and prognosis is discussed.


[Effects of piracetam on Na(+)-K(+)-ATPase and monoamine oxidase in rat brain and its antioxidation effect]. Piracetam, ig 600 mg.kg-1.d-1 for 30 d, caused a 20% decrease in the activity of Na(+)-K(+)-ATPase and monoamine oxidase (MAO) in vivo. In vitro, it presented an inhibitory effect on MAO, but had no direct effect on Na(+)-K(+)-ATPase at a concentration of 100 mmol.L-1. Piracetam had a potential action in scavenging free radicals. This action may be related to its clinical effects on amnesia and Alzheimer's disease.


[3,4-Diaminopyridine-evoked norepinephrine release from hippocampal in the absence of extracellular calcium]. Slices of rat hippocampus, preincubated with [3H]norepinephrine ([3H]NE), were superfused and stimulated by addition of 3,4-diaminopyridine (3,4-DAP) for 10 min to the superfusion medium. 3,4-DAP-evoked [3H]NE release both in the absence and presence of extracellular Ca2+. 3,4-DAP-evoked Ca(2+)-independent release of [3H]NE was highly sensitive to sodium channel blocker, tetrodotoxin (TTX, 100% inhibition), suggesting that Na+ per se entered into nerve terminals but not depolarization of membrane followed by Ca2+ channel opening is necessary for 3,4-DAP-evoked Ca(2+)-independent release of [3H]NE. Phorbol ester PDB increased by 480% of control and polymyxin B (PMB) decreased 3,4-DAP-evoked [3H]NE release by 94% of control in the absence of extracellular Ca2+ indicating that the alterations of voltage-dependent Ca(2+)-currents into the cells are not involved in the mechanism of the modulation of 3,4-DAP-evoked [3H]NE release by protein kinase C.


[Quantitative studies on dauricine block of cardiac sodium channels]. Quantitative studies on kinetics of dauricine (Dau) blockade of cardiac sodium channels based on the model of gate-related receptor hypothesis were performed by using computer simulation. The time constant of recovery from blocking of Dau at holding potential -80 mV was 9.8 s and increased with membrane potential hyperpolarization, which suggested that Dau might be trapped in channel by activation gate. The apparent rate of onset of blocking of Dau was 0.33/pulse at stimulation frequency of 1.0 Hz. These results showed that Dau blocks sodium channel in a fast-in-slow-out (FISO) fashion. The binding rate of drug-receptor at clamping potential -50 mV was 753 kL.mol-1.s-1, the unbinding rate was 32.86/s, with Kd of 44 mumol.L-1 which coincided reasonably with documented IC50 value of 46 mumol.L-1 for inhibition of sodium current. The studies also showed a shift by -4 mV on the mid-point of h infinity curve. All these studies lead us to suggest that the binding site of Dau in cardiac sodium channel is activation gate-related receptor site.


The modulatory effects of prostaglandins on both excitatory and inhibitory non-adrenergic non-cholinergic neurotransmission in guinea-pig airways. Guinea-pig trachea and bronchi were used to investigate the effects of indomethacin and prostaglandin E2 (PGE2) on non-adrenergic non-cholinergic excitatory (e-NANC) and inhibitory (i-NANC) neurotransmission evoked by electrical field stimulation. Indomethacin potentiated e-NANC responses in bronchi with intact epithelium but had no effect on epithelium-denuded preparations. Inhibitory NANC responses were increased by indomethacin independent of the epithelium. Both i-NANC and e-NANC neurotransmission were suppressed by PGE2 in a dose-dependent manner. These results indicate that endogenous prostaglandins (e.g. PGE2) generated from the epithelium have an inhibitory effect on i-NANC and e-NANC nerve responses in airways. The epithelium is presumably not the only source for generation of prostaglandins that are involved in i-NANC neurotransmission.


Voltage-sensitive calcium channels in a human small-cell lung cancer cell line. Utilizing the whole-cell patch-clamp method we assessed the Ca2+ current (ICa) in well-established cell lines from human small-cell carcinoma (SCC) of the lung, NCI-H209 and NCI-H187. The Ca2+ current was readily observed in H209 tumour cells (90% of the cells tested), whereas H187 tumour cells only occasionally expressed Ca2+ channels (26% of the cells tested). H209 Ca2+ current was evoked by potentials greater than -30 mV and exhibited partial inactivation over the duration of a 40 ms command potential. This inward current was unchanged by alteration of the holding potential from -80 to -40 mV and the activation phase of the Ca2+ current was best fitted by Hodgkin-Huxley m(t)2 kinetics. H209 Ca2+ current was reduced over 80% by verapamil (100 microM), whereas w-conotoxin (5 microM) appeared to be without effect. In contrast, H209 Ca2+ current was rapidly abolished by nifedipine (10 microM), strongly suggesting the presence of L-type Ca2+ channels. Voltage-gated Ca2+ channels may be important to the secretion of ectopic hormones and the etiology and pathogenesis of Lambert-Eaton syndrome, an autoimmune disorder of the motor nerve terminal in which autoantibodies directed against voltage-gated Ca2+ channels are produced.


Water flux and early signs of entrance into phase III of fasting in grey seal pups. Body water loss and turnover rate were measured in fasting newly weaned grey seal (Halichoerus grypus) pups without access to water for 52 days, by use of bolus injections of deuterated water. Total body water (N) was 41.8 +/- 2.3 (SD)% of total body weight at day 3 and 44.5 +/- 5.9 (SD)% at day 51 of fasting (P greater than 0.05), while the water content of body core decreased from 73 to 61% over the same period. The average water efflux rate in the same period was 362 +/- 17 (SD) ml d-1, the reduction in N over the same period contributing 35% to this end. The biological half-time of deuterated water was 38.2 +/- 3.3 (SD) d. Plasma osmolality increased from a stable level of 329 +/- 11 (SD) mosmol kg-1 to 445 +/- 11 (SD) mosmol kg-1 after day 38 of fasting. Plasma urea was the major contributor to this increase, in spite of a steadily decreasing urine urea concentration (Nordøy et al. 1990) throughout the entire experimental period. It is concluded that grey seal pups endure 52 d of fasting without intake of water with a minor dehydration of body core, while they become hyperosmotic due to increased catabolism of protein after 38 d when sparing of the insulating blubber layer in preparation for life in cold water seems to occur.


Effect of ginsenoside Rgl on the release of enzymes by cultured endothelial cells. The effects of ginsenoside Rgl, isolated from Ginseng Radix, on the secretion of plasminogen activator and angiotensin-converting enzyme from cultured human umbilical vein endothelial cells were investigated in vitro. Ginsenoside Rgl significantly increased the secretion of plasminogen activator from the cells both with and without stimulation of the cells by thrombin. Ginsenoside Rgl also remarkably induced the secretion of angiotensin-converting enzyme from the cells. Furthermore, ginsenoside Rgl showed some morphological alteration in the surface membrane of the cells. In addition, survival-promoting effect of CPAE cell line by ginsenoside Rgl was observed.


A survey of Epstein-Barr virus gene expression in sporadic non-Hodgkin's lymphomas. Detection of Epstein-Barr virus in a subset of peripheral T-cell lymphomas. This study analyzes the association of Epstein-Barr virus (EBV) with non-Hodgkin's lymphoma (NHL) arising in patients without pre-existing overt immunodeficiency. The authors examined 201 lymphomas (105 high-grade B-cell, 82 peripheral T-cell, 7 high-grade non-B-cell, non-T-cell, and 7 hairy-cell leukemia) for EBV gene expression by immunohistologic procedures using monoclonal antibodies to EBV latent, immediate early, and replicative infection antigens. Transformation-associated EBV latent membrane protein 1 (LMP 1) was detected in 13 (6%) NHL, comprising 4 (4%) high-grade B-cell, 8 (10%) peripheral T-cell, and 1 non-B-cell, non-T-cell lymphomas. Anaplastic large-cell lymphoma of T-cell type was consistently LMP 1-negative. EBV nuclear antigen 2 was demonstrated in only three (1%) cases. Induction of replication as defined by expression of the immediate early BamHI Z leftward reading frame 1 (BZLF1) protein was detected in five cases, but early (EA) and late (VCA and MA) lytic cycle antigens were only found in two cases and in one case, respectively. The presence of EBV was confirmed by in situ DNA hybridization in 9 of 11 EBV antigen-positive lymphomas. This study shows the surprisingly frequent presence of EBV in peripheral T-cell NHL in European patients without pre-existing overt immunodeficiency. Interestingly, most sporadic B-cell NHL are not associated with the virus. Furthermore, the usefulness of selected monoclonal antibodies for the routine immunohistological diagnosis of EBV infection was confirmed.


Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction. To examine the association of human papillomavirus (HPV) infection with anal squamous cell carcinoma, the authors applied the highly sensitive polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques to detect HPV DNA in formalin-fixed, paraffin-embedded tissues from 18 patients. The presence of HPV types 16/18 in 3 (16.7%) of 18 patients with anal carcinoma was found, using a colorimetric ISH technique for HPV types 6, 11, 16, 18, 31, 35, and 51. Results from one of these three patients were also positive for HPV 31, 35, 51 by ISH techniques. When the same series was analyzed using the PCR and consensus primers to the L1 open reading frame of the HPV genomes, the frequency of positive patients rose to 14 (77.8%) of 18. PCR analysis of the 14 lesions containing HPV DNA, using type-specific primers and probes for HPV 6, 11, 16, 18, and 33, showed that 1 contained HPV 6, 1 contained HPV 11, 4 contained HPV 16, 1 contained HPV 18, 1 contained HPV 33, 5 contained HPV of unclassified type(s), and 1 contained a mixture of three HPV types. There was concordance between typing of cases that were positive by ISH and PCR methods. These data agree with the concept that HPV, in particular type 16, is implicated in the pathogenesis of anal cancer.


Cellular localization of retinoic acid receptor-gamma expression in normal and neoplastic skin. Retinoids profoundly affect the normal growth and differentiation of epithelial tissues. Retinoic acid receptor-gamma (RAR-gamma) is a member of a family of retinoid receptors, and has been shown to be expressed almost exclusively in skin. However, little is known about the cellular localization of this receptor in human skin. The authors studied the expression of RAR-gamma in normal skin and human skin tumors by Northern blot analysis and in situ hybridization. RAR-gamma mRNA was detected in normal skin as well as in cultures of neonatal keratinocytes. Using an oligonucleotide specific for the RAR-gamma cDNA isoform 1 (RAR-gamma 1), RAR-gamma 1 mRNA was localized to all layers of the epidermis, the outer root sheath of hair follicles, follicular hair bulbs, eccrine and sebaceous glands. Basal cell carcinoma constitutively expressed gamma-1 mRNA and one of seven squamous cell carcinomas showed loss of gamma-1 mRNA expression, relative to adjacent epithelium. By contrast, normal melanocytic nevi and tumor-associated lymphocytes expressed little or no RAR-gamma mRNA. These results suggest that RAR-gamma 1 may play an important role in the maintenance and differentiation of normal epidermis and skin appendages.


Immunohistological analysis in the distribution of cells in the fetal porcine adenohypophysis. Adenohypophyses of porcine fetuses from 25 to 110 days of gestation were studied by immunohistochemical staining to ascertain the ontogeny of specific cell types and their spatial distribution in the pars distalis. No hormone-containing cells were found before 30 days of gestation. ACTH cells were observed first at 40 days, while GH and LH cells appeared first at 60 days. PRL cells were initially detected at 105 days. ACTH immunoreactive cells were also observed in the pars intermedia at 40 days. Blood capillaries were interposed between cell cords of the pars distalis after 40 days of gestation. ACTH cells were evenly distribution in all areas of the pars distalis except the rostral area (sex zone). GH cells were densely distributed in lateral wings of the pars distalis and immediately anterior to Rathke's lumen. PRL cells resembled GH cells in their distribution pattern, but PRL cells were fewer in number. LH cells were scattered in the sex zone of the pars distalis from 60 to 80 days of gestation. After 90 days, they became scattered throughout the pars distalis but were more numerous in the sex zone than in other areas. The inductive elements of adenohypophysial cells from Rathke's pouch epithelia are discussed. We hypothesize that cell cords of specific areas facing Rathke's lumen may differentiate into specific cell types of the pars distalis during fetal life.


Novel inclusion bodies in Betz cells of cortical area 4 of aged rhesus monkeys. Seven adult rhesus monkeys ranging in age from 5 to 35 years were studied in an ultrastructural investigation of cortical area 4. Some Betz cells of layer 5 contained a perikaryal inclusion body (IB) of a unique and characteristic ultrastructural configuration that has not previously been described. The IBs consist of a regular array of three sets of equidistant parallel sheets and are not membrane bound. The distance between the sheets measures approximately 80 nm. The three sets of sheets intersect at an invariable angle of 60 degrees, forming a regular geometric structure. Each sheet comprises a single layer of parallel and densely packed straight filaments, which are approximately 10 nm thick. Thus the IBs present highly ordered crystalline arrays of filaments. The occurrence and number of IBs may be age related, since they appeared only in monkeys in mid to late adulthood and were most numerous in the oldest monkey.


Topographical mapping of GnRH receptors on dispersed mouse pituitary cells by backscattered electron imaging. Mouse anterior pituitary cells cultured for 2 days were stimulated with one of three biotinylated-GnRH probes ([biotinyl-Lys6]-[D-Lys6]GnRH, [biotinyl-Ser4]-[D-Lys6]GnRH, [biotinyl-Ser4]-[D-Trp6, des-Gly10]GnRH) in the cold (4 degrees C) for 1 hr. These cells were subsequently fixed and an avidin-gold complex was conjugated to the bound GnRH. After a second fixation, the gold label was silver-enhanced for viewing with a scanning electron microscope. Gonadotropes were identified as a result of the labeling procedure, measured for size, and the number of GnRH receptor sites counted. Gonadotropes ranged from 3 to 13 microns in diameter and contained from 23.2 +/- 3.3 to 338.4 +/- 25.2 sites per cell depending upon the size of the cell and the ligand employed. The methods described should be applicable for studying the topographical distribution of a variety of cellular receptors.


Nerve growth factor receptor (NGFR)-like immunoreactivity in the perineurial cell in normal and sectioned peripheral nerves of rats. The occurrence of nerve growth factor receptor (NGFR)-like immunoreactivity in the perineurial cells of sympathetic and sensory nerves was analyzed in normal controls and after surgical severance. In the normal nerve trunk, no cellular elements exhibited NGFR-like immunoreactivity. Immunoreactivity was induced in the innermost perineurial cells and in all Schwann cells distal to a severed nerve. The acquisition of immunoreactivity in perineurial cells was also seen in deafferented sympathetic ganglia. On the other hand, the perineurial cells as well as axonal endings showed NGFR-like immunoreactivity consistently in the terminal region of normal nerves, and this immunoreactivity was enhanced by nerve section. The present study clearly showed that different types of nerve terminals are ensheathed to different extents by perineurial cells which were first clearly identified by their NGFR-like immunoreactivity: 1) nerve terminals were free of the perineurium far away from targets in the iris, 2) they were free of the perineurium at sites close to targets in the lingual epithelium, and 3) the perineurium ensheathed nerve terminals together with targets in Meissner's sensory corpuscle. The functional significance of the three different patterns of perineurial enclosure requires further examination.


Long-term outcome of patients with relapsed and refractory germ cell tumors treated with high-dose chemotherapy and autologous bone marrow rescue. To review the long-term outcome of patients with recurrent and refractory germ cell tumors treated with high-dose chemotherapy and autologous bone marrow rescue. Cohort study. A university hospital. Forty consecutive patients with recurrent or refractory germ cell tumors treated at Indiana University between September 1986 and June 1989. Patients were treated with high-dose carboplatin (900 to 2000 mg/m2 body surface area) and etoposide (1200 mg/m2). Three patients also received ifosfamide (10 g/m2). All patients had autologous bone marrow rescue. Of the 40 study patients, 26 received two full courses of therapy. Patient charts were reviewed to determine the rate and duration of complete and partial remission and the number of long-term, disease-free survivors. The influence of cisplatin-refractory disease and the site of the primary tumor on the incidence of remission and survival were also investigated. Of the 40 study patients, 26 (65%) responded to treatment; 12 (30%) achieved a complete response, and 14 (35%) achieved a partial response. Of the 12 complete responders, 5 relapsed, and 1 died of treatment-related acute leukemia 27.5 months after treatment without evidence of germ cell cancer. Six (15%) of the original 40 patients, of whom 3 were refractory to cisplatin, remained in complete remission after at least 24 months of follow-up. Eight of 40 patients had primary mediastinal germ cell tumors with no complete remissions and a median survival of 2 months (range, 0.5 to 9.0 months). Treatment with high-dose carboplatin and etoposide in conjunction with autologous bone marrow rescue in patients with relapsed or refractory germ cell tumors is a potentially curative therapeutic option, even for heavily pretreated or cisplatin-refractory patients. Some degree of disease resistance to cisplatin can be overcome with dose escalation of platinum compounds. Patients with multiple recurrences of relapsed or refractory primary mediastinal germ cell tumors were not helped by this approach.


[Neuropathology of the brain in 174 patients who died of AIDS in a Paris hospital 1982-1988]. Between 1982 and 1988, 174 brains were systematically collected from consecutive, autopsied AIDS patients in a Parisian general hospital without neurology and psychiatry departments. The data obtained under these conditions provide reliable information on the frequency of central nervous system (CNS) involvement in a non-selected population of AIDS patients, most of whom were homosexuals (75.9%). One or several lesions were observed in 148 cases (85%). HIV encephalitis and/or leucoencephalopathy with multinucleated giant cells was found in 33 cases (18.9%). Opportunistic infections were identified in 91 patients (52.3%): toxoplasmosis (65 cases; 37.3%), cytomegalovirus encephalitis (25 cases; 14.3%), cryptococcosis (9 cases; 5.8%), progressive multifocal leukoencephalitis (5 cases; 2.8%), candidosis (1 case) and tuberculosis (1 case). Neoplasias were observed in 23 patients: primary (16 cases; 17.9%) or secondary malignant non Hodgkin's large B-cell lymphoma (3 cas; 1.1%), Kaposi's sarcoma (1 case) and glioma (3 cases; 1.1%). Non-specific lesions (vasculitic, hemorrhagic, metabolic and especially microglial nodules) were common. During the 6 years of study, the rate of CNS involvement was constant. The number of toxoplasmosis cases per year was stable, however, evolutive forms were more prevalent between 1982 and 1986, whereas treated inactive lesions were seen most frequently thereafter. The opportunistic complications were often associated and it should be noted that HIV encephalitis was associated with one of several such infections in 85% of the patients. This high rate of association suggests that these opportunistic infections may play a role in the pathogenesis of HIV encephalitis.


Possible effects of the kallikrein-kinin system on male reproductive functions. All four components of the kallikrein-kinin system--kininogens, tissue kallikreins, kinins, and kininases--have been found in human male genital secretions. Kinins are continuously released from seminal plasma kininogens through limited proteolysis by kininogenases like tissue kallikrein from prostate and sperm acrosin. Kinins are the terminal effectors of the kallikrein-kinin system and increase sperm motility and sperm metabolism at nanomolar concentrations. Recent investigations indicate that these effects are possibly mediated by a specific sperm membrane integrated bradykinin receptor, subtype B2. The two major kininase that are present in seminal plasma are kininase II and neutral metallo-endopeptidase. Kininase II, which is identical with angiotensin-converting enzyme, is also involved in the renin-angiotensin system as it converts angiotensin I into angiotensin II and thus is the connecting enzyme of both systems. Apart from the observed effects of kinins on sperm motility, the kallikrein-kinin system is thought to be involved in the regulation of spermatogenic functions of the testis: in the rat, kallikrein activates Sertoli cell function, increases the relative number of spermatocytes and the [3H] thymidine incorporation of testicular tissue, enhances glucose-intake, and increases testicular blood flow. Clinical trials showed that systemic administration of kallikrein may be particularly useful for treatment of infertile men suffering from asthenozoospermia and/or oligozoospermia. During kallikrein therapy, the number of spermatozoa and both quantitative and qualitative sperm motility increased, and a significant improvement of the conception rate was achieved. An increased sperm number was also observed after application of the specific kininase II inhibitor captopril.(ABSTRACT TRUNCATED AT 250 WORDS)


Age-related alterations of isoproterenol-stimulated adenylyl-cyclase activity are partially corrected by thymic graft. Previous experimental results have demonstrated progressive impairments in beta-adrenergic responsiveness with advancing age. Beta-adrenoceptors are involved in the alterations as their density progressively decreases during aging. Alterations in both in vivo responsiveness and receptor density are corrected by neonatal thymic grafts. In the present paper adenylyl-cyclase (AC) activity has been studied in the same animal models used before. Results show that no statistically significant changes can be observed when AC is assayed in absence of beta-adrenergic stimulation. On the contrary, when assayed after Isoproterenol stimulation, AC activity shows a shift of the peak and a decrease of its height in aged animals. A neonatal thymus grafted into old recipients one month before the experiment was performed, is capable of correcting the altered height of the peak but not the peak concentration.


Association of human sperm nuclear decondensation and in vitro penetration ability. Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize an egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head (r = 0.43; P less than 0.005). The fertilizing ability of sperm processed with Test-Yolk buffer was correlated with the percentage of sperm at the fully decondensed stalk stage (r = 0.51; P less than 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive (greater than 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or whole-wash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.


Immunoenhancing effects of alprazolam, a benzodiazepine receptor agonist. The effects of alprazolam (ALP), a triazolobenzodiazepine with high affinity for "central" benzodiazepine receptors, were examined on several parameters of immune function in mice. NK, MLR, and mitogen-induced lymphocyte proliferation were all significantly increased 2 hr after administration of low doses (0.02-1.0 mg/kg) of ALP. Twenty four hr later, similar but less robust immunoenhancing effects were observed. These measures of immune functions were not affected by higher doses of ALP (5-10 mg/kg). The immunoenhancing effects of ALP did not appear related to serum corticosterone levels. These and other findings demonstrate that the GABA/benzodiazepine receptor chloride channel complex can bidirectionally modulate immune function.


Induction of adrenocorticotropic hormone release by interleukin-6 in vivo and in vitro. Recent reports show that cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and intravenously administered interleukin-6 (IL-6) stimulate adrenocorticotropic hormone (ACTH) release. Both IL-1 and TNF are known to be potent inducers of IL-6, a monokine produced by activated monocytes and folliculo-stellate cells of the pituitary gland and released from the hypothalamus. To determine the site(s) of action of IL-6 in the control of ACTH release, we injected human recombinant IL-6 into the third brain ventricle (3V) of freely moving, conscious male rats and measured ACTH by RIA. Both 0.05 pmole and 0.25 pmole doses of IL-6 were ineffective to change plasma ACTH in comparison to the values in controls. The maximal IL-6 dose tested of 1.25 pmole increased plasma ACTH within 15 min and the response lasted over 180 min. The effects of IL-6 on plasma ACTH were only partially paralleled by increased rectal temperature which suggests that hypothalamic temperature regulating centers were independent of these actions. To evaluate a possible direct effect on the pituitary, IL-6 was incubated in vitro with hemipituitaries under an atmosphere of 95% O2/5% CO2. After 1 hr of incubation IL-6 failed to cause any change in the secretion of ACTH throughout a concentration range of 10(-15) to 10(-9) M. Increased ACTH secretion into the incubation medium was found only with 10(-13) M IL-6 after a 2-hr incubation. The results support a possible role for IL-6 at both hypothalamic and/or pituitary levels to stimulate ACTH release.


Cholesterol-rich rabbit serum modulates beta-adrenergic receptor density of human lymphocytes. A possible role of LDL-cholesterol. The effect of in vitro treatment of human lymphocytes with rabbit cholesterol-rich serum (RCS) on the membrane microviscosity as well as on the beta-adrenergic receptor density has been investigated. RCS treatment of cells resulted in a 30% decrease of receptor density without any effect on membrane microviscosity. A complete recovery was observed incubating the RCS cells either with the "Active Lipids" (AL) or with heparin. The AL are a mixture of neutral lipids, phosphatidylcholine and phosphatidylethanolamine from hen egg yolk known to fluidify the cell membrane. The AL modified membrane microviscosity of control lymphocytes without altering their beta-receptor number. These observations support the proposition that beta-receptor density of human lymphocytes is not regulated by membrane microviscosity and suggest that probably low density lipoprotein-cholesterol complex is involved in such a regulation.


Mobilization of cutaneous immunity for systemic protection against infections. This laboratory reported that a single subcutaneous (SC) injection of the natural steroid hormone dehydroepiandrosterone (DHEA) resulted in significant protection against a lethal herpes virus type 2 encephalitis or a systemic coxsackievirus B4 infection. Our previous results have shown that SC injection of DHEA resulted in upregulation of the specific host immune response resulting in protection against a lethal infection. This hormone did not have any direct antiviral effects in vitro. Furthermore, results indicate that, in vivo, DHEA is not the agent directly mediating the upregulation of the immune response. In the skin, DHEA is converted to androstenediol (AED) and it, in turn, is converted to androstenetriol; this is a metabolic process which appears unique to the skin. This report demonstrates that SC injection of AED results in markedly greater resistance against both viral and bacterial infection. Both DHEA and AED appear to function by facilitating and upregulating host immune responses via mobilization of cutaneous immunity to obtain systemic protection against infections. Because these steroids are native to the host and are regulated by the central nervous system, it is suggested that they may be an integral element of neuroimmunomodulation.


[Primary Aspergillus endocarditis. Apropos of a case and review of the international literature]. The authors report a case of primary aspergillus endocarditis with endophthalmitis and vertebral osteomyelitis. No underlying disease and no predisposing factors were found. Valve replacement plus combined antifungal chemotherapy proved to be effective as the patient is asymptomatic 18 months after the first symptoms. 48 cases of aspergillus endocarditis, without prior cardiac surgery have been reported in the literature. Aspergillus endocarditis was valvular or mural. Extracardiac dissemination was common but endophthalmitis and osteomyelitis were infrequent. In 11 cases, the diagnosis was made by histologic examination of embolectomy or ocular, skin biopsy tissue. All patients were febrile. Blood cultures showed no Aspergillus species. Clinical manifestations of endocarditis were described in less than fifty per cent of cases. Echocardiographic visualization of vegetations was obtained in 5 cases. Many patients experienced embolic phenomena. Mortality from Aspergillus endocarditis is extremely high (96%). Surgery is the main treatment, consisting of valve replacement. Antifungal chemotherapy should be combined. The proper duration and dosage and the combination of antifungal drugs have not been clearly defined.


Selective naltrexone-derived opioid receptor antagonists. Progress in opioid research relies heavily on ligands as probes to evaluate selectivity of action. The design of such ligands using naltrexone as a precursor has afforded a number of highly selective antagonists. These include the kappa opioid receptor antagonist, norBNI, and delta opioid receptor antagonists, NTI and NTB. The unifying concept in the development of these antagonists was the enhancement of selectivity through simultaneous occupation of two neighboring recognition sites by a single ligand. These selective naltrexone-derived antagonists have been used widely to study the involvement of kappa and delta opioid receptors in a variety of pharmacologic, physiologic, and biochemical effects.


In vitro activity of sparfloxacin (AT-4140), a new quinolone agent, against invasive isolates from pediatric patients. Sparfloxacin is a new oral fluoroquinolone agent with putative activity against common pediatric pathogens. Using the broth microdilution method, we evaluated sparfloxacin activity in comparison with those of other antimicrobial agents against 383 pediatric isolates derived from cultures of blood and other normally sterile body fluids. MICs were assessed in Mueller-Hinton broth, serum, and urine, as well as at inoculum sizes of 10(4) to 10(8) CFU/ml. The emergence and stability of resistance and cross-resistance of Pseudomonas aeruginosa (mucoid and nonmucoid) and Staphylococcus aureus to sparfloxacin and ciprofloxacin were evaluated. Inhibitory activity of sparfloxacin against most test organisms was within achievable serum levels. Sparfloxacin was greater than or equal to 2- to 4-fold more active than other quinolones against gram-positive pathogens and 2- to 4-fold less active than ciprofloxacin against P. aeruginosa. Sparfloxacin activity was unaffected by urine and was enhanced by two- to eightfold in human serum. Its potency was not affected by inocula of less than or equal to 10(7) CFU/ml. The frequency of development of spontaneous resistance was similar to that found for other new quinolone agents, and stable resistance emerged only in P. aeruginosa. Sparfloxacin merits additional study against invasive pediatric pathogens.


Comparison of enterococcal and staphylococcal beta-lactamase-encoding fragments. A restriction map of a 13.5-kb EcoRI fragment encoding beta-lactamase from a plasmid isolated from enterococcal strain PA was prepared and found to differ markedly from the published maps of beta-lactamase-encoding EcoRI fragments of two staphylococcal plasmids and the plasmid from enterococcal strain HH22. This comparison also showed that one of two contiguous EcoRV fragments that encompass the beta-lactamase gene differs in size in the PA strain from that found in the other strains. However, restriction sites in the beta-lactamase structural gene (blaZ) were identical in all four plasmids. Further studies compared the beta-lactamase genes of four clinical enterococcal isolates from various geographic locations with those described above. Isolates from Virginia and Florida generated EcoRV fragments identical to those from the plasmid from strain PA, while isolates from Lebanon and Argentina showed EcoRV fragments analogous to those from HH22 and the staphylococci studied. Although there is evidence suggesting that some of these beta-lactamase-producing enterococcal isolates represent a single strain, this study indicates that there is significant variation in the plasmids that encode beta-lactamase.


Transport of the anti-varicella-zoster virus agent 6-methoxypurine arabinoside and its 2'-O-valerate prodrug into human erythrocytes. The transport of the anti-varicella-zoster virus agent 6-methoxypurine arabinoside and its 2'-O-valerate prodrug, 170U88, was investigated by using the human erythrocyte model. The influx of 6-methoxypurine arabinoside was found to occur primarily by means of the nucleoside transporter. (i) Influx was nonconcentrative and saturable (Km = 106 +/- 2 microM). (ii) The inhibitors of nucleoside transport, nitrobenzylthionosine, dipyridamole, and dilazep, inhibited the influx of 10 microM 6-methoxypurine arabinoside by greater than 94%. (iii) Influx was inhibited by nucleosides but not by nucleobases. (iv) 6-Methoxypurine arabinoside was a competitive inhibitor (Ki = 129 +/- 10 microM) of adenosine influx, and adenosine (Km = 160 +/- 9 microM) was found to be a competitive inhibitor (Ki = 134 +/- 9 microM) of 6-methoxypurine arabinoside influx. By contrast, the influx of 170U88 occurred by means of nonfacilitated diffusion. (i) Influx was linearly dependent on the 170U88 concentration. (ii) Influx was not inhibited by nucleobases, nucleosides, or inhibitors of nucleoside transport.


In vitro evaluation of activities of azithromycin, erythromycin, and tetracycline against Chlamydia trachomatis and Chlamydia pneumoniae. The in vitro activities of azithromycin (CP-62,993; Pfizer), erythromycin, and tetracycline were evaluated by inhibiting Chlamydia trachomatis and Chlamydia pneumoniae, formerly TWAR, propagation in vitro in McCoy cells, HeLa cells, and HL cells. Eleven clinical isolates of C. trachomatis (serovars D, E, F, J, K, and L2) and four strains of C. pneumoniae were tested with an inoculum of 10(3) inclusion-forming units in a 96-well microtiter plate. The MIC ranges of these antimicrobial agents against C. trachomatis were as follows: azithromycin, 0.125 to 0.5 microgram/ml; erythromycin, 0.25 to 0.1 microgram/ml; and tetracycline, 0.0625 to 1.0 microgram/ml. The MBC ranges, calculated from passage into antibiotic-free medium, were as follows: azithromycin, 0.125 to 4.0 micrograms/ml; erythromycin, 0.5 to 8.0 micrograms/ml; and tetracycline, 0.0625 to 4.0 micrograms/ml. The MIC ranges for C. pneumoniae in both HeLa and HL cells were as follows: azithromycin, 0.125 to 1.0 micrograms/ml; erythromycin, 0.0625 to 1.0 microgram/ml; and tetracycline, 0.125 to 1.0 microgram/ml. The MBC ranges were as follows: azithromycin, 0.25 to 1.0 microgram/ml; erythromycin, 0.25 to 1.0 microgram/ml; and tetracycline, 0.125 to 4.0 micrograms/ml. From the results of this in vitro study, azithromycin appears to be an effective antibiotic comparable to tetracycline and erythromycin for use in the treatment of both C. trachomatis and C. pneumoniae infections.


Single-dose pharmacokinetics and antibacterial activity of daptomycin, a new lipopeptide antibiotic, in healthy volunteers. Three separate single-dose studies were performed to define the disposition and pharmacokinetics of daptomycin in healthy volunteers. Daptomycin was administered as a single 14C-labeled dose (1.0 mg/kg of body weight) and as single doses between 0.5 and 6.0 mg/kg. All doses were intravenous. Antibacterial activity was determined from doses of 2.0, 3.0, 4.0, and 6.0 mg/kg against two strains of Staphylococcus aureus (one methicillin resistant) and one Enterococcus strain. After administration of 14C-labeled daptomycin, recovery of 14C in urine and feces accounted for 83% of the administered dose, with the greatest fraction (78%) appearing in the urine. Specific analysis for daptomycin in both urine and plasma indicated that metabolic products were present in urine, but total 14C in plasma consisted of daptomycin only. Doses between 0.5 and 6 mg/kg were linear, with a limited total body clearance (0.13 to 0.21 ml/min/kg) and a small volume of distribution (0.10 to 0.15 liter/kg). The small volume of distribution may be a factor of the high plasma protein binding (90 to 95%). Renal clearance made up 34 to 54% of total body clearance. Daptomycin demonstrated in vivo antibacterial activity against all three test strains, with the greatest activity observed against methicillin-resistant S. aureus. The predicted MIC for all three strains was approximately 13 micrograms/ml, corresponding to total (bound plus unbound) drug. On the basis of the drug's pharmacokinetics and antibacterial activity, doses of 4 to 6 mg/kg/day, possibly in divided doses, are predicted to be effective.


Metabolism and pharmacokinetics of the anti-varicella-zoster virus agent 6-dimethylaminopurine arabinoside. The metabolism of 6-dimethylaminopurine arabinoside (ara-DMAP), a potent inhibitor of varicella-zoster virus replication in vitro, was studied in rats and cynomolgus monkeys. Rats dosed intraperitoneally or orally with ara-DMAP excreted unchanged ara-DMAP and one major metabolite, 6-methylaminopurine arabinoside (ara-MAP), in the urine. They also excreted allantoin and small amounts (less than 4% of the dose each) of hypoxanthine arabinoside (ara-H) and adenine arabinoside (ara-A). The relative amount of each urinary metabolite excreted remained fairly constant for intraperitoneal ara-DMAP doses of 0.3 to 50 mg/kg of body weight. Rats pretreated with an inhibitor of microsomal N-demethylation, SKF-525-A, excreted more unchanged ara-DMAP and much less ara-MAP than did rats given ara-DMAP alone. Rats pretreated with the adenosine deaminase inhibitor deoxycoformycin excreted more ara-MAP and much less ara-H and allantoin. ara-MAP was shown to be a competitive alternative substrate inhibitor of adenosine deaminase (Ki = 16 microM). Rats given ara-DMAP intravenously rapidly converted it to ara-MAP and purine metabolism end products; however, ara-A generated from ara-DMAP had a half-life that was four times longer than that of ara-A given intravenously. In contrast to rats, cynomolgus monkeys dosed intravenously with ara-DMAP formed ara-H as the major plasma and urinary end metabolite. Rat liver microsomes demethylated ara-DMAP much more rapidly than human liver microsomes did. ara-DMAP is initially N-demethylated by microsomal enzymes to form ara-MAP. This metabolite is further metabolized by either adenosine deaminase, which removes methylamine to form ara-H, or by microsomal enzymes, which remove the second methyl group to form ara-A.


Pharmacokinetics of temafloxacin in humans after multiple oral doses. The multiple-dose pharmacokinetics and tolerance of temafloxacin, a new fluoroquinolone antibacterial agent, were evaluated in healthy volunteers. Temafloxacin was found to be well tolerated when administered orally every 12 h for 7 days at doses of 100, 200, 300, 400, 600, and 800 mg. Steady-state maximum and minimum concentrations in plasma were proportional to dose, averaging slightly over 1.0 and 0.5 microgram/ml/100 mg administered. Analyses of variance found no significant differences among the dosage groups in total apparent clearances (CLT/F), renal clearances (CLR), or nonrenal clearances, which averaged 197, 119, and 78 ml/min, respectively. The half-life increased slightly with dose, averaging 8.4 h overall. The extent of absorption of temafloxacin was quite reproducible, with day-to-day intrasubject variability in minima averaging under 10%. Renal glomerular filtration of unbound drug was the dominant elimination process; however, tubular secretion and reabsorption also appear to occur. Secretion was estimated to account for about 12% of CLT/F during a regimen of 600 mg every 12 h. CLR was relatively constant for urine flow rates above 1 ml/min, but reabsorption appeared to occur under low-flow conditions, resulting in day-versus-night differences in CLR. Intersubject variability in CLT/F over the eightfold range in dosage was only 20%, and 60% of this variance was accounted for by differences in body surface area (or lean body mass), concentration in plasma, and urine flow rate. Overall, it appears that the pharmacokinetics of temafloxacin are essentially linear, reproducible within a subject, and predictable among subjects.


Pentoxifylline modulates activation of human neutrophils by amphotericin B in vitro. The antifungal agent amphotericin B (AmB) alters neutrophil (polymorphonuclear leukocyte [PMN]) function, and this may be the mechanism for some of the adverse effects caused by AmB. AmB is a potent inhibitor of PMN migration, increases PMN adherence and aggregation, and primes PMN for increased oxidative activity in response to a second stimulus. AmB also stimulates mononuclear leukocytes (MNLs) to release inflammatory mediators which augment the effects of AmB on PMN function. In the present study, we observed that the methylxanthine derivative pentoxifylline decreased the effects of AmB on PMN function. AmB (2 micrograms/ml) priming doubled PMN chemiluminescence stimulated by fMet-Leu-Phe. In the presence of MNLs, AmB priming increased fMet-Leu-Phe-stimulated PMN chemiluminescence to 622% of unprimed PMN activity. Pentoxifylline (100 microM) blunted the rise in AmB-augmented PMN chemiluminescence in the presence of MNLs to 282% of unprimed PMN activity, and pentoxifylline metabolites were active at 10 microM. Pentoxifylline (100 microM) also blocked AmB-augmented PMN oxidative activity in whole blood, as measured by nitroblue tetrazolium reduction. In the presence of MNL, AmB (2 micrograms/ml) doubled the expression of the important PMN adherence factor Mac-1. Pentoxifylline (1 mM) decreased AmB-stimulated PMN Mac-1 expression back to unstimulated amounts. In the presence of MNLs, AmB (2 micrograms/ml) decreased PMN nondirected and directed migration to fMet-Leu-Phe to 40 and 38% of control PMN migration, respectively. Pentoxifylline (300 microM) counteracted AmB inhibition of nondirected and directed migration to fMet-Leu-Phe, resulting in migration that was 71 and 87% of control PMN migration, respectively. In contrast, the methylxanthine caffeine (100 muM) increased AmB-enhanced chemiluminescence but did not affect AmB-inhibited PMN migration. Pentoxifylline should be evaluated as adjunctive therapy to lessen the inflammatory damage caused by AmB.


Study of the parameters of binding of R 61837 to human rhinovirus 9 and immunobiochemical evidence of capsid-stabilizing activity of the compound. The binding of the antiviral compound R 61837 to human rhinovirus 9 (HRV 9) was studied quantitatively and compared with binding of R 61837 to HRV 9H, a semiresistant variant. For both strains, radiolabelled R 61387 bound to native particles only. The Kd values obtained by Scatchard analysis of saturation binding data were 37 nM for HRV 9 and 172 nM for HRV 9H, whereas the concentrations resulting in a 50% reduction of cytopathic effect were 42 nM and 840 nM, respectively. Reversibility experiments showed that 65% of the compound could be extracted with chloroform from HRV 9H but less than 5% could be extracted from HRV 9. Dissociation studies demonstrated that in the presence of excess unlabelled compound, the half-lives of the virus compound complex HRV 9 and HRV 9H were 385 and 15 min, respectively. The effect of this antirhinoviral compound on the formation of subviral particles induced by low pH or heat was also investigated. Rate zonal centrifugation experiments using [35S]methionine-labelled HRV 9 showed that binding of R 61837 protected the virus against heat (56 degrees C) and acid (pH 5.0) and that at the same concentration of R 61837 the semiresistant strain was stabilized to a lesser extent. This observation was confirmed immunochemically with nonneutralizing and neutralizing monoclonal antibodies. Both 80S and 130S subviral particles have C antigenic determinants, whereas native particles (150S) have been designated D. R 61837 prevented the switch from D to C antigenicity which can be induced by exposure of rhinoviruses to mild denaturing conditions. These findings indicate that the compound is able to prevent a conformational change of the capsid which may be a prerequisite for infection.


Treatment of Coxsackievirus A9 myocarditis in mice with WIN 54954. The therapeutic efficacy of an experimental antiviral agent, WIN 54954, was evaluated in murine myocardial infection with coxsackievirus A9 (CVA9). Eight-month-old male Swiss Webster mice were inoculated with 1.5 x 10(4) PFU of CVA9, Boston strain 13. WIN 54954, a broad-spectrum antipicornavirus agent, was administered orally in a dose of 0.25, 2.5, 25, 50, 100, or 200 mg/kg of body weight per day on days 1 to 3 after virus inoculation. Control animals received xanthan gum carrier only. Mice were sacrificed on day 4. Myocardial titers of virus were determined and found to be significantly lower in the four highest dose treatment groups (P less than 0.001 for all groups) compared with controls. Heart weights were also significantly lower compared with controls in these four groups (P less than 0.001 for all groups). When mice received 50 mg of WIN 54954 per kg daily beginning at either 48 or 72 h postinoculation, myocardial titers were once again significantly reduced compared with those of controls (P less than 0.001 for both groups). Neurological toxicity was observed in the 100- and 200-mg/kg/day groups but not in the lower-dose groups. Thus, WIN 54954 effectively reduced myocardial CVA9 replication in a murine model.


Cytotoxicity of acridine compounds for Leishmania promastigotes in vitro. The effect of mammalian and bacterial topoisomerase II inhibitors on Leishmania promastigotes was studied in vitro. Parasites were incubated with drugs, and cytotoxicity was assessed on the basis of the loss of flagellar motility and cell lysis after 48 h. 9-Aminoacridines, which are structurally related to the known antileishmanial compounds quinacrine and chlorpromazine, showed activity against the parasite at concentrations in the range of 10 to 20 microM. Adriamycin showed far less activity, while etoposide and several quinolones were inactive at 100-microM concentrations. These results demonstrate that a particular structural class of compounds is cytotoxic to Leishmania species. The unique structure-activity relationship discovered suggests that leishmanial topoisomerase II could be a useful target for chemotherapy.


Antibacterial activity of sparfloxacin against experimental renal infections in mice. In a murine model of renal infection (Staphylococcus aureus and Escherichia coli), sparfloxacin was compared with ciprofloxacin and fleroxacin. After intrarenal inoculation, mice were treated orally for 5 days. The drugs were administered at five different dosages, ranging from 3.125 to 50 mg/kg of body weight per day for S. aureus and from 0.78 to 12.5 mg/kg/day for E. coli. Evaluation of efficacy was based on the proportional reduction of bacterial counts in the kidney tissues of treated animals compared with those of untreated control animals. For S. aureus, the doses required to clear the infection in 50% of mice were as follows: sparfloxacin, 10 mg/kg/day; ciprofloxacin, 33 mg/kg/day; and fleroxacin, 16 mg/kg/day. For E. coli renal infection, the corresponding dosages were as follows: sparfloxacin, 1.5 mg/kg/day; ciprofloxacin, 2.45 mg/kg/day; and fleroxacin, 1.8 mg/kg/day. Sparfloxacin and fleroxacin have a lower effective dose than ciprofloxacin in these models, probably because ciprofloxacin has a shorter serum half-life than the other two compounds.


Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells. The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.


Heavy chain binding protein (BiP/GRP78) and endoplasmin are exported from the endoplasmic reticulum in rat exocrine pancreatic cells, similar to protein disulfide-isomerase. Previously we found that in rat exocrine pancreatic cells, protein disulfide-isomerase (PDI), one of the major resident proteins in the lumen of the endoplasmic reticulum (ER) of many cells, is localized not only in the ER but also in the Golgi apparatus, secretory granules, plasma membranes, and even in the glandular lumens, despite possessing the ER retention signal KDEL (Lys-Asp-Glu-Leu) at the carboxyl terminus. In this report, we examined whether other ER luminal proteins bearing the KDEL signal at their C-termini, such as BiP/GRP78 and endoplasmin/GRP94 are also exported from the ER. We prepared two kinds of affinity-purified polyclonal antibodies; one against a synthetic peptide with 12 amino acids which is identical to the carboxyl terminus of BiP and another against purified endoplasmin. Immunoblot analysis using these two antibodies showed that BiP and endoplasmin exist in both the plasma membrane and the microsomal fractions, similar to the intracellular distribution of PDI in rat exocrine pancreas. The ratios of the amount of the three proteins in the two fractions, however, were variable, suggesting that the KDEL-bearing proteins such as PDI, BiP, and endoplasmin are exported from the ER with different efficiencies. Postembedding protein A-immunogold electron microscopy revealed that endoplasmin was exported from the ER and secreted to the extracellular space. The secretion of PDI in rat pancreatic lobules was inhibited by Brefeldin A (BFA) and by guanidino acid esters (FOY-305), which are known to be the inhibitors of the intracellular transport. Taken together with the previous immunogold electron microscopic analyses by Akagi et al. (1988), it is strongly suggested that in rat exocrine pancreatic cells PDI and the other KDEL-bearing proteins found in the extracellular space were not artificially released by cell damage during incubation but were secreted via the normal secretory pathway.


Insulin mediator stimulation of pyruvate dehydrogenase phosphatases. A two stage assay for detecting insulin mediator based upon its stimulation of soluble pyruvate dehydrogenase (PDH) phosphatase to activate soluble pyruvate dehydrogenase complex (PDC) has been developed. This coupled assay determines the activation of PDC by monitoring production of [14C]CO2 from [1-14C]pyruvic acid. In addition to being more sensitive than the rat liver mitoplast assay previously used, it allows for the separation and investigation of the effects of mediator on the PDH phosphatases individually. It has been previously shown that the insulin mediator stimulates the most abundant PDH phosphatase, the divalent cation dependent PDH phosphatase, by decreasing the phosphatase's metal requirement (1). A metal independent PDH phosphatase has been found in bovine heart mitochondria. This phosphatase is not immunoprecipitated by antiphosphatase 2A antibody, it is not inhibited by okadaic acid, and it is not stimulated by spermine. However, it is stimulated (more than threefold) by insulin mediator prepared from isolated rat liver membranes. It is inhibited by Mg-ATP, with half-maximal inhibition at 0.3 mM; however, this inhibition is overcome by the insulin mediator.


Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes. Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.


Alpha-oxidation of 3-methyl-substituted fatty acids in rat liver. 3-Methyl-substituted fatty acids are first oxidatively decarboxylated (alpha-oxidation) before they are degraded further via beta-oxidation. We synthesized [1-14C]phytanic and 3-[1-14C]methylmargaric acids in order to study their alpha-oxidation in isolated rat hepatocytes, rat liver homogenates and subcellular fractions. alpha-Oxidation was measured as the production of radioactive CO2. In isolated hepatocytes, maximal rates of alpha-oxidation amounted to 7 and 10 nmol/min x 10(8) cells with phytanic acid and 3-methylmargaric acid, respectively. At equimolar substrate concentrations, alpha-oxidation of branched fatty acids was approximately 10- to 15-fold slower than the beta-oxidation of the straight chain palmitate. In whole liver homogenates, rates of alpha-oxidation that equaled 60 to 70% of those observed in the hepatocytes were obtained. Optimum rates required O2, NADPH, Fe3+, and ATP. Fe3+ could be replaced by Fe2+ and ATP could be replaced by a number of other phosphorylated nucleosides and even inorganic phosphate without loss of activity. NADH could substitute for NADPH but not always with full restoration of activity. A variety of other cofactors and metal ions was either inhibitory or without effect. Scavengers of reactive oxygen species, known to be formed during the NADPH-dependent microsomal reduction of ferric-phosphate complexes, were without effect on alpha-oxidation. No evidence was found for the accumulation of NADPH-dependent or Fe(3+)-dependent reaction intermediates. Subcellular fractionation of liver homogenates demonstrated that alpha-oxidation was located predominantly, if not exclusively, in the endoplasmic reticulum. alpha-Oxidation, measured in microsomal fractions, was not inhibited by CO, cytochrome c, or ferricyanide, indicating that NADPH cytochrome P450 reductase and cytochrome P450 are not involved in alpha-oxidation. Our results indicate that, contrary to current belief, alpha-oxidation is catalyzed by the endoplasmic reticulum. The cofactor requirements suggest that alpha-oxidation involves the reduction of Fe3+ by electrons from NADPH and that it is stimulated by phosphate ions and nucleotides.


Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipocytes. The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been investigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal 32P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3-month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the Km for ATP was not modified. In contrast, the insulin-stimulated Vmax value was decreased by two-fold in 24-month-old rats. The decrease in Vmax does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor beta-subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor.


Myeloperoxidase-catalyzed iodination and coupling. Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, I-, and H2O2, the pH optimum of MPO-catalyzed iodination was markedly acidic (approximately 4.0), compared to LPO (approximately 5.4) and TPO (approximately 6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the H2O2 generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased by 0.1 N Cl-. Under these conditions iodination and thyroxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 microM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination.


Processing of the intermediate form of the iron-sulfur protein of the BC1 complex to the mature form after import into yeast mitochondria. The Rieske iron-sulfur protein of the cytochrome bc1 complex is synthesized in the cytosol as a precursor with an additional 30 amino acids at the amino terminus. After import into the mitochondrial matrix, the precursor is processed to the mature form by two distinct proteolytic cleavages. Addition of 2.5 mM EDTA and 0.5 mM o-phenanthroline to the incubation mixture during import of the iron-sulfur protein precursor in vitro resulted in the selective inhibition of the second processing step with the concomitant accumulation of the intermediate form. The intermediate form was chased to the mature form in the presence of antimycin and oligomycin (to block the formation of a membrane potential) provided that 0.5 nM ATP and a metal ion such as Ca2+, Mn2+, or Mg2+ were added. Ca2+ ion was the most effective and at a concentration of 2.5 mM resulted in the complete cleavage of the intermediate to the mature form. Addition of Zn2+, Co2+, Mo2+, and Fe2+ was not effective in restoring the second cleavage. The pH optimum for the processing of the intermediate form of the iron-sulfur protein to the mature form was between 6.8-8.0. Processing of the intermediate form of the iron-sulfur protein to the mature form was observed at temperatures ranging from 12 to 27 degrees C in a temperature-dependent manner. The time course during the chase indicated that the second processing step was completed within 2 min after addition of Ca2+ ions. Attempts to isolate the second processing enzyme by sonication of mitochondria or by solubilization with detergents such as digitonin, Triton X-100, dodecyl-maltoside, or octyl-glucoside were unsuccessful as only the first cleavage was observed. Hence, the second processing enzyme may be present in the inner membrane or matrix in a conformation disrupted by detergents or alternatively the enzyme may be very labile.


Exogenous quinones directly inhibit the respiratory NADH dehydrogenase in Escherichia coli. The ability of naphthoquinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress. However, excess superoxide dismutase and catalase failed to protect Escherichia coli in rich medium against growth inhibition by plumbagin, indicating that its toxic effect was not due to the production of partially reduced oxygen species. Respiration failed immediately upon the addition of growth-inhibitory levels of plumbagin. Studies in vitro showed that plumbagin and other redox-active quinones intercept electrons from NADH dehydrogenase, the primary respiratory dehydrogenase in glucose-containing media. An excess of oxidative substrate, such as plumbagin, inactivates this enzyme, which appears to be redox-regulated. The resultant respiratory arrest is a cautionary example of metabolic dysfunction from redox-cycling drugs that cannot be attributed to superoxide or hydrogen peroxide.


Horseradish peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid. II. Oxygen uptake and chemiexcitation. Light emission from the horseradish peroxidase-catalyzed aerobic or anaerobic oxidation of indole-3-acetic acid has been investigated under opposite extreme conditions of enzyme/substrate ratio. The O2-dependent chemiluminescent processes represent a minor part of the total oxygen consumption. Superoxide is involved in chemiexcitation as is evident from the observed inhibitory effect of superoxide dismutase. At high enzyme/substrate ratio, only a part of the emission is dependent on superoxide ion; at low ratio the dependence is extensive. At high ratio, some of the emission is independent of superoxide and O2. The identical quenching effects of D- and L-tryptophan are consistent with the formation of the quenching species only in bulk solution. The similarity of the emission spectra under extreme conditions indicates that the same main emitters are formed. This is also supported by the effect of quenchers. Possibly some of the emitters originate in the oxidative cleavage of the 2,3-double bond of the indole ring.


Spin-labeling studies of rat liver NADPH-cytochrome p450 reductase: conformation and function relationship. ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.


[Pilot phase II trial of high-dose etoposide combined with cisplatin in the treatment of refractory lung cancer]. The efficacy of combined high-dose etoposide with standard dose cisplatin was evaluated in patients who had refractory lung cancer after standard chemotherapy. Each patient was given etoposide at 500 mg/m2/day on day 1 to 3 continuously (total dose 1,500 mg/m2) and cisplatin at 80 mg/m2 on day 1. Fifteen patients (7 adenocarcinoma, 5 small cell lung cancer, 2 squamous cell lung cancer and 1 sarcoma, which latter was difficult to distinguish from giant cell carcinoma) were entered in this study. The overall response was 41.7% (5 of 12); five partial response, 6 no change, and 1 progressive disease. Three treatment-related deaths were observed; one resulted from sepsis and two from respiratory failure because of tumor progression. All of the patients developed severe myelosuppression; the mean nadir white blood cell count was 400, and the mean nadir platelet count was 24,000 in 28 evaluable courses. The range of maximum concentration of etoposide determined by HPLC was from 17.4 to 39.1 micrograms/ml. These results suggest that high-dose etoposide combined with a standard dose of cisplatin is effective against refractory lung cancer.


[A phase II clinical study of cis-diammine glycolato platinum, 254-S, for primary lung cancer]. A phase II clinical study of 254-S, a new anticancer platinum complex, for primary lung cancer was conducted by the 254-S Lung Cancer Study Group consisting of 15 institutions nation-wide. Considering the results of the phase I clinical study, 254-S was administered at 100 mg/m2 by intravenous drip infusion and this administration was repeated at least 2 times at 4-week intervals. Of 75 patients registered, 61 patients consisting of 22 with small cell lung cancer (SCLC) and 39 with non-small cell lung cancer (NSCLC) were evaluable for complete tumor response. Partial response (PR) was obtained in 17 patients, for a 27.9% response rate. The response rate for SCLC was 40.9% (9 PR in 22 patients) and that for NSCLC was 20.5% (8 PR in 39 patients). In SCLC patients with no prior chemotherapy, a 50.0% (5 PR in 10 patients) response rate was obtained. In those with prior chemotherapy, the response rate was 33.3% (4 PR in 12 patients). In NSCLC patients with no prior chemotherapy, a 22.6% (7 PR in 31 patients) response rate was obtained. In hose with prior chemotherapy, the response rate was 12.5% (1 PR in 8 patients). Major toxic effects observed were hematotoxicity such as thrombocytopenia and leukopenia, and gastrointestinal toxicity such as nausea, vomiting and anorexia. Nephrotoxicity observed was mild and infrequent in spite of the low-volume hydration performed. Based on these results, it was concluded that 254-S is a useful anticancer agent for the treatment of primary lung cancer.


[A case report of postoperative recurrent hepatocellular carcinoma effectively treated with HCFU administration combined with TAE]. A 62-year-old man was found to have recurrent hepatocellular carcinoma (HCC) 2 cm in diameter in the left lobe in June 1990. After right lobectomy of the liver which contained the tumors of S6 2 cm and S8 3 cm in diameter 7 months before, the patient was treated with hepatic arterial embolization (TAE) combined with infusion of anti-cancer drug (ADM, CDDP) four times since June 1990. HCFU 300 mg per day was administered orally since June 1990. Since the 3rd TAE in December, 1990, the tumor stain in the left lobe disappeared for 10 months till October, 1991. No remarkable side effect of HCFU was noticed during this period. HCFU administration combined with TAE effectively prevented post-surgical recurrence of HCC in this case.


HPV 16 DNA in autopsy material of a metastatic cervical carcinoma. Human papillomavirus (HPV) DNA has been regularly detected in primary cervical carcinomas and in some metastatic lesions. Using Southern blot hybridization on autopsy material we found HPV 16 DNA in a primary cervical carcinoma and in multiple metastases therefrom.


[Colloid carcinoma of the prostate].

[Colloid carcinoma of the prostate]. We report a case of colloid carcinoma of the prostate, a rare tumor type whose diagnosis is exclusively based on the anatomopathological findings. The case described herein is a primary prostatic tumor. Patient work up revealed no tumor localized to other organs. The total acid phosphatase and prostate acid phosphatase levels were elevated. Subsequent analyses revealed metastases.


[Malignant mixed mesodermal tumor of the ovary. A review of the literature apropos of a case with immunohistochemical study]. A very large left ovarian tumor in a 76 year old woman, on microscopic study, consisted of an epithelial component: both glandular and squamous, and a sarcomatous component: both cartilage and striated muscle. Epithelial and sarcomatous patterns were intermingled in a spindle-cell stroma. Immunohistochemical study revealed, in these spindle cells, cytokeratin-positive anaplastic epithelial structures and vimentin-positive mesenchymal structures. A review of the literature concerning this rare ovarian tumor especially including cases with a immunohistochemical study is added. However, immunohistochemical study does not allow histogenetic interpretation.


Adnexal tumour of probable Wolffian origin. Report of a case. An adnexal tumour of probable Wolffian origin occurring in a 36 year old woman is reported. The lesion was asymptomatic and incidentally discovered during laparotomy. No recurrences have been demonstrated to date. Its origin from Wolffian remnants is briefly.


Synthesis and binding properties to GABA receptors of 3-hydroxypyridinyl- and 3-hydroxypiperidinyl-analogues of baclofen. The synthesis of 3-(3-hydroxy-2-pyridinyl)propanoic acid, 3-(3-hydroxy-2-pyridinyl)-4-aminobutanoic acid, their corresponding piperidine compounds, and of some cyclized derivatives is described. In in vitro assays none of the new compounds shows any noteworthy affinity for GABAA or GABAB receptors; only (R,S)-3-(3-hydroxy-2-pyridinyl)-4-aminobutanoic acid and its lactam inhibited in some degree [3H]GABA binding, at 10(-4) M concentration, with low specificity as regards the two receptors.


Inhibition in vitro of the replication of murine cytomegalovirus or reovirus type 3 by the glutamine analogue acivicin. The replication of synchronised cultures of Balb/c L-3T3 fibroblasts was shown to require glutamine at a concentration of at least 0.12 mM, but the replication of murine cytomegalovirus (MCMV) or reovirus type 3 in the cells required at least 0.25 mM glutamine. In the presence of 2 mM glutamine, the glutamine analogue acivicin inhibited DNA synthesis at a minimum concentration of 2 microM in uninfected cells but partially inhibited the replication of MCMV at 0.12 microM and reovirus at 0.03 microM, and totally inhibited MCMV production at 1.0 microM and reovirus at 0.5 microM. Although the therapeutic index in this instance is not great, it does offer encouragement to explore other amino acid analogues as possible broad-spectrum antiviral agents.


Altered chromatin higher-order structure in cells infected by herpes simplex virus type 1. In cells infected by herpesviruses chromosomal aberrations such as breaks and constrictions are commonly observed. Previous studies have shown that chromatin modifications in cells infected by herpes simplex virus type 1 (HSV-1) are not due to extensive breakdown of host-cell DNA or disruption of nucleosomal structure. We have previously shown that infection by HSV-1 induces single-stranded breaks in the host-cell DNA in a time-dependent fashion. Here we report that the early DNA damage observed in virus-infected cells is related to modifications in the higher-order structure of host-cell chromatin. Such modifications seem to be of a more permanent nature than the DNA single-stranded breaks.


A family outbreak of gastroenteritis caused by group C rotavirus. A family outbreak of gastroenteritis caused by group C rotavirus is described. All five members of the family, with children between 8 and 15 years of age, fell ill with diarrhea. The diagnosis was initially based on the detection of rotavirus RNA showing a typical group C profile in gel electrophoresis in stool samples, and it was serologically verified from patient sera using a cell culture adapted porcine group C rotavirus as a source of standard antigen. All collected serum samples from the family contained IgM and/or IgG class antibodies to group C rotavirus measurable by immunofluorescence antibody test (IFAT). Group C rotavirus specific IgM class antibodies were present in the early serum samples in 3/4 patients. A roller tube neutralization test (NT) was established to demonstrate neutralizing antibodies to porcine group C rotavirus in human sera. These methods can be used to detect serologically group C rotavirus infections.


Induction of protective immunity and neutralizing antibodies to pseudorabies virus by immunization of anti-idiotypic antibodies. Xenogenic anti-idiotypic antibodies (anti-Id) were prepared in rabbits against three murine neutralizing monoclonal antibodies (MAbs) directed to pseudorabies virus glycoproteins. These anti-Id were highly specific to idiotopes on the corresponding MAb molecules. Because the binding of MAb to the corresponding anti-Id was inhibited by the addition of viral envelope protein, these anti-Id seemed to contain a subpopulation of antibodies against the antigen-combining site (paratope) or the region related to the paratope of the MAb molecules. One of the anti-Id to a MAb directed against glycoprotein gp50 induced neutralizing antibodies to PrV. Mice immunized with the anti-Id were protected from lethal infection of PrV.


Coxsackie B1 virus-induced changes in cell membrane-associated functions are not responsible for altered sensitivity to bacterial invasiveness. To analyze the possible mechanisms by which coxsackie B1 virus infection affects the invasiveness of Shigella flexneri, we have studied the influence of intracellular levels of Na+ and K+, ATPase activity, cytoplasmic membrane potential, cAMP level and cell communication through gap junctions. 3h after adsorption of viable or UV-inactivated coxsackie B1 virus the Na(+)-K+ gradient of the cell collapsed, ATPase activity decreased, the cytoplasmic membranic potential-dependent tetraphosphonium ion uptake were reduced. No changes in cAMP or intercellular cell communication were observed. S. flexneri invasiveness in HEp-2 cell pretreated with viable or UV-inactivated coxsackie B 1 virus was enhanced, but bacterial invasiveness was unchanged in K(+)-depleted HEp-2 cells, cell cultures with high intracellular Na+ content or ouabain pre-treated cells compared to control cells. We found no correlation between the enhanced bacterial invasiveness in the early phase of coxsackie B 1 virus infection in HEp-2 cell cultures and intracellular K+ depletion, high intracellular Na+ content, inhibited Na(+)-K+ ATPase activity or membranic depolarization.


Detection of anti-gag antibodies of feline immunodeficiency virus in cat sera by enzyme-linked immunosorbent assay. Using gag protein of feline immunodeficiency virus (FIV) expressed in Escherichia coli, an enzyme-linked immunosorbent assay (ELISA) system was developed for detection of antibodies to FIV gag protein in cat sera. With serum samples from cats experimentally infected with several strains and an infectious molecular clone of FIV, increases of the antibody titers to FIV gag protein were observed in all cases by the ELISA at early stage of infection. When we examined a total of 415 field cat sera which were previously tested by an indirect immunofluorescence assay (IFA), 9 (12.9%) out of 70 IFA positive sera were judged as negative by the ELISA. However, all 3 serum samples tested among the 9 IFA positive sera had antibodies to gp130 but not to p26 by a radioimmunoprecipitation assay. The results indicated that some IFA positive sera did not have antibodies to the p26 though they have antibodies to other proteins specific for FIV.


European brown hare syndrome in the U.K.; a calicivirus related to but distinct from that of viral haemorrhagic disease in rabbits. The virus recovered from cases of European brown hare syndrome in the U.K. contains one major capsid protein of approximately 60 k molecular weight and morphologically resembles known caliciviruses. It has been compared with a European isolate of rabbit haemorrhagic disease calicivirus and, although it shows some antigenic similarity, it is not identical. In transmission and protection studies the virus from U.K. hares failed to produce disease in rabbits and did not effectively protect against subsequent challenge with the rabbit calicivirus.