Cytomegalovirus infection in a cyclosporine-treated burn patient: case report. We report the case of a 45-year-old burned man (55% total body surface area full-thickness burn) who developed symptomatic cytomegalovirus infection during cyclosporine (CSA) therapy (3 mg/kg orally) for skin transplantation. During the sixth hospital week the patient developed signs compatible with CMV infection, and CMV was recovered from the urine and sputum. Examination of skin biopsy specimens from the transplanted cadaver allograft revealed inclusion bodies compatible with CMV infection, and CMV antigens were detected by immunohistochemical testing. The CMV infection of the skin was confirmed by recovery of infectious virus and by detection of CMV nucleic acids using in situ hybridization with a biotinylated HCMV DNA probe. Restriction enzyme analysis of a urine CMV isolate and two isolates from skin demonstrated differences in DNA patterns, suggesting that the patient was infected simultaneously with two different CMV strains.


Seroprevalence of anti-HCV in an urban child population: a pilot survey in a developing area, Cameroon. Hepatitis C Virus (HCV) is the principal agent of non-A, non-B hepatitis and its spread in the community is not well defined. We evaluated the prevalence of anti-HCV (ortho ELISA System) in 696 children (4-14 years) in Kumba, Cameroon. Children were selected by systematic random sampling in six primary schools. A seroprevalence of 14.5% (101 children) was found which increased steadily with age. No significant differences were observed with respect to sex or to family size. There was a highly significant association both with parents' social class, the lowest class presenting a 2.2-fold risk factor, and with area of residence, suburban children showing a significantly higher prevalence (P less than 0.01). These results suggest that Cameroon is endemic for HCV infection, that children are infected at an early age and that infection increases with age. We can therefore hypothesize that transmission of HCV infection in this population is from child to child. Furthermore, the infection seems to be influenced by social factors but not by demographic ones.


HIV-infected professionals, patient rights, and the 'switching dilemma'. The ethical issues surrounding the Centers for Disease Control and American Medical Association guidelines for health professionals infected with the human immunodeficiency virus are examined and discussed. Although human immunodeficiency virus transmission risks during surgery are lower than many risks we routinely face, it is not irrational for a patient to want to switch from an infected professional to an uninfected one. The American Medical Association claim that physicians have a duty to avoid imposing any identifiable risks is implausible. Knowing the Centers for Disease Control estimate of risks gives us no way to decide whether the rights of patients or those of handicapped (infected) workers should be given priority. Granting priority to patient rights, either by giving patients the opportunity to know the risks they face and to switch to another provider, or by removing infected providers (compulsory switching), makes us all worse off. This gives us reason to reject these guidelines and emphasize other infection control measures.


Superior results of ileoanal pull through (IAPT) in polyposis coli vs ulcerative colitis patients. Colectomy, mucosal proctectomy, and ileoanal pull through (IAPT) have replaced proctocolectomy as the operation of choice in most patients with the colorectal mucosal diseases ulcerative colitis and polyposis coli. This study was conducted to determine whether there is any difference in postoperative morbidity and long-term functional results when IAPT is performed for polyposis coli than when it is performed for ulcerative colitis patients. Between 1982 and 1989, 277 IAPT procedures were performed at the University of Utah Medical Center, 239 for ulcerative colitis and 38 for polyposis coli syndromes. A prospective study compared polyposis and colitis patients after IAPT with regards to age, family history, preoperative findings, operative findings, postoperative complications, and long-term complications. Functional results were compared including day and night stool frequency, incidence of nighttime incontinence, and incidence of pouchitis. The incidence of postoperative complications was lower in the polyposis group than in the colitis group, but the numbers were low enough in both diseases that significant differences were not detected. However, the incidence of pouchitis (0% vs 19%), day (4.2 vs 6.0) and night (0.4 vs 0.9) stool frequency, and nighttime incontinence (5% vs 25%) was significantly lower in the polyposis group when compared to the colitis group. We conclude that IAPT performed for polyposis conditions is associated with superior results when compared with results from patients who undergo the procedure for ulcerative colitis.


Erythromycin stimulates ileal motility by activation of dihydropyridine-sensitive calcium channels. Erythromycin, a macrolide antibiotic, is a potent stimulant of small bowel motor activity (MA) which may motility either via the peptide motilin receptor or neural mechanisms. We hypothesized that erythromycin stimulates directly stimulates smooth muscle cells by a calcium-mediated event. Thus, we evaluated the effect of neuronal blockade with tetrodotoxin, muscarinic blockade with atropine, and opiate blockade with naloxone on erythromycin-stimulated MA in isolated perfused segments of rabbit terminal ileum. We also tested the effect of nonspecific calcium channel blockade (verapamil and cadmiun) and specific blockade (dihydroxypyridine and nichol) on erythromycin-stimulated MA. MA was measured with a multichannel continuous perfusion manometry catheter. Erythromycin caused a concentration-dependent increase in MA (ED100 5 x 10(-4) M). Tetrodotoxin, atropine, and naloxone did not effect erythromycin-stimulated MA (P greater than 0.05). Both verapamil (10(-7) M) and cadmium (10(-2)-10(-4) M) inhibited erythromycin-stimulated MA. Selective blockade of "l" type calcium channels using dihydropyridine (10(-6) M) and "t" channels with nickel (10(-2)-10(-4) M) both reversed erythromycin-stimulated MA. Since the isolated segments of terminal ileum were free of exogenous humoral and neural effects, these studies indicated that erythromycin directly stimulated MA in the terminal ileum. Furthermore, since tetrodotoxin, atropine, and naloxone did not inhibit this increase in MA, erythromycin acted by a mechanism which was independent of the intrinsic nervous and opiate systems. In conclusion, these data are consistent with the model that erythromycin stimulates ileal motility by a mechanism involving activation of dihydroxypyridine and nickel-sensitive calcium channels.


Functional expression of VIP receptors in normal, immortalized and transformed mammary epithelial cells. The effect of VIP and its related peptides on cAMP production has been characterized: 1) in long term culture of normal human mammary epithelial cells (HMEC); 2) in immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40; 3) in the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1D, described to exhibit normal morphogenesis in vivo and functional differentiation in vitro. Basal cAMP levels were increased 1.5- to 8.7-fold in mammary epithelial cells (p less than 0.001-0.05), with a potency EC50 = 0.02-0.6 nM VIP. The pharmacological specificity of the VIP receptors coupled to cAMP generation was established according to the following potency sequence: VIP greater than PACAP-38 greater than helodermin greater than PHM, PHV greater than helospectin 1 much greater than hpGRF, secretin in HMEC, VIP greater than PACAP-38 greater than helodermin greater than helospectin 1, PHM, PHV greater than hpGRF greater than secretin in S1T3 cells, and VIP, PHI, helodermin greater than PHV greater than rhGRF greater than secretin in HC-11 cells. Our data demonstrate the presence of functional, highly sensitive and specific VIP receptors in normal, immortalized and transformed mammary epithelial cells, suggesting a regulatory role for this neuropeptide on the growth, differentiation and function in normal and neoplastic breast tissue.


Neurotensin enhances plasma adrenocorticotropin concentration by stimulating corticotropin-releasing hormone secretion. The systemic administration of neurotensin (NT) dose-dependently increased plasma adrenocorticotropin (ACTH) concentration in rats, and this effect was annulled by (alpha-helical)-CRH9-41, an antagonist of corticotropin-releasing hormone (CRH). The systemic administration of [D-Trp11]-neurotensin (NT-A), a specific NT antagonist, dose-dependently reduced the basal level of circulating ACTH, and this effect was blunted by NT injection. The ACTH inhibitory action of NT-A was completely overcome by the administration of CRH. Taken together, our findings suggest that NT plays a physiologic role in rats, as ACTH secretagogue, and that the mechanism underlying this action of NT involves the stimulation of CRH release.


[Polysyndactylia with brachymetacarpia (Type Bonola)]. Four cases of polysyndactyly from Japan, Taiwan and Turkey are described, which show similar short thumbs, metacarpals and middle phalanges as well as analogous malformations of the feet. A new type of polysyndactyly can be defined. The occurrence in brother and sister from a consanguineous marriage is consistent with autosomal recessive inheritance.


Kinetoplast DNA and molecular karyotypes of Trypanosoma evansi and Trypanosoma equiperdum from China. We compared 12 stocks of Trypanosoma evansi and 1 recently isolated stock of Trypanosoma equiperdum from different regions of China by analysis of kinetoplast DNA (kDNA), nuclear DNA and molecular karyotypes. The T. equiperdum stock was remarkably similar to the T. evansi stocks, except for the possession of kDNA maxi-circles, suggesting a very close evolutionary relationship between T. evansi and T. equiperdum. The maxi-circles of the Chinese T. equiperdum stock were approximately 14.3 kb in size, i.e., about half the size of those of Trypanosoma brucei. This stock is thus similar to an old laboratory stock of T. equiperdum, which also has maxi-circles with a sizeable deletion. Both T. equiperdum and T. evansi kDNA mini-circles hybridised with a T. evansi-specific mini-circle fragment isolated from a Kenyan T. evansi stock. Our results extend the generality that T. evansi and T. equiperdum mini-circles are microheterogeneous rather than homogeneous. Molecular karyotypes obtained by pulsed field gradient gel electrophoresis provided a more sensitive way of distinguishing the T. evansi stocks than isoenzymes or restriction fragment length polymorphisms in kDNA mini-circles, genes for ribosomal RNAs and variant surface glycoproteins. Our results fit the general idea that T. evansi stocks worldwide have a single origin.


Multiple forms of chromosome I, II and V in a restricted population of Leishmania infantum contrasting with monomorphism in individual strains suggest haploidy or automixy. We have resolved the molecular karyotypes of 22 Leishmania infantum strains isolated between 1980 and 1988 in a restricted geographic area and belonging to zymodemes MON-11, -29 and -33. Three strains were isolated from sandflies and all the others from human cutaneous lesions. A high degree of karyotypic homology is observed among these strains, contrasting with the highly polymorphic MON-1 strains isolated in the same area. We have analysed the time-dependent evolution of size variants of chromosomes I to V, each identified by chromosome-specific DNA probes. More evidence is given for the role of subtelomeric regions in chromosomal size variation in Leishmania for both chromosomes I and II. At the population level, the chromosomes I, II and V are present in respectively 8, 4 and 3 distinct sizes. Furthermore, and despite the small size of the sample, various combinations were observed among these different chromosomal forms. These results could be explained by the occurrence of a high rate of recurrent mutations or of genetic exchange. In contrast, only one chromosomal form was observed in individual karyotypes for the chromosomes I-V. These results could tally with the hypothesis of a haploid organisation for these chromosomes and strains, or, in the frame of a diploid organisation, with the hypothesis of a predominantly automictic sexuality giving rise to 2 identical forms of the homologues in the same strain.


The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes. We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.


Signalling by the sevenless protein tyrosine kinase is mimicked by Ras1 activation. Cell-fate specification of R7 photoreceptors in the developing Drosophila eye depends on an inductive signal from neighbouring R8 cells. Mutations in three genes, sevenless (sev), bride-of-sevenless (boss) and seven-in-absentia (sina) cause the R7 precursor to become a non-neural cone cell. The sev gene encodes a receptor protein tyrosine kinase (Sev) localized on the R7 surface, activated by a boss-encoded ligand presented by R8. The sina gene encodes a nuclear factor required in R7. Reduction in the dosage of the Ras1 gene impairs Sev-mediated signalling, suggesting that activation of Ras1 may be an important consequence of Sev activation. We report here that Ras1 activation may account for all of the signalling action of Sev; an activated Ras1Va112 protein rescues the normal R7 precursor from transformation into a cone cell in sev and boss null mutants and induces the formation of supernumerary R7 cells. Similar activation of the Drosophila Ras2 protein does not produce these effects, demonstrating Ras protein specificity.


Dynamin is a GTPase stimulated to high levels of activity by microtubules. Dynamin was initially identified in calf brain tissue as a protein of relative molecular mass 100,000 which induced nucleotide-sensitive bundling of microtubules. Purified dynamin showed only trace ATPase activity. But in combination with an activating factor removed during the purification, it exhibited microtubule-activated ATPase activity and dynamin-induced bundles showed evidence of ATP-dependent force production. Dynamin is the product of the Drosophila gene shibire, which has been implicated in synaptic vesicle recycling and, more generally, in the budding of endocytic vesicles from the plasma membrane. Dynamin also shows extensive homology with proteins that participate in vacuolar protein sorting and spindle pole-body separation in yeast, and in interferon-induced viral resistance in mammals. All members of this family contain consensus sequence elements consistent with GTP binding near their amino termini, although none has been shown to have GTPase activity. We report here that dynamin is a specific GTPase which can be stimulated to very high levels of activity by microtubules.


Crystal structure of a dUTPase.

Crystal structure of a dUTPase. The enzyme dUTPase catalyses the hydrolysis of dUTP and maintains a low intracellular concentration of dUTP so that uracil cannot be incorporated into DNA. dUTPase from Escherichia coli is strictly specific for its dUTP substrate, the active site discriminating between nucleotides with respect to the sugar moiety as well as the pyrimidine base. Here we report the three-dimensional structure of E. coli dUTPase determined by X-ray crystallography at a resolution of 1.9 A. The enzyme is a symmetrical trimer, and of the 152 amino acid residues in the subunit, the first 136 are visible in the crystal structure. The tertiary structure resembles a jelly-roll fold and does not show the 'classical' nucleotide-binding domain. In the quaternary structure there is a complex interaction between the subunits that may be important in catalysis. This possibility is supported by the location of conserved elements in the sequence.


[Acyclovir-resistant herpes simplex virus infections in immunocompromised patients]. In order to determine the sensitivity of herpes simplex virus (HSV) isolates from immunocompromised patients treated with antiviral compounds, a retrospective study was carried out in the Clinical Virology Department of the University Medical Centre, Amsterdam. Virus isolates from four AIDS patients and one bone marrow transplant recipient were examined for their sensitivity for the antiviral compounds used by means of plaque reduction assay. In some of the virus isolates, from patients in whom resistance was assumed on clinical grounds, in vitro resistance of the HSV to acyclovir (ACV) could be demonstrated, both after oral and after parenteral administration. There was a clear correlation between the clinical course of the HSV infection and in vitro resistance. ACV resistant virus isolates were sensitive to foscarnet, both clinically and in vitro. In immunocompromised patients treated for some time with ACV for HSV infection, resistance should be considered at lack of results or progression of the lesion and when necessary be demonstrated in vitro. Alternative therapy then consists of intravenous foscarnet treatment.


Activation of the colony-stimulating factor 1 receptor leads to the rapid tyrosine phosphorylation of GTPase-activating protein and activation of cellular p21ras. We have previously reported that platelet-derived growth factor (PDGF) induced tyrosine phosphorylation of GTPase-activating protein (GAP) in intact quiescent fibroblasts under conditions in which insulin and basic fibroblast growth factor (bFGF) were ineffective (Molloy et al., 1988). In the present study, we have provided evidence that colony-stimulating factor 1 (CSF-1) is capable of inducing tyrosine phosphorylation of GAP and its associated cellular proteins, p62 and p190, in NIH3T3 cells overexpressing the human CSF-1 receptor (CSF-1R). However, the extent of GAP tyrosine phosphorylation induced by CSF-1 was approximately 10% of that induced by PDGF-BB in the NIH3T3 fibroblasts. Despite this significant difference, both PDGF-BB and CSF-1 increased the activation of p21ras, the extent of which correlated well with the mitogenic response induced by each growth factor in these cells. Taken together, our findings provide evidence for a possible role of tyrosine phosphorylation of GAP and GAP-associated phosphoproteins in regulating transduction of CSF-1-induced mitogenic signals through p21ras activation.


p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. We screened 77 non-small-cell lung cancer (NSCLC) cell lines for mutations of the p53 gene using a single-strand conformation polymorphism (SSCP) assay. We found that 57 cell lines (74%) had mutations of the p53 gene. Three cell lines had a deletion of the p53 gene. Of the remaining 54 cell lines, 49 cell lines were sequenced and 52 mutations were confirmed. In contrast to previously published p53 mutations in other human tumors, the p53 gene mutations in NSCLC were diverse with regard to the location and nature of the mutations. The region corresponding to codons 144-166, which is outside the evolutionarily conserved regions, was a frequent site of p53 gene mutations in NSCLC. The presence of a p53 gene mutation was not associated with age, sex, histological types, culture site, treatment intent, presence of prior cytotoxic treatment, neuroendocrine differentiation, median culture time or patient survival. The prevalence of p53 mutations in cell lines with ras mutations did not differ from that in cell lines without ras mutations. However, p53 gene mutations in NSCLC cell lines with ras mutations tended to cluster in exon 8, suggesting the presence of a functional domain of the p53 gene relating to interaction with the ras gene. We conclude that p53 and ras mutations are frequent and apparently independent genetic alterations which play different roles in the pathogenesis, progression and prognosis of NSCLC.


The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus. Although human papillomavirus type 16 (HPV-16) is believed to be a major etiological agent in the development of cervical cancer, the biological function of several of its early genes remains to be established. In the present study, we have defined some of the biological properties of the E5 gene from HPV-16. Expression of the HPV-16 E5 gene in 3T3-A31 cells induced transformation to anchorage-independent growth (colony formation in soft agar). Addition of epidermal growth factor (EGF) to the soft-agar medium caused the E5-expressing cells to form larger colonies than those formed in the absence of EGF. Parental 3T3-A31 cells did not form colonies in soft agar either in the presence or in the absence of EGF. Analysis of clones expressing high levels of E5 mRNA revealed that these cells also expressed higher levels of c-fos mRNA in response to serum, EGF and platelet-derived growth factor (PDGF) than did the parental 3T3-A31 cells. Cells expressing the E5 gene were also capable of accelerated growth in low serum and were more tumorigenic in nude mice than were control cells. We conclude that the E5 gene from HPV-16 is an oncogene which transforms cells in part through enhancing signal transduction from growth factors to the nucleus.


Human papillomavirus type 16 E5 gene stimulates the transforming activity of the epidermal growth factor receptor. We have until recently made several unsuccessful attempts to assign any activity to the human papillomavirus type 16 (HPV-16) E5 gene product. However, studies with the bovine papilloma virus 1 (BPV-1) E5 protein indicated an interaction with the epidermal growth factor receptor (EGFR). In light of the overall similarity between the HPV and BPV E5 proteins we attempted to determine whether the HPV-16 E5 gene had any common activity. In cells expressing high levels of EGFR plus HPV-16 E5 we found a dramatically increased proliferative activity in soft-agar assays in the presence of EGF. The specificity of this activity was monitored by the addition of other mitogenic agents. The phorbol ester phorbol 12-myristate 13-acetate (PMA) had no effect on the E5-containing cells, although insulin weakly stimulated their growth in soft agar. Further analysis revealed the same number of EGF receptors were present on the E5-containing cells as on the control cells, although the E5 cells were more sensitive to lower concentrations of EGF. These results imply that E5 is amplifying the mitogenic signals from the EGFR in an as yet unknown manner, but which may form the basis of interactions with a variety of growth factor receptors. This report brings to three the number of transforming genes encoded by HPV-16.


Expression of genes encoding type IV collagen-degrading metalloproteinases and tissue inhibitors of metalloproteinases in various human tumor cells. Uncontrolled expression of matrix metalloproteinases 2, 3 and 9 (MMP-2, -3 and -9) is believed to be a critical part of the invasive potential of tumor cells because of their ability to degrade type IV collagen, a major structural component of basement membranes. Availability of proteolytic activity in the vicinity of the cell surface is further affected by a local balance between the enzymes and their inhibitors produced by the cell. To determine how frequently deregulated expression of the MMPs and tissue inhibitors of metalloproteinases (TIMPs) is associated with tumor cells, 26 human tumor cell lines were examined by Northern blotting. Transcripts for MMP-2 and MMP-9 were more frequently expressed in mesenchymal tumor cells (9/9 for MMP-2 and 6/9 for MMP-9) than in epithelial tumor cells (4/17 for MMP-2 and 2/17 for MMP-9). Although expression of MMP-2 mRNA was clearly cell type-specific, MMP-9 mRNA expression in mesenchymal cells correlated well with the reported tumorigenicity of the cells. Enhanced expression of MMP-9 mRNA was also associated with the tumorigenic transformation of cells by an activated c-H-ras gene in human embryonic fibroblasts. Only 3 of the 26 tumor cells expressed MMP-3 mRNA, and 2 of the 3 were epithelial tumor cells which coordinately expressed MMP-9 and TIMP-1 mRNAs. TIMP-1 mRNA was almost undetectable in 50% of the tumor cells, but TIMP-2 mRNA was expressed in the majority of the cells. These findings provide comprehensive information about mRNA expression of the MMPs and TIMPs in tumor cells, the deregulation of which is thought to be an integral part of the invasive potential of tumor cells.


Inhibition of in vitro myogenic differentiation by a polyomavirus early function. In the present work we report on the role of a polyomavirus (Py) early function in interfering with both morphological and biochemical differentiation of the myogenic C2 cell line. The analysis of cell clones stably transfected with a plasmid carrying an ORI- Py genome showed that in the presence of the whole viral early region myogenesis is blocked and a transformed phenotype is evident. By using a plasmid that only encodes large-T function, the involvement of this individual early viral gene product was determined. Inhibition of myogenic differentiation by Py large T is proportional to the level of its expression. This inhibition does not appear to require alteration of cell growth properties. The analysis of muscle-specific functions expressed at different steps in the myogenic pathway showed that Py large T blocks the expression of terminal differentiation markers without altering the expression of the regulatory gene MyoD.


Symptomatic congenital cytomegalovirus infection: neonatal morbidity and mortality. Knowledge of the natural history of symptomatic congenital cytomegalovirus (CMV) infection in the newborn is essential in order to anticipate complications and assess the potential benefit from antiviral therapy. To define the disease course we reviewed data on 106 neonates with symptomatic congenital CMV infection diagnosed and managed by the investigators. Petechiae, jaundice and hepatosplenomegaly were each noted in 70% or more patients. Microcephaly was noted in 54 of 102 (53%) at birth. Elevated alanine aminotransferase, conjugated hyperbilirubinemia and thrombocytopenia were seen in 83, 81 and 77%, respectively. Eighty-six percent had at least two of the manifestations highly suggestive of congenital infection. Platelet count fell to its nadir during the second week of life whereas elevated alanine aminotransferase and direct bilirubin persisted past the first month. In spite of the difficulty in assessing central nervous system function in the newborn, evidence of damage was present in the majority. Seventy-two had microcephaly, poor suck, lethargy/hypotonia or seizures. Abnormal computerized tomographic scan was present in 16 of 20 (80%) and decreased hearing in 20 of 39 (56%). Cerebrospinal fluid protein was greater than 120 mg/dl in 24 of 52 (46%) and this elevation was associated with neurologic abnormalities as well as hearing loss. The mean length of hospital stay was 13 and 22.4 days for term and preterm infants, relatively. Thirteen infants (12%) died during the first 6 weeks of life. Disseminated CMV infection with multiorgan involvement was evident in 7 of 9 at postmortem examination. We conclude that neonates with symptomatic congenital CMV infection have a multi-system disease with significant morbidity and mortality.


Prophylactic oral acyclovir in outbreaks of primary herpes simplex virus type 1 infection in a closed community. Oral acyclovir was given prophylactically to 37 children in the early stages of three outbreaks of herpes simplex virus type 1 (HSV-1) infection and the results were compared with those in untreated control subjects in two other outbreaks. The rates of seroconversion to HSV were significantly reduced in children treated with acyclovir compared with control subjects (91% vs 27%, P less than .001). The incidence of symptomatic disease was also significantly reduced (82% vs 0%, P less than .001). In some children receiving prophylactic acyclovir, anti-HSV antibody titers did not rise despite the presence of replicative HSV on throat swabs just before the start of treatment. Restriction endonuclease analysis of isolated HSV-DNA confirmed that one strain was responsible for the five outbreaks. No resistance to acyclovir was detected during the study, and no adverse effects of treatment were noted. In conclusion, short-term prophylactic acyclovir may limit the spread and reduce clinical manifestations of HSV infections in closed communities, although this use should be restricted to communities where severe symptoms are observed.


Optimal tRNA((Ser)Sec) gene activity requires an upstream SPH motif. The X. laevis tRNA((Ser)Sec) gene is different from the other tRNA genes in that its promoter contains two external elements, a PSE and a TATA box functionally equivalent to those of the U6 snRNA gene. Of the two internal promoters governing classical tRNA gene transcription, only subsists the internal B box. In this report, we show that the tRNA((Ser)Sec) contains in addition an activator element (AE) which we have mapped by extensive mutagenesis. Activation is only dependent on a 15 bp fragment residing between -209 and -195 and containing an SPH motif. In vitro, this element forms a complex with a nuclear protein which is different from the TEF-1 transcriptional activator that binds the SV40 Sph motifs. This AE is versatile since it shows capacity of activating a variety of genes in vivo, including U1 and U6 snRNAs and HSV thymidine kinase. Unexpectedly for an snRNA-related gene, the tRNA((Ser)Sec) is deprived of octamer or octamer-like motifs. The X.laevis tRNA((Ser)Sec) gene represents the first example of a Pol III snRNA-type gene whose activation of transcription is completely octamer-independent.


Sequence specificity of cyclobutane pyrimidine dimers in DNA treated with solar (ultraviolet B) radiation. Cyclobutane pyrimidine dimers were quantified at the sequence level after irradiation with solar ultraviolet (UVB) and nonsolar ultraviolet (UVC) light sources. The yield of photoproducts at specific sites was dependent on the nucleotide composition in and around the potential lesion as well as on the wavelength of ultraviolet light used to induce the damage. Induction was greater in the presence of 5' flanking pyrimidines than purines; 5' guanine inhibited induction more than adenine. UVB irradiation increased the induction of cyclobutane dimers containing cytosine relative to thymine homodimers. At the single UVC and UVB fluences used, the ratio of thymine homodimers (T mean value of T) to dimers containing cytosine (C mean value of T, T mean value of C, C mean value of C) was greater after UVC compared to UVB irradiation.


During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element. The principal early promoter of human papillomaviruses (HPVs), designated P97 in the case of HPV-16, contains four characteristically aligned cis-responsive elements, namely one binding site for Sp1, two for the viral E2 proteins, and the TATA box. The Sp1 binding site is needed to mediate activation of P97 by the remote epithelial-specific enhancer, and the two E2 binding sites contribute to a negative feedback-loop of viral gene expression. The Sp1 consensus motif and the TATA-box distal E2 binding site are spaced in all genital papillomaviruses by a single nucleotide. We show here that at physiological concentrations, the binding of E2 proteins and Sp1 are mutually exclusive events, since a bandshift analysis with nuclear extracts from ID13, a mouse cell line transformed by BPV-1, showed only the E2 or the Sp1 bandshift, but no complex indicative of the concomitant binding of both factors. Increasing concentrations of in vitro translated E2 protein compete efficiently with the Sp1 factor for binding to an oligonucleotide containing both binding sites. Interference between Sp1 and E2 protein binding is apparently relevant for P97 repression in vivo, since a mutational analysis revealed that both E2 binding sites are necessary for negative transcriptional regulation: Alone, neither the distal site, where E2 protein can induce Sp1 displacement, nor the proximal site, where E2 protein interferes with formation and function of the pre-initiation complex, have a significant effect, but two functional E2 binding sites lead to repression of P97.


The transcriptionally-active MMTV promoter is depleted of histone H1. We have used an ultraviolet light cross-linking and immunoadsorption assay to demonstrate that histones H1 and H2B are bound to the repressed MMTV promoter. Hormone activation results in reduced H1 content with little or no change in H2B. High resolution analysis of the glucocorticoid-inducible DNaseI hypersensitive region demonstrates an NF-1 footprint as well as specific sites of enhanced cleavage on nucleosome B and in the nucleosome B/nucleosome A linker. These results are consistent with a model in which binding of the glucocorticoid receptor to glucocorticoid regulatory elements on the surface of nucleosome B induces a chromatin transition that is necessary for transcription factor (NF-1 and TFIID) recruitment to the MMTV promoter. We hypothesize that association of histone H1 with important cis-elements on the promoter masks these sites, and glucocorticoid-induced displacement of H1 is necessary to expose factor binding sites at the 3' edge of nucleosome B, in the nucleosome B/nucleosome A linker and at the 5' edge of nucleosome A.


Identification of a complete P-element in the genome of Drosophila bifasciata. A full-size P-element (IbifM3) was isolated from a genomic library of Drosophila bifasciata. The sequence has a length of 2935 bp and is flanked by 8 bp duplications of the target site. The termini are formed by 31 bp inverted repeats. The four exons have intact reading frames and possess the coding capacity for a protein of 753 amino acids and a molecular weight of 86.4 kd. The sections of the D. melanogaster transposase presumed to be functionally important (three leucine zippers and a helix turn helix motif) are conserved in the D. bifasciata P-element. Copy number and genomic distribution resemble the situation in true P-strains of D. melanogaster. Both findings support the idea that IbifM3 represents an active transposon. The sequence comparison between the P-elements of D. bifasciata, D. melanogaster and Scaptomyza pallida reveals relationships not in accordance with the phylogeny of the species. This result suggests a further case of horizontal transmission involving mobile elements in the genus Drosophila.


The recognition of DNA cleavage sites by porcine spleen topoisomerase II. The cutting sites specificity of topoisomerase II from porcine spleen were determined by a modified Sanger's DNA sequencing method. The topoisomerase II prefers to cut DNA at the 3' side of A and leave 5' protruding end with two staggering bases. Through the free energy analysis for DNA duplex, we also found that the topoisomerase II seemed cut DNA preferably at energetically unstable regions. So it is concluded that the specific DNA cutting by porcine spleen topoisomerase II has two structural recognition factors: one is to localize around the energetically unstable region and another is to act at the 3' side of A base.


Directional recombination is initiated at a double strand break in human nuclear extracts. The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast.


Transcriptional enhancer related DNA sequences: anomalous 1H NMR NOE crosspeaks. A dynamic heterogeneity which correlates with the function of the operator DNA in the lactose operon of E. coli. was previously observed (1) as a local minimum in the thymine imino proton T1 centered at a GTG/C-CAC sequence. Since this triplet occurs frequently in DNA regulatory regions, it was proposed that these sequences may be part of a structural element for specific protein interaction. We examine here three additional biologically significant 17 base pair duplexes containing GTG/CAC triplets: (1) a sequence from the mouse heavy chain immunoglobulin enhancer, (2) a sequence from the critical core of the Simian Virus 40 (SV40) enhancer, and (3) a sequence from pBR322 plasmid used as control for experiments with the SV40 DNA sequences. The 1H NMR resonance assignment for nearly all the nonexchangeable protons for both eukaryotic enhancer duplexes with the exception of the H5'/H5" protons was accomplished to use for structural analysis of these duplexes. The data presented show several NOE's associated with the GTG/CAC triplets which suggest structural variation from uniform B-DNA. In addition, anomalous broad crosspeaks for the fixed thymine methyl to its own H6 proton in combination with the imino proton kinetics associated with these triplets reinforces the original observation of a sequence dependent dynamic variation.


[Treatment of venous thrombosis with low molecular weight heparin and fluindione]. Thirty-one consecutive patients with deep vein thrombosis were treated with Fraxiparine, a low molecular weight heparin, and fluindione, an oral anticoagulant prescribed at an early stage. Despite the weight/biological effectiveness ratio, the dose of Fraxiparine had to be adjusted in 46 percent of the patients to remain within the therapeutic range selected (0.5 to 1 antiXa/ml units). The early administration of fluindione reduced the duration of heparin therapy to 5.75 days. Using a prescription guide on days 2 and 4 might improve the safety of the fluindione induction.


Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A. The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.


Nonconventional opioid binding sites mediate growth inhibitory effects of methadone on human lung cancer cells. Methadone was found to significantly inhibit the in vitro and in vivo growth of human lung cancer cells. The in vitro growth inhibition (occurring at 1-100 nM methadone) was associated with changes in cell morphology and viability detectable within 1 hr and was irreversible after a 24-hr exposure to the drug. These effects of methadone could be reversed in the first 6 hr by naltrexone, actinomycin D, and cycloheximide, suggesting involvement of opioid-like receptors and the requirement for de novo mRNA and protein synthesis. The inhibitory effects of methadone on the growth of lung cancer cells also could be achieved by the less addictive (+) isomer of methadone. Characterization of the methadone binding to lung cancer cell membranes revealed high-affinity (nM), saturable binding sites for (+/-)-[3H]methadone, which cross-reacted with ligands for kappa, phencyclidine, sigma, but not mu, and delta opioid receptors, and the binding characteristics appeared to be different from methadone sites present in rat brain. Methadone decreases cAMP levels in lung cancer cells, but the receptors are not coupled to a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein. We conclude that the lung cancer growth inhibitory effects of methadone are significant, occur at low concentrations, and are mediated by a nonconventional type of opioid binding site distinct from methadone receptors found in the brain.


Synthesis of receptor antagonists of neuropeptide Y. We report the synthesis of receptor antagonists of neuropeptide Y (NPY) by a strategy based on synthesis of mixtures of analogs and the subsequent isolation and identification of receptor antagonists from these mixtures. After screening a series of mixtures of NPY analogs by using an NPY antagonist assay, two potent receptor antagonists, designated PYX-1 and PYX-2, were isolated from an antagonist-containing mixture. Structural analysis revealed these analogs to be Ac-[3-(2,6-dichlorobenzyl)Tyr27, D-Thr32]NPY-(27-36) amide and Ac-[3-(2,6-dichlorobenzyl)Tyr27,36,D-Thr32]NPY-(27-36) amide, respectively. The receptor antagonists inhibited release of intracellular calcium elicited by NPY in human erythroleukemia cells and displaced 3H-labeled NPY from NPY receptors in rat brain membrane. The approach of screening and identifying useful analogs from synthetic mixtures may significantly reduce the time and resources previously required for development of receptor antagonists.


Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. The murine Pax-2 gene contains a protein coding domain homologous to the Drosophila paired-box, first described in certain developmental control genes of the segmentation type. Polyclonal antibodies recognize two Pax-2 proteins that are encoded by differentially spliced mRNAs. The Pax-2 proteins can bind a DNA sequence known to interact with the paired domain of a Drosophila protein. By immunocytochemistry, expression of Pax-2 could be localized to the nuclei of condensing mesenchyme cells and their epithelial derivatives in the developing kidney. Expression is abruptly down-regulated as the tubular epithelium differentiates. High levels of Pax-2 expression could also be detected in the epithelial cells of human Wilms tumors. These data suggest that Pax-2 is a transcription factor active during the mesenchyme-to-epithelium transition in early kidney development and in Wilms tumor.


Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice. Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.


Association of the halobacterial 7S RNA to the polysome correlates with expression of the membrane protein bacterioopsin. The sedimentation behavior of the halobacterial 7S RNA and bacterioopsin mRNA was assessed after application of total cell lysates to sucrose gradients. These two RNAs cosedimented predominantly with membrane-bound polysomes, and the quantity of 7S RNA bound to the ribosomes was directly correlated with the expression of bacterioopsin. Puromycin treatment released the 7S RNA from the polysomes, indicating that it is transiently associated with protein translation. We suggest that halobacteria contain a signal-recognition-like particle involved in translation of membrane-associated proteins.


A negative retinoic acid response element in the rat oxytocin promoter restricts transcriptional stimulation by heterologous transactivation domains. Retinoic acid receptors are ligand-dependent transcription factors that stimulate gene transcription from promoters containing retinoic acid or thyroid hormone response elements. We describe a high-affinity binding site from the rat oxytocin promoter that mediates negative transcriptional regulation by the retinoic acid receptor. To examine whether strong, constitutive transactivation domains would be capable of stimulating gene transcription when bound to this DNA binding site that normally mediates transcriptional repression, we fused the transactivation domain of the herpes simplex viral protein VP16 to the amino terminus of the retinoic acid receptor and tested the activity of the chimeric protein on the negative retinoic acid response element. This chimeric retinoic acid receptor acted as a strong, constitutive transactivator when bound to promoters containing palindromic thyroid hormone/retinoic acid response elements but surprisingly it still repressed gene transcription when bound to promoters containing the oxytocin-negative retinoic acid response element. These results suggest that a negative DNA binding site itself can inhibit the function of even potent constitutive transactivation domains, and provide evidence that tethering of a constitutive transactivation domain to DNA is insufficient to activate gene transcription.


Immortalization of primary human smooth muscle cells. Primary human aortic and myometrial smooth muscle cells (SMCs) were immortalized using an amphotropic recombinant retroviral construct containing the E6 and E7 open reading frames (ORFs) of human papillomavirus type 16. The SMCs expressing the E6/E7 ORFs have considerably elevated growth rates when compared with nonimmortalized control cells and show no signs of senescence with long-term passage. The first SMC line derived in this study has been maintained in continuous tissue culture for greater than 1 year (greater than 180 population doublings). The immortalized SMCs have decreased cell size and decreased content of muscle-specific alpha-actin filaments as determined by indirect immunofluorescence. Southern blot analysis has demonstrated the stable integration of the E6/E7 ORFs in the retrovirally infected cells, and radioimmunoprecipitation has confirmed the continued expression of the E6 and E7 genes. Cytogenetic studies of the SMC lines have revealed essentially diploid populations except for the myometrial clonal line, which became aneuploid at late passage (greater than 125 doublings). These cell lines were not tumorigenic in nude mice.


IS5: a mobile enhancer of transcription in Escherichia coli. The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element IS5 into various positions both upstream and downstream of the bgl promoter P0. Activation of the operon was in all cases attributable to enhancement of P0 activity. Introduction of internal deletions into IS5 almost completely abolished P0 enhancement, demonstrating that enhancement is not simply the result of mutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by IS5 gene ins5A, an essential transposition function. Activation of gene expression by means of interaction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.


A strategy for the generation of conditional mutations by protein destabilization. Conditional mutations such as temperature-sensitive (ts) mutations are important for the analysis of protein function but are often difficult, or impossible, to obtain. Here we present a simple method for generating conditional mutations based on the use of a protein-destabilizing genetic element in combination with systems allowing the induction and repression of gene expression. This genetic cassette can be fused to other protein-coding sequences, and once transcription is turned off and synthesis of the gene product ceases, the preexisting protein is rapidly degraded. We have applied this method to the analysis of the yeast ARD1 gene product, a subunit of an N-terminal acetyltransferase, and show that a complete loss of ARD1 product can be achieved in less than one generation. Despite the rapid loss of ARD1 protein, there is a prolonged delay in the expression of the ard1 mutant phenotype, suggesting that the acetylated substrates of ARD1 are metabolically stable and/or exert a long-lasting effect on processes such as the repression of the silent mating type cassettes.


Restricted heterogeneity in T-cell antigen receptor V beta gene usage in the lymph nodes and arthritic joints of mice. We have used PCR to study the expression of T-cell antigen receptor beta RNA containing particular variable region (V) elements from transcripts directly in the cells isolated from joints and lymph nodes of B10.Q mice (H-2q) immunized with chicken type II collagen. Our data show that the T cells present in arthritic joints expressed only a few V beta transcripts--V beta 2, -6, -7, -8.2, -9, -10, and -15. V beta 6 and -8.2 were expressed predominantly (six out of seven animals) while others were expressed at a relatively low level in different animals. In lymph node cells, transcripts for V beta 6, -8.2, and -9 were detected in four out of seven animals. The data indicate that in collagen-induced arthritis there is a restrictive usage of TCR V beta elements and that V beta 6 and -8.2 are probably used preferentially.


The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors. Interactions among growth factors, cells, and extracellular matrix are critical to the regulation of directed cell migration and proliferation associated with development, wound healing, and pathologic processes. Here we report the association of PDGF-AB and -BB, but not PDGF-AA, with the extracellular glycoprotein SPARC. Complexes of SPARC and 125I-labeled PDGF-BB or -AB were specifically immunoprecipitated by anti-SPARC immunoglobulins. 125I-PDGF-BB and -AB also bound specifically to SPARC that was immobilized on microtiter wells or bound to nitrocellulose after transfer from SDS/polyacrylamide gels. The binding of PDGF-BB to SPARC was pH-dependent; significant binding was detectable only above pH 6.6. The interaction of SPARC with specific dimeric forms of PDGF affected the activity of this mitogen. SPARC inhibited the binding of PDGF-BB and PDGF-AB, but not PDGF-AA, to human dermal fibroblasts in a dose-dependent manner. The expression of SPARC and PDGF was minimal in most normal adult tissues but was increased after injury. Enhanced expression of both PDGF-B chain and SPARC was seen in advanced lesions of atherosclerosis. We suggest that the coordinate expression of SPARC and PDGF-B-containing dimers following vascular injury may regulate the activity of specific dimeric forms of PDGF in vivo.


Viral small nuclear ribonucleoproteins bind a protein implicated in messenger RNA destabilization. Herpesvirus saimiri (HVS) is one of several primate viruses that carry genes for small RNAs. The five H. saimiri-encoded U RNAs (HSURs) are the most abundant viral transcripts expressed in transformed marmoset T lymphocytes. They assemble with host proteins common to spliceosomal small nuclear ribonucleoproteins (snRNPs). HSURs 1, 2, and 5 exhibit sequences at their 5' ends identical to the AUUUA motif, which targets a number of protooncogene, cytokine, and lymphokine mRNAs for rapid degradation. We show that a 32-kDa protein previously demonstrated to bind to the 3' untranslated region of several unstable messages can be UV crosslinked specifically to HSUR 1, 2, and 5 transcripts in vitro, as well as to endogenous HSUR snRNPs. Our results suggest an unusual role for these viral snRNPs: HSURs may function to attenuate the rapid degradation of certain cellular mRNAs, thereby facilitating viral transformation of host T lymphocytes.


Human carbonic anhydrase IV: cDNA cloning, sequence comparison, and expression in COS cell membranes. We have isolated a full-length cDNA for human carbonic anhydrase IV (CA IV) from a lambda gt10 human kidney cDNA library. The 1105-base-pair (bp) cDNA contains a 47-bp 5' untranslated region, a 936-bp open reading frame, and a 122-bp 3' untranslated region. The deduced amino acid sequence is colinear with the N-terminal sequence and the sequence of several tryptic peptides of human lung CA IV. It includes an 18-amino acid signal sequence, a 260-amino acid region that shows 30-36% similarity with the 29-kDa cytoplasmic CAs (CA I, CA II, and CA III), and an additional 27-amino acid C-terminal sequence that ends in a 21-amino acid hydrophobic domain. Of the 17 "active site" residues that are highly conserved in other human CAs, 16 are also present in CA IV. Expression of the cDNA in COS cells produced a 35-kDa enzyme that was membrane associated, resistant to inactivation by SDS, contained no carbohydrate, and reacted on Western blots with antiserum to the 35-kDa CA IV from human lung. Treatment of membranes from transfected COS cells with phosphatidylinositol-specific phospholipase C released 20-30% of the expressed enzyme from membranes, indicating that at least 20-30% of the expressed enzyme was anchored to membranes by a glycosyl-phosphatidylinositol linkage.


Cytotoxic and viral neutralizing antibodies crossreact with streptococcal M protein, enteroviruses, and human cardiac myosin. The development of autoimmunity in certain instances is related to infectious agents. In this report, cytotoxic monoclonal antibodies (mAbs) that recognize epitopes on both enteroviruses and the bacterium Streptococcus pyogenes are described. Murine anti-streptococcal mAbs that were crossreactive with streptococcal M protein, human cardiac myosin, and other alpha-helical coiled-coil molecules were found to neutralize coxsackieviruses B3 and B4 or poliovirus type 1. The viral-neutralizing anti-streptococcal mAbs were also cytotoxic for heart and fibroblast cell lines and reacted with viral capsid proteins on a Western immunoblot. Alignment of amino acid sequences shared between streptococcal M protein, coxsackie-virus B3 capsid protein VP1, and myosin revealed 40% identity in a 14- to 15-amino acid overlap. Synthetic peptides containing these sequences blocked mAb reactivity with streptococcal M protein. The data show that antibodies against alpha-helical structures of bacterial and viral antigens can lead to cytotoxic reactions and may be one mechanism to explain the origin of autoimmune heart disease.


A BamHI repeat element is predominantly associated with the degenerating neo-Y chromosome of Drosophila miranda but absent in the Drosophila melanogaster genome. In Drosophila miranda, females have two X1 and two evolving X2 chromosomes, and males have one of each of these two X chromosomes and a Y chromosome. In males, the homologue of the X2 chromosome, the neo-Y chromosome, is attached to the Y chromosome and is under the process of degenerative evolution. We have examined a developmentally regulated X2/neo-Y chromosome-linked gene, 549mr, of D. miranda and found that the neo-Y chromosome-linked copy of this gene (549mr-NY) contains an insertional DNA. We discovered that sequences similar to those in the insertional DNA are present in multiple copies in the genome of both sexes of D. miranda but are more abundant in the males. The insertional DNA also identified a 1.1-kilobase BamHI repeat that is present in at least 6-fold excess in the male genome as compared to the female. This BamHI repeat and similar DNA sequences are predominantly concentrated on the evolving neo-Y chromosome, but very few are found on the homologous X2 and other chromosomes. The BamHI repeat also hybridizes with 2.0- and 1.8-kb RNAs and many other RNA species, which together are also approximately 6-fold greater in males. No sequences similar to the BamHI repeat are found in Drosophila melanogaster. Moreover, the BamHI repeat is not homologous to P, copia, or other D. melanogaster transposable elements. This repeat, named the NY element, may be involved in gene disruption and the process of degenerative evolution of the neo-Y chromosome.


(125I)iodoazidococaine, a photoaffinity label for the haloperidol-sensitive sigma receptor. A carrier-free radioiodinated cocaine photo-affinity label, (-)-3-(125I)iodo-4-azidococaine [(125I)IACoc], has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM (125I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. Covalent labeling of the 26-kDa polypeptide was inhibited by 1 microM haloperidol, di(2-tolyl)guanidine (DTG), 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP), dextromethorphan, and carbetapentane. Stereoselective protection of (125I)IACoc photolabeling by 3-PPP [(+)-3-PPP more potent than (-)-3-PPP] was observed. (125I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 microM imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the (125I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-[3H]DTG. Kinetic analysis of (125I)IACoc binding to rat liver microsomes revealed two sites with Kd values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, (125I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.


The third gamma subunit of the gamma-aminobutyric acid type A receptor family. Cloned cDNAs encoding a member of the gamma-aminobutyric acid type A receptor gamma-subunit class were isolated from rat-brain-mRNA-derived libraries. The gamma 3 mRNA is present in cortex, claustrum, caudate putamen, and some thalamic nuclei, particularly the medial geniculate nucleus, where it is the predominant gamma-subunit transcript. The gamma 3 gene is expressed at very low levels in cerebellum and hippocampus. In coexpression experiments with the alpha 1 and beta 2 subunits, gamma 3 imparts benzodiazepine binding to gamma-aminobutyric acid type A receptors and forms gamma-aminobutyric acid-gated benzodiazepine-modulated chloride channels that exhibit a larger conductance than alpha 1 beta 2 receptor channels. Furthermore, the presence of gamma 3 in place of gamma 2 in alpha 1 beta 2 gamma x receptors generates a marked decrease in the affinity of agonists while leaving the affinity of antagonists or negative modulators largely unaffected.


Carbachol-activated calcium entry into HT-29 cells is regulated by both membrane potential and cell volume. Intracellular Ca2+ ([Ca2+]i) was measured in single Cl(-)-secretory HT-29/B6 colonic carcinoma cells with the Ca2+ probe fura-2 and digital imaging microscopy. Resting [Ca2+]i was 63 +/- 3 nM (n = 62). During treatment with the muscarinic agonist carbachol, [Ca2+]i rapidly increased to 901 +/- 119 nM and subsequently reached a stable level of 309 +/- 23 nM, which depended on Ca2+ entry into the cells from the extracellular solution. The goal of this study was to characterize the Ca2+ entry pathway across the cell membrane with respect to its dependence on membrane potential and cell volume. Under resting conditions [Ca2+]i showed no apparent dependence on either potential or cell volume. After stimulating Ca2+ entry with carbachol (100 microM), [Ca2+]i increased with hyperpolarization (low-K+ or valinomycin treatment) and decreased with depolarization (high-K+ or gramicidin treatment) of the cell, as expected from changes in driving force for Ca2+ entry. In stimulated cells, hypotonic solutions caused [Ca2+]i to increase, whereas hypertonic solutions blocked Ca2+ entry. The shrinkage-induced decreases in [Ca2+]i were only slightly affected when the membrane potential was increased with valinomycin, suggesting that shrinkage directly affects the carbachol-activated Ca2+ conductance. In contrast, the swelling-induced increase in [Ca2+]i was significantly reduced in valinomycin-treated cells, suggesting an indirect dependence on a swelling-activated K+ conductance. Thus, carbachol-stimulated Ca2+ entry is under the dual control of membrane potential and cell volume. This mechanism may serve as a regulatory influence that determines the extent of Ca2+ influx during cholinergic stimulation.


Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors. A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCR analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists with the following specificity sequence: domoate greater than kainate much greater than quisqualate greater than or equal to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid greater than or equal to L-glutamate much greater than N-methyl-D-aspartate. The kainate-elicited currents were specifically blocked by 6-cyano-7-nitroquinoxaline-2,3-dione but were insensitive to 2-amino-5-phosphonovalerate and kynurenic acid. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.


Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling. We have recently described the properties of direct repeats (DRs) of the half-site AGGTCA as hormone response elements (HREs). According to our results, spacing the half sites by 3, 4, or 5 nucleotides determines specificity of response for vitamin D3, thyroid hormone, and retinoic acid receptors, respectively. This so-called 3-4-5 rule led to the prediction that remaining spacing options of 0, 1, and 2 might serve as targets for other nuclear receptors. A concurrent prediction is that receptors recognizing common sites might display more complex or combinatorial interactions. In exploring these predictions, we discovered that both the retinoid X receptor (RXR) and COUP-TF bind preferentially to a DR-1 motif. In vivo, RXR and COUP-TF display antagonistic action such that RXR-mediated activation is fully repressed by COUP-TF. In vitro studies reveal that COUP-TF and RXR form heterodimers on DR-1. Thus, these results support a general proposal in which the half-site spacing preferences may be used as a means to decipher potentially complex and interactive regulatory circuits.


Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart. Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.


Effect of DuP 753, a nonpeptide angiotensin II receptor antagonist, on the drinking responses to acutely administered dipsogenic agents in rats. The present studies examine the effect of the nonpeptide angiotensin II (AII) type 1 receptor antagonist, DuP 753, on water intake in rats treated with dipsogenic stimuli, which are thought to induce drinking via release of renin and subsequent formation of AII. Subcutaneous administration of DuP 753 in doses that are known to inhibit drinking induced by AII failed to inhibit the water intake of rats following subcutaneous administration of the beta-adrenoceptor agonist isoproterenol. The peptide antagonist1 Sar, 8Ileu-AII, which blocks both AII type 1 and AII type 2 receptors, also failed to inhibit isoproterenol-induced drinking, suggesting that neither subtype is involved in this drinking response. Additional studies verified previous reports that acute subcutaneous administration of both the beta-adrenoceptor antagonist propranolol and the angiotensin I-converting enzyme inhibitor captopril could block the drinking response to subcutaneous administration of isoproterenol. Subcutaneous administration of DuP 753 also failed to inhibit the drinking responses to subcutaneous administration of serotonin, 5-hydroxytryptophan, hypertonic saline, and polyethylene glycol. However, central intraventricular administration of DuP 753 inhibited the drinking response to subcutaneous administration of isoproterenol. The results are discussed in terms of the importance of AII in mediating isoproterenol-, serotonin-, and 5-hydroxytryptophan-induced water intake and suggest a need to readdress this mechanism.


Electrogenic anion absorption in rabbit distal colon. The mechanisms of anion transport in the rabbit distal colon were investigated in vitro under short-circuit conditions by examining the effects of transport inhibitors (the stilbene derivatives SITS and DIDS) under a variety of conditions. These agents consistently inhibited Jm-sCl: SITS (10(-3) M) reduced both unidirectional chloride fluxes to the same degree and did not alter JnetCl. In contrast, 10(-4) M DIDS had no effect on Js-mCl and had a significant chloride antiabsorptive effect. DIDS had no effect on either tissue cyclic AMP levels or on basal flux of potassium. The effects of SITS and the cyclic AMP-related secretagogue theophylline on Isc were independent. Additionally, there was no significant alteration of intracellular potential difference or apical membrane fractional resistance elicited by SITS during microelectrode impalement of colonic surface epithelial cells. These results suggest a complex mechanism of anion transport in the distal colon, with a component of electrogenic anion absorption inhibited by the stilbenes. The subsequent changes in current, conductance, and chloride fluxes are dependent upon additional, independent anion transport processes. These pharmacologic agents exhibit an antiabsorptive effect, rather than a stimulation of electrogenic chloride secretion.


delta 9-Tetrahydrocannabinol suppresses macrophage extrinsic antiherpesvirus activity. The effect of delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, on macrophage intrinsic and extrinsic antiherpesvirus activities was examined. THC had no effect on the capacity of the macrophage-like cells RAW264.7, J774A.1, and P388D1 to take up virus. In addition, replication of virus within macrophages did not occur regardless of drug treatment, indicating that THC had no effect on macrophage intrinsic antiviral activity. In contrast, the cannabinoid exerted a dose-dependent inhibition of macrophage extrinsic antiviral activity. This activity describes that macrophage function by which these cells suppress virus replication within xenogeneic cells in an interferon-independent manner. The inhibitory effect of THC on extrinsic antiviral activity was greatest on RAW264.7 and J774A.1 cells, followed by P388D1 cells. These macrophage-like cells regained their extrinsic antiviral activity in a time-related fashion following removal of the drug. These results indicate that THC inhibits macrophage extrinsic antiherpesvirus activity, but has no effect on intrinsic antiviral activity. However, the suppressive effect of THC on extrinsic antiviral activity is reversible upon removal of the drug.


Effects of respiratory motion on dose uniformity with a charged particle scanning method. A three-dimensional spot-scanning technique for radiotherapy with protons is being developed at the Paul Scherrer Institute. As part of the effort to optimize the design and ensure clinically useful dose distributions, a computer simulation of the dose deposition in the presence of respiratory motion was performed. Preliminary experiments have characterized the proton beam and the scanning procedure. Using these parameters, the computer program calculated the dose within a uniform volume of water in the presence of respiratory motion. Respiration amplitude, respiration period, respiration direction, number of fractions, size and position of the beamspots and rescanning multiplicity were systematically varied and the effect on the dose distribution determined. The dose uniformity is very dependent on the direction of the respiration relative to the three independent beam scanning directions. The dose uniformity decreases with increasing respiration amplitude, but has little response to changes in respiration frequency. Rescanning the volume, such as with fractionation, improves the dose uniformity roughly as the square root of the number of fractions. Broad, Gaussian beams result in better dose uniformity than narrow, sharply delineated ones, but produce slower dose fall-off at the edges of the scanned volume. Results of this work are being incorporated into the design of the system.


Conditioned facilitatory modulation of the response to an aversive stimulus in the crab Chasmagnathus. A mild electrical shock delivered to the walking legs of the crab Chasmagnathus granulatus through a fine layer of sea water, induces a running response that declines after repeated stimulation. Herein, a first series of experiments was aimed at conditioning the response to a light or a dark pulse, but no detectable CR was disclosed during training nor in a later test with the CS alone, even though several stimulus parameters were assayed. In a second series, crabs were repeatedly exposed during training to a light pulse (CS) immediately followed by shock (UCS), and after a 6-h rest interval, tested with either CS-UCS or UCS. The CS-UCS presentation at testing elicited a higher recovery (potentiated recovery, circa 70%) than that evoked by the UCS alone (unconditioned recovery, circa 35%) (Experiment 3). An experiment including the explicitly unpaired control procedure (Experiment 4), confirmed that the enhanced response (CR) to the shock (UCS) (measured as potentiated recovery) is conditioned to an illumination signal (CS). An alternative explanation of the potentiated recovery in terms of retardation of habituation proved hardly tenable (Experiment 5). In Experiment 6, crabs were exposed to a variety of light pulse shock and dark pulse shock temporal relationships, and results were found consistent with a delay of reinforcement gradient when light termination was considered as CS. Thus a decrease in luminance is able to become associated with shock and to condition an enhanced response regardless of light termination being the offset of a light pulse or the onset of a dark pulse.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of mesencephalic morphine analgesia by methysergide in the medial ventral medulla of rats. The neural substrates of endogenous supraspinal opioid pain inhibition are mediated in part by connections between the midbrain periaqueductal gray (PAG) and the ventral-medial medulla, including the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis (NRGC). To ascertain whether a serotonergic synapse participated in this pathway, the present study determined whether microinjections of methysergide into the NRM or NRGC would alter analgesia elicited by morphine microinjections into the PAG. Morphine (2.5 micrograms) in the PAG and immediately adjacent areas produced significant analgesia on the tail-flick and jump tests in rats. Pretreatment with the serotonin receptor antagonist methysergide (0.5-5 micrograms) in either the NRM or NRGC significantly reduced morphine analgesia elicited from the PAG by 69% on the tail-flick and by 50% on the jump tests without altering basal nociceptive thresholds. Medullary placements ventral or lateral to the NRM/NRGC failed to support this antagonistic effect. These data indicate that a ventro-medial medullary serotonergic synapse participates in the transmission of opioid pain-inhibitory signals from the PAG.


Lithium chloride and immunomodulation in taste aversion conditioning. Lithium chloride has been used in many studies of conditioning to induce taste aversion behaviour, and in some experiments investigating conditioning effects on immunity it has been used on the assumption that it is immunologically neutral. The studies reported here, however, indicate that LiCl is not immunologically neutral and when used to endow a UCS with noxious properties to enhance the behavioural response in taste aversion conditioned immunosuppression, it may antagonize the residual immunosuppression following initial UCS administration and also the conditioned immunosuppression occurring after CS reexposure. Therefore, conclusions drawn from studies of behaviourally conditioned immunomodulation where LiCl is used as part of either the CS or UCS may require reevaluation.


Enhanced dopamine metabolism in accumbens leads to motor activity and concurrently to increased output from nondopamine neurons in ventral tegmental area and substantia nigra. We previously have reported that nondopamine (non-DA) neurons in substantia nigra (SN) and ventral tegmental area (VTA) of the rat show increased discharge rates during amphetamine (AMPH) and apomorphine (APO)-induced motor activity. The present study represents an attempt to determine the contribution of nucleus accumbens (ACC) dopaminergic activity to these effects, and to ascertain whether the effects in VTA differ from those seen in SN when dopaminergic activity is enhanced locally in ACC. The experiments were carried out in male albino rats (300-400 g) chronically implanted with multiple fine wire electrodes (62 microns) aimed at the pars reticulata of SN (SNR) and VTA. Unit activity was recorded extracellularly in the behaving rat, from neurons identified on the basis of the properties of their action potentials as representing the output of the non-DA neurons in these two structures. In each drug session, unit activity was recorded in parallel from several probes, while motor activity was measured with the open-ended wire technique. But with the recording technique used, a unit represented in most instances the output of a small family of neurons (3-10). Each animal underwent a series of tests given on consecutive days. During these tests, motor and unit activity were measured for 90 min before the drug was administered, and for 135 min after. The first test was of the effects of AMPH, 5 mg/kg, given by the systemic route. The second was of the effects of saline containing 0.1% ascorbic acid (the vehicle) injected bilaterally in ACC, in a volume of 2 microliters per side. The third and all subsequent tests were of the effects of a mixture containing 40 micrograms AMPH, 20 micrograms DA, and 20 micrograms pargyline (P) dissolved in 2 microliters of the vehicle, injected bilaterally in ACC. The results showed that systemic AMPH made the animal hyperactive and at the same time, increased the discharge rate of the non-DA neurons. The bilateral injections of the vehicle in ACC, increased motor activity for about 7 min, an effect interpreted as a rebound from the restraint of the animal during the intracerebral injections, and then depressed motor throughout the 135 min of the postinjection recording period. The effect of the vehicle was to depress unit activity. The effects of injecting the mixture in ACC was to increase motor activity, but with the magnitude and duration of the increase depending on the number of treatments received.(ABSTRACT TRUNCATED AT 400 WORDS)


NGC-evoked nociceptive behaviors: I. Effect of nucleus gigantocellularis stimulation. The role of the nucleus gigantocellularis (NGC) in nociception was examined by investigating behavioral responses that are supported by NGC stimulation in rats. Analyses indicated that NGC stimulation will support escape, active avoidance, and increases in behaviors that are thought to reflect fear in rats. A variety of behavior patterns were elicited by NGC stimulation, including gross peripheral motor movements such as walking, circling and rearing, and specific orofacial movements such as eye closure, ear movement, jaw opening and facial muscle contractions. No major differences in avoidance, affect, or escape behaviors were revealed between the various electrode placements, suggesting functional homogeneity within this neural region. The results of this study suggest that NGC stimulation generates several components of nociception, including behavioral arousal, autonomic nervous system-mediated responses, aversive affect, and motor responses.


NGC-evoked nociceptive behaviors: II. Effect of midbrain and thalamus lesions. Alterations in nociceptive behaviors evoked by stimulation of the nucleus gigantocellularis (NGC) were observed following lesions of the dorsal central gray (DCG) or parafascicularis nucleus (PF) in rats. Lesions of the DCG decreased affective responses and facilitated extinction of avoidance responding associated with NGC stimulation. Lesions of the PF disrupted escape and avoidance responding to NGC stimulation, although postlesion training improved avoidance but not escape performance. The NGC, without its rostral connections to the DCG or PF, does not generate sufficient information for either aversive affective responses or locomotor escape responses that are associated with stimulation of this structure. In contrast, the elicited motor reactions do not depend upon these rostral structures. This supports a role for NGC neurons in functions related to motor, rather than to affective or complex behavioral components of nociception. The differential disruption of the more complex behaviors by DCG and PF lesions provides behavioral evidence for functional differentiation of a nociceptive system involving the relay of information from the NGC to these rostral areas.


Bacterial resistances to inorganic mercury salts and organomercurials. Environmental and clinical isolates of mercury-resistant (resistant to inorganic mercury salts and organomercurials) bacteria have genes for the enzymes mercuric ion reductase and organomercurial lyase. These genes are often plasmid-encoded, although chromosomally encoded resistance determinants have been occasionally identified. Organomercurial lyase cleaves the C-Hg bond and releases Hg(II) in addition to the appropriate organic compound. Mercuric reductase reduces Hg(II) to Hg(O), which is nontoxic and volatilizes from the medium. Mercuric reductase is a FAD-containing oxidoreductase and requires NAD(P)H and thiol for in vitro activity. The crystal structure of mercuric ion reductase has been partially solved. The primary sequence and the three-dimensional structure of the mercuric reductase are significantly homologous to those of other flavin-containing oxidoreductases, e.g., glutathione reductase and lipoamide dehydrogenase. The active site sequences are the most conserved region among these flavin-containing enzymes. Genes encoding other functions have been identified on all mercury ion resistance determinants studied thus far. All mercury resistance genes are clustered into an operon. Hg(II) is transported into the cell by the products of one to three genes encoded on the resistance determinants. The expression of the operon is regulated and is inducible by Hg(II). In some systems, the operon is inducible by both Hg(II) and some organomercurials. In gram-negative bacteria, two regulatory genes (merR and merD) were identified. The (merR) regulatory gene is transcribed divergently from the other genes in gram-negative bacteria. The product of merR represses operon expression in the absence of the inducers and activates transcription in the presence of the inducers. The product of merD coregulates (modulates) the expression of the operon. Both merR and merD gene products bind to the same operator DNA. The primary sequence of the promoter for the polycistronic mer operon is not ideal for efficient transcription by the RNA polymerase. The -10 and -35 sequences are separated by 19 (gram-negative systems) or 20 (gram-positive systems) nucleotides, 2 or 3 nucleotides longer than the 17-nucleotide optimum distance for binding and efficient transcription by the Escherichia coli sigma 70-containing RNA polymerase. The binding site of MerR is not altered by the presence of Hg(II) (inducer). Experimental data suggest that the MerR-Hg(II) complex alters the local structure of the promoter region, facilitating initiation of transcription of the mer operon by the RNA polymerase. In gram-positive bacteria MerR also positively regulates expression of the mer operon in the presence of Hg(II).


Capsular hematoma as a late complication in breast reconstruction with silicone gel prostheses. This paper presents a rare complication of breast reconstruction with silicone gel prostheses in which formation of intracapsular hematomas in the mammary region occurred 3 years after surgery. Treatment by bilateral capsular excision was successful. The rarity of this occurrence and the progress of the case are the major reasons for this publication.


Therapeutic effect of transcatheter oily chemoembolization therapy for encapsulated nodular hepatocellular carcinoma: CT and pathologic findings. An emulsion of iodized oil and doxorubicin hydrochloride was intraarterially injected in 27 patients with encapsulated single nodular hepatocellular carcinoma (HCC). Computed tomography (CT) was performed 1-3 weeks after injection, and the lesions were resected 1-4 weeks thereafter. The percentages of tumor necrosis were evaluated in cut surfaces of resected specimens and were compared with the findings at iodized-oil CT. Six tumors with complete intratumor retention of iodized oil had 98% necrosis, and 21 tumors with incomplete retention had 64% necrosis. Two tumors with complete retention of iodized oil in the surrounding liver had 100% necrosis, while 16 tumors with partial retention and nine tumors without retention of iodized oil in surrounding liver had 74% and 62% necrosis, respectively. Complete retention of iodized oil in the tumor and surrounding liver demonstrated the best therapeutic effects. Evaluation of the pattern and distribution of iodized oil in the tumor and surrounding liver with iodized-oil CT is useful in the assessment of the therapeutic effects of transcatheter arterial embolization in encapsulated nodular HCCs.


Postprocedural symptoms in children who undergo imaging studies of the urinary tract: is it the contrast material or the catheter? The frequency, nature, and duration of postprocedural symptoms in 100 children who underwent voiding cystourethrography (VCUG) after administration of 17.2% wt/vol iothalamate meglumine, 100 children who underwent radionuclide cystography (RNC) after administration of saline and technetium-99m pertechnetate, and 28 children catheterized before diuretic renal scintigraphy (DRS) were prospectively assessed with telephone follow-up. All children were aged 2 years or older; 61 were boys, 167 were girls. Postprocedural symptoms occurred in 80 children (35.1%). The frequency of postprocedural symptoms was nearly identical in the VCUG group and the two other groups. Boys (n = 33 [54%]) had symptoms significantly more often than girls (n = 47 [28%]) (P less than or equal to .0005). Dysuria was the most common symptom (n = 75 [32.9%]) and was frequently accompanied in younger children by anxiety over going to the bathroom. Symptoms disappeared within 24 hours in 32 of 80 children (40%) and lasted 4-10 days in eight children. It is concluded that most postprocedural symptoms in children who undergo VCUG, RNC, or DRS are secondary to catheterization rather than to the use of iodinated contrast material.


Breast cancer: reliability of mammographic appearance as a predictor of hormone receptor status. The association of mammographic appearance with hormone receptor status was investigated in 397 patients with primary breast cancers. The mammographic appearance was classified as type 1, spiculated (n = 159); type 2, structural changes (density) (n = 102); type 3, calcifications (n = 30); type 4, circumscribed opacity (n = 65); and type 5, not visible on mammogram (n = 41). Univariate analysis showed a significant association with estrogen receptor (ER) status for age (less than 50 vs greater than or equal to 50 years), tumor TNM category (those in category 1 vs those in higher categories), and mammographic appearance; with progesterone receptor status, the association was significant only for age. Multivariate analysis adjusted for potential confounders confirmed a significant association between ER status and mammographic appearance (ER status was more likely with type 1 than with the other mammographic types), but the strength of the association was limited. The mammographic appearance of breast cancer is not a reliable method to predict hormone receptor status for clinical purposes.


Liver tumors: follow-up with P-31 MR spectroscopy after local chemotherapy and chemoembolization. The authors examined and quantified the changes observed in the phosphorus-31 magnetic resonance (MR) spectra of liver tumors after chemotherapy and chemoembolization to investigate the suitability of P-31 MR spectroscopy for follow-up. A 1.5-T unit was used before and at specific times during therapy to obtain spectra of liver tumors in 10 patients with liver metastases from colorectal carcinoma and two patients with hepatocellular carcinoma. A marked increase in inorganic phosphate and a decrease in the alpha- and beta-nucleotide phosphate portions of the spectra were observed during the first few hours after local chemotherapy or chemoembolization. Later, the phosphomonoester signals increased markedly and the phosphodiester signals decreased slightly. The effects of successful chemoembolization or local chemotherapy become apparent in the P-31 MR spectrum during the first few hours after the start of therapy. The results demonstrate that P-31 MR spectroscopy is a suitable method for follow-up. However, long-term studies are needed to determine whether it also yields prognostic information.


Brain mechanisms of drug-induced reinforcement. The results of the experiments described above suggest that although the abused psychomotor stimulants and opioids have independent actions that contribute to their reinforcing effects, there are common neuronal substrates for some of their rewarding effects. Additionally there is considerable evidence implicating dopamine systems in these rewarding effects. The neuronal systems involved in these pharmacological actions may be similar to those involved in the rewarding effects of electrical stimulation to the brain. Although both classes of compounds cause euphoria in humans and are reinforcing in animals, the opioids are also central nervous system depressant drugs. These depressant properties influence the subjective effects in humans and possibly the nature of the rewarding effect in animals. Experiments using the 2-deoxyglucose procedure indicate that BSR to either the ventral tegmental area or the medial forebrain bundle results in functional activation throughout the mesocorticolimbic system. The major effects are found in the nucleus accumbens, olfactory tubercle, and the medial prefrontal cortex. Cocaine produces increases in metabolic rates similar in distribution to BSR. Morphine, however, only causes significant increases in functional activity in the olfactory tubercle. Further, only in this brain site did the combination of BSR plus morphine or cocaine cause increases in functional activity over that of stimulation alone. These findings suggest that the olfactory tubercle plays a major role in the pharmacological actions of both the psychomotor stimulants and the opioids as well the rewarding effects of electrical stimulation of the brain.


[Hepatitis-C virus and hepatoma].

[Hepatitis-C virus and hepatoma]. To clarify the relationship between hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC), frozen serum samples from 213 patients with histologically proven liver cirrhosis alone (96 alcoholics, 59 HBsAg positive, 29 non-A, non-B hepatitis, 29 cryptogenic) and 40 patients with liver cirrhosis and HCC (12 alcoholics, 7 HBsAg positive, 7 non-A, non-B hepatitis, 14 cryptogenic) were analyzed for antibodies to hepatitis C virus (anti-HCV) with the ortho-HCV-ELISA. The results were as follows. 50 of 253 (20%) patients were anti-HCV positive. The prevalence of anti-HCV was significantly higher in patients with HCC than in patients without HCC (14 of 40 [35%] vs 36 of 213 [17%]; p less than 0.001). In anti-HCV-positive patients HCC were significantly more frequent than in anti-HCV-negative patients (14 of 50 [28%] vs 26 of 203 [13%]; p less than 0.001). The significantly higher occurrence of HCC in anti-HCV-positive patients was not related to other known risk factors such as alcoholism or chronic hepatitis B virus (HBV) infection. Patients with HCV infection as the only risk factor also had a significantly higher occurrence of HCC (12 of 38 [32%] vs 26 of 203 [13%]; p less than 0.001). Our data suggest that chronic HCV infection plays an important role in the pathogenesis of HCC, in particular in patients with cirrhosis unrelated to alcohol or HBV infection.


[Anti-HCV test in patients with chronic non-A, non-B hepatitis]. Our study was designed to evaluate the clinical significance of the ELISA-anti-HCV test. 90 patients with histologically proven chronic non-A, non-B hepatitis (NANBH) and a control group consisting of 36 patients with primary biliary cirrhosis (PBC) were analyzed for antibodies to hepatitis C virus (anti-HCV). Frozen serum samples were tested with the ortho-HCV-ELISA. 67 of 90 (74%) patients with chronic NANBH showed antibodies to the hepatitis C virus. The prevalence of anti-HCV in patients with parenterally transmitted and sporadic chronic NANBH was 70% (23 of 33) and 77% (44 of 57) and in patients with chronic active and chronic persistent NANBH 73% (27 of 37) and 76% (40 of 53) respectively. No significant difference in the prevalence of anti-HCV in the 4 subgroups of patients with chronic NANBH was found. 2 of 36 patients (6%) with PBC were anti-HCV positive without evidence of previous HCV infection. Our results bear out the experience of other authors, that HCV is the commonest pathogen of chronic NANBH. The low prevalence of anti-HCV in patients with PBC suggests good specificity. There have been reports in the literature of "false positive results", particularly in autoimmune hepatitis, and it has been shown that borderline positive test results are often unspecific and therefore to be interpreted with care.


Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na(+)-Ca2+ exchange. In cardiac myocytes, calcium influx through the calcium channel is the primary pathway for triggering calcium release. Recently it has been suggested that the calcium-induced calcium release mechanism can also be activated indirectly by the sodium current, which elevates the sodium concentration under the cell membrane, thereby favoring the entry of "trigger" calcium via the sodium-calcium exchanger. To test this hypothesis, sodium current was suppressed by reducing the external sodium concentration or applying tetrodotoxin. At potentials positive to -30 millivolts, calcium release was unaffected. A small calcium release at more negative potentials could be attributed to partial activation of calcium channels, because it was unaltered by replacement of sodium with lithium and was blocked by cadmium. Thus, sodium influx or its accumulation does not initiate calcium release. In addition, sodium-calcium exchange-related calcium release at potentials positive to +80 millivolts has slower kinetics than calcium channel-induced release. Therefore, only the calcium channel gates the fast release of calcium from the sarcoplasmic reticulum in the range of the action potential.


Tyrosyl phosphorylation and activation of MAP kinases by p56lck. T cell signaling via the CD4 surface antigen is mediated by the associated tyrosyl protein kinase p56lck. The 42-kilodalton mitogen-activated protein (MAP) kinase (p42mapk) was tyrosyl-phosphorylated and activated after treatment of the murine T lymphoma cell line 171CD4+, which expresses CD4, with antibody to CD3. Treatment of the CD4-deficient cell line 171 with the same antibody did not result in phosphorylation or activation of p42mapk. Purified p56lck both tyrosyl-phosphorylated and stimulated the seryl-threonyl phosphotransferase activity of purified p44mpk, a MAP kinase isoform from sea star oocytes. A synthetic peptide modeled after the putative regulatory phosphorylation site in murine p42mapk (Tyr185) was phosphorylated by p56lck with a similar Vmax, but a fivefold lower Michaelis constant (Km) than a peptide containing the Tyr394 autophosphorylation site from p56lck. MAP kinases may participate in protein kinase cascades that link Src family protein-tyrosyl kinases to seryl-threonyl kinases such as those encoded by rsk and raf, which are putative substrates of MAP kinases.