Mycological findings in feline immunodeficiency virus-infected cats. Thirty-five FIV-seropositive cats and 55 FIV-seronegative matched cats were examined for yeasts (oropharyngeal swabs) and dermatophytes (hair brushings). The frequency of isolation of Candida albicans and Cryptococcus neoformans was significantly higher in the former group. The only dermatophyte isolated was Microsporum canis. Its prevalence was three times higher among FIV-infected cats than among control animals.


The HIV-1 surface protein gp120 has no effect on transmembrane signal transduction in T cells. The ability of HIV-1 envelope glycoprotein gp120 to induce transmembrane signaling processes in human T cells and tumor T-cell lines was investigated. Differently glycosylated gp120 preparations were characterized with respect to their purity, the fraction of native gp120, and the affinity of the gp120-CD4 interaction. These data were used to establish experimental conditions that allow a substantial fraction of the CD4 receptor to be complexed with gp120 in the course of the experiments. The results are in contrast to several previous studies since no effect of gp120 on the intracellular Ca2+ concentration, the metabolism of inositol phosphates and arachidonic acid, protein kinase C translocation, and tyrosine phosphorylation was found. Cross-linking of the gp120:CD4 complex by anti-gp120 antibodies did not elicit additional effects.


[The study of Chinese herbal medicinal prescription with enzyme inhibitory activity. VI. The study of makyo-kanseki-to with adenosine 3',5'-cyclic monophosphate phosphodiesterase]. A Chinese herbal medicinal prescription, Makyo-kanseki-to, was studied for the inhibitory activity of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase. The effect for the inhibitory activity of cAMP phosphodiesterase by combination with constituent crude drugs of the prescription was studied. Gypsum acted as a mitigatory component for Ephedra herb and Glycyrrhiza in cAMP phosphodiesterase inhibition test.


The isolation and characterisation of a major outer-membrane protein from Bacteroides distasonis. An outer-membrane protein (OMP) was isolated from a clinical strain of Bacteroides distasonis. Changes in growth media did not appreciably affect the appearance of this protein in crude outer-membrane preparations examined by SDS-PAGE. However, the proportion of the protein relative to other OMPs was greater in 24-h cultures than in 48-h cultures. The protein could not be readily solubilised by various conventional detergent extraction techniques but treatment of the insoluble material at 100 degrees C with SDS released the protein, as did overnight extraction at 37 degrees C with SDS. This OMP was heat-modifiable, and thus was similar to the OmpA protein of Escherichia coli, with a faster mobility on SDS-PAGE when solubilised at 25 degrees C than at 100 degrees C. The critical temperature for conversion was between 80 degrees C and 90 degrees C. Because of the characteristic heat-modifiability, the protein was called B. distasonis HMP-1 (heat modifiable protein-1). Overnight exposure to EDTA or NaCl at 37 degrees C favoured conversion of the 25 degrees C form to the 100 degrees C form. In intact cells, the protein was labelled by a cell-surface radio-iodination procedure, and thus is at least partially exposed at the cell surface. No reactions between the B. distasonis HMP-1 and antibodies to either E. coli OmpA or E. coli porin were found by Western blot analysis. A B. distasonis OM preparation containing predominantly HMP-1 had pore-forming ability in a liposome assay. This study is the first report of the isolation and characterisation of a heat-modifiable OMP in Bacteroides, and it is the first description of pore-forming activity in a Bacteroides OM fraction.


Intra- and inter-species mobilisation of non-conjugative plasmids in staphylococci. The ability of Staphylococcus aureus conjugative plasmids to mobilise non-conjugative resistance plasmids from clinical isolates of S. aureus and S. epidermidis was studied. Plasmids which could not be transferred by transduction or mixed-culture transfer were transferred from phage-typable and non-typable S. aureus and from S. epidermidis. Plasmids encoding single resistance determinants were transferred by mobilisation whereas multiple-resistance plasmids were transferred as co-integrates between the conjugative and non-conjugative plasmids. This study demonstrates that mobilisation is a useful tool for the transfer and study of staphylococcal plasmids and illustrates how antibiotic resistance could be transferred between staphylococci in vivo.


Sequence of a second human KDEL receptor. Retention of luminal endoplasmic reticulum (ER) proteins is mediated via a conserved carboxy-terminal tetrapeptide that serves as a signal for their retrieval from subsequent compartments of the secretory pathway. The signal is recognized by a receptor molecule that is believed to cycle between the Golgi apparatus and the ER. This receptor in Saccharomyces cerevisiae is encoded by the ERD2 gene, and a human cDNA homologue of the gene has been isolated. Binding of ligand by the product of this gene results in a shift of its steady-state location from Golgi to ER, suggesting that retrograde transport has been triggered. Here we report the identification of a related human protein with similar properties. This indicates that there are at least two distinct genes in humans that encode functional KDEL receptors.


RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa. Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.


dnaR function of the prs gene of Escherichia coli in initiation of chromosome replication. A new Escherichia coli mutant named dnaR, which was temperature sensitive in initiation of DNA replication, has been characterized through identification of the mutant gene. A 1.65 x 10(3) base-pair chromosomal DNA fragment isolated from wild-type cells, but not the corresponding fragment from the dnaR mutant, exhibited an activity that reversed temperature-sensitive growth of the mutant. The DNA fragment was found to include the entire prs-coding sequence and specify a 34,000 M(r) protein with phosphoribosylpyrophosphate synthetase activity. The dnaR mutation resided within the prs-coding segment and made the synthetase thermolabile. The coding segment for the dnaR product was determined, by introduction of various mutations into the cloned fragment, to be the same as that for the synthetase. The dnaR function of the prs gene product in DNA replication is discussed on the basis of an observation that thermal treatment of the dnaR mutant caused a delay in initiation of chromosome replication after the downshift, despite the presence of the synthetase activity at the preheat level.


Modification of fatty acid composition of the phospholipids of cultured rat ventricular myocytes and the rate of phosphatidylinositol-4,5-bisphosphate hydrolysis. Cultured neonatal cardiac myocytes have been utilized as a model for the study of the role of fatty acids in the alpha 1-adrenoceptor mediated phosphatidylinositol turnover. Experiments were started 24 h after seeding, when there was a confluent monolayer of beating cardiomyocytes. The cells were incubated for 3-4 days in sera containing culture medium with (1) no additives or (2) a mixture of 107 microM 18:0 and 18:1n-9, or (3) only 214 microM 18:2n-6 or (4) 214 microM 20:5n-3. No differences in the cellular content of the various phospholipid classes among the different groups of fatty acid treated cells were found. The predicted elevations of 18:1n-9, 18:2n-6 and 20:5n-3 associated with a partial depletion of 20:4n-6 were confirmed in all phospholipid classes, except for sphingomyelin. The mol% of 18:0, 18:2n-6, 20:4n-6 and 20:5n-3 in the phosphatidylinositol fraction were respectively 39, 4, 30 and 0.6 for the control treated cells, 34, 3, 15 and 0 for 18:0/18:1n-9 treated cells, 40, 17, 24 and 0.2 for the 18:2n-6 treated cells and 41, 3, 13 and 21 for the 20:5n-3 treated cells. Apart from the observed reductions in the basal rates, the phenylephrine (30 microM) stimulated production of inositolphosphates was reduced by 51% and 71%, respectively in the 18:2n-6 and 20:5n-3 treated cardiomyocytes. The basal rate of inositolphosphate formation was 37% increased in the 18:0/18:1n-9 treated cells. The [3H]-inositol incorporation into phosphatidylinositol 4,5-bisphosphate was only slightly reduced by 18:2n-6 and 20:5n-3 treatments (respectively 12 and 28% compared to control treated cells). Prolonged (30 min) alpha 1-adrenergic stimulation did not affect the contents and fatty acid profiles of any class of phospholipid, not even phosphatidylinositol. In conclusion, variations in the polyunsaturated fatty acid composition of membrane phospholipids do affect the basal and the alpha 1-adrenoceptor stimulated rate of phosphatidylinositol-4,5-bisphosphate hydrolysis. The reducing effects of 18:2n-6 and 20:5n-3 treatment on the rate of inositolphosphate production may be partially ascribed to altered levels of phosphatidyl-inositol 4,5-bisphosphate.


Phylogenetic evidence for multiple Alu source genes. A member of the young PV Alu subfamily is detected in chimpanzee DNA showing that the PV subfamily is not specific to human DNA. This particular Alu is absent from the orthologous loci in both human and gorilla DNAs, indicating that PV subfamily members transposed within the chimpanzee lineage following the divergence of chimpanzee from both gorilla and human. These findings and previous reports describing the transpositional activity of other Alu sequences within the human, gorilla, and chimpanzee lineages provide phylogenetic evidence for the existence of multiple Alu source genes. Sequences surrounding this particular Alu resemble known transcriptional control elements associated with RNA polymerase III, suggesting a mechanism by which cis-acting elements might be acquired upon retrotransposition.


Does the VP1 gene of foot-and-mouth disease virus behave as a molecular clock? We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts.


[Analysis of T-cell receptor delta chain gene in hematological malignancies]. We analyzed the rearrangement of T-cell receptor (TcR) delta chain gene in 196 cases of hematological malignancies. This rearranged band (s) was observed in 15% of the total cases investigated. All T-ALL patients and cell lines, except for P30/Okubo, had a new band (s) or deletion of J delta 1 gene locus, indicating the gamma delta T-cell type or the alpha beta T-cell type. In the other T-cell malignancies, the delta rearranged band (s) was recognized in 5% of T-cell lymphomas, 20% of AILD but not in ATL, Hodgkin's disease, T-CLL. Inappropriate delta rearrangement was frequently recognized in 63% of B-ALL and 50% of CML-BC but none or few (5% less) in B-CLL, B-lymphoma and AML. Southern blotting, using J delta 1 and V delta gene probes or Pst I enzyme digestion, indicated that the inappropriate delta rearranged band in B-ALL and CML-BC is V delta 2D or DD without a J delta locus. The rearranged band (s) involved J delta locus, was mostly recognized in 5/6 cases of CD7 (+) stem cell leukemia. Therefore, the TcR delta gene is useful in evaluating clonality for the most immature T-cell neoplasms, not showing rearrangement of the other TcR genes. Moreover, this delta gene may be a useful tool for distinguishing T-lineage from the other lineages, using the characteristic rearrangement pattern (V delta 2D as a inappropriate pattern, or (D) DJ and V (D) DJ as the T-lineage pattern (s)).


[Retinoic acid receptor alpha gene in t (15; 17) APL]. A t(15; 17) (q22; q21) translocation is identified in most patients with acute promyelocytic leukemia (APL). This translocation constructs fusion genes between retinoic acid receptor alpha (RARA) at 17q21 and PML at 15q22. These rearrangements can be detected in the majority of patients with APL but not in other types of leukemias, by Southern blotting. Breakpoints cluster in limited regions of RARA and PML, and PML/RARA and RARA/PML transcripts can also be detected by RT-PCR. Although PML/RARA and RARA/PML fusion products are transcribed in APL, PML/RARA may play an important role in the etiology of APL. Clinically, most APL patients achieve remission by oral administration of high dose all-trans retinoic acid.


Growth behavior of low passage cultured cells derived from human non-small cell lung carcinoma in deepithelialized rat tracheas transplanted into nude mice. Proliferation behavior of six low passage cell lines derived from human non-small cell lung carcinoma was investigated both by cell inoculation into deepithelialized Fisher 344 rat tracheas xenotransplanted into nude mice and by direct cell injection into the subcutis of nude mice. In the tracheal transplants, all the cell lines showed a comparable or higher degree of differentiation than in the primary lesion, while most cell lines showed a lower degree of differentiation in the mouse subcutis. The surfaces of the tracheal transplants repopulated with the cells derived from squamous cell carcinomas were initially lined with a layer of flattened cells, two to three cells thick, while those repopulated with the cells from the three adenocarcinoma cases (HKT-3, HKT-5, HKT-6) were initially lined with simple epithelia comprising cuboidal-, round-and columnar-shaped cells, respectively. Subsequently, the cells from HKT-3 increased in height and proliferated in a pseudostratified pattern, those from HKT-5 proliferated in a papillary growth pattern and those from HKT-6 proliferated in a cribriform pattern. Ultrastructurally, they resembled ciliated bronchial epithelial cells, non-ciliated bronchiolar epithelial cells and bronchial goblet cells, respectively. Histological examination of cultured cells using this system was considered to be advantageous in the evaluation of differentiation since it provided unique proliferation patterns unobserved in primary tumors or tumors formed after s.c. direct cell injections.


Reconstruction of a segmental clavicular defect with a Marlex mesh-enveloped composite graft consisting of a hydroxyapatite prosthesis and autogenous bone: a case report. The present study reports a case of a 38-year-old man with a malignant lymphoma of the right clavicle who underwent reconstruction after a segmental resection of the clavicle. A hydroxyapatite prosthesis in the shape of the clavicle was placed across the defect in conjunction with Marlex mesh which entirely enveloped the prosthesis and iliac crest autogenous bone. The mesh also acted as a stabilizer to the segments. The postoperative management was not a burden to the patient. Postoperative radiographs indicated bony union to have been completed within six months of the operation. The patient returned to work as a driver eight months after surgery. The range of motion of the shoulder joint was not limited. The patient had no trouble in carrying out his daily life without pain. The appearance of the area operated was excellent. At the postoperative 39 months follow-up visit, a radiograph reconfirmed the stable reconstruction with a composite graft and bone bridging at the gap left in the prosthesis to have been maintained with little bone absorption. The results indicate a reconstruction using a Marlex mesh-enveloped composite graft consisting of a hydroxyapatite prosthesis and autogenous bone to be a useful alternative to other types of reconstruction for segmental clavicular defects.


[So-called "gastric ileus" induced by the use of topical ice slush: report of two cases]. Of 167 patients, who underwent cardiac surgery using topical ice slush, 21 (12.6%) were found to have phrenic nerve palsy postoperatively. In addition, two patients showed severe epigastric discomfort, such as nausea, vomiting and gastric distension. These symptoms were consistent with so called "gastric ileus", first reported by Spencer et al. The injury of thoracic vagi induced by topical ice slush might be related to the development of this complication.


Pre- and postoperative sequential study on the serum gastrin level in patients with lung cancer. Serial changes in serum gastrin level were detected by radioimmunoassay in 58 lung cancer patients before and after operation. In comparing these tests with those of 40 cases of noncancerous thoracic lesions and 151 normal adults, the serum gastrin from lung cancer patients is significantly higher than that of noncancerous thoracic lesions and normal individuals (P less than 0.01). The gastrin level is closely related to stage of cancer, size of primary tumor, presence of lymph node metastasis, and type of histological classification. The serum gastrin was found to decrease gradually after the removal of the tumor and to return to normal on the 14th postoperative day. Those patients whose serum gastrin level can return to normal on the 14th postoperative day will have a good prognosis; if not, their prognosis will be very poor. These results suggest that serum from patients with lung cancer contains a high concentration of gastrin that can help differentiate benign from malignant thoracic lesions and evaluate prognosis of patients with lung cancer. Therefore, the cause of high serum gastrin in patients with lung cancer is likely due to the gastrin-producing property of the lung cancer cells.


Investigation of colon cancers for human papillomavirus genomic sequences by polymerase chain reaction. Two previous studies have reported the presence of human papillomavirus (HPV) genomic sequences in colorectal cancers. We examined DNAs from 50 colorectal tumors for HPV sequences by polymerase chain reaction (PCR), using HPV consensus L1 primers for amplification. The PCR products were screened with a generic HPV probe consisting of amplimers of HPV-16 and HPV-18. All tumor DNAs were negative for HPV sequences. These results call into the question the reported association of colorectal cancers with HPVs.


Mucinous component in colorectal carcinoma--prognostic significance: a study in a south Indian population. One hundred eighteen specimens of colorectal carcinoma have been studied with a view to assess the clinicopathological significance of the mucinous component in these neoplasms. When 50% or more of high-power fields examined consisted of "mucinous" tumour tissue, the term mucinous carcinoma was applied. Such mucinous carcinomas (MCa) constituted 19% of the total neoplasms studied. Predilection for the younger age group, a higher incidence in the proximal colon as against the rectosigmoid, and a lower distribution in the rectosigmoid as against nonmucinous carcinomas in that region were some of the features that characterised MCa. Clinical and histological features suggestive of aggressive behaviour and poor prognosis were more frequently observed in MCa. These features correlated with the percentage of mucinous component independently of the histological grade. It is concluded that colorectal mucinous carcinomas form a distinct group of neoplasms with certain clinical and pathological characteristics. These neoplasms tend to follow an aggressive clinical course, which is directly influenced by the mucinous component.


Mullerian adenosarcoma of the uterus with sarcomatous overgrowth. A case of mullerian adenosarcoma of the uterus with sarcomatous overgrowth is described. The patient was also found to have bilateral polycystic ovaries. This 25 year-old white female presented with irregular vaginal bleeding and was diagnosed as having mullerian adenosarcoma with sarcomatous overgrowth by dilatation, curettage, and biopsy. Total abdominal hysterectomy, bilateral salpingo-oophorectomy with lymph node sampling were performed.


Criteria for the definition of Epstein-Barr virus association in Hodgkin's disease. There is a clear association between the Epstein-Barr virus (EBV) and Hodgkin's disease (HD). EBV is not, however, detectable within the affected tissues of all cases. The proportion of positive cases varies from 15-79% depending on the assay used to detect EBV. The techniques utilised vary not only in sensitivity but in their ability to detect viral DNA, RNA, or protein and in their ability to demonstrate the cellular localisation of the virus. Thus, the biological significance of a positive result will vary depending on the method of analysis. In the present study, four different methods of detecting EBV were compared. RNA in situ hybridization was found to be the most practical method of detecting EBV in tumour cells. Using this assay EBV was detected in the Reed-Sternberg cells of 33% and 45% of the two series of HD cases examined in this study. We believe that these cases should be considered EBV-associated.


Lung cancer: a review of current therapeutic modalities. This article describes the current approach to the systematic management of both small cell and non-small cell lung cancer (NSCLC). The treatment of stages I, II, and IIIa NSCLC is surgical resection. Although adjuvant chemotherapy in stage I disease offers no survival benefit, the role of adjuvant chemotherapy in stage II and IIIa NSCLC remains controversial. Results of pilot studies using neoadjuvant chemotherapy in stage IIIa NSCLC are encouraging and data from ongoing randomized trials are awaited with interest. For locally advanced NSCLC, chest irradiation remains the standard of care. However, the addition of systemic chemotherapy holds promise. The impact of cisplatin-based regimens on overall survival in stage IV NSCLC remains disappointing. The introduction of newer agents, such as 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11), a topoisomerase-I inhibitor, has shown early favorable results. Chemotherapy is the most important therapeutic modality in the management of small cell lung cancer because of this cancer's propensity for early dissemination. In limited stage small cell lung cancer, chest radiotherapy, particularly if used early and concurrently with chemotherapy, may improve survival, but at the expense of increased toxicity. The role of prophylactic brain irradiation remains controversial in limited-stage disease. Chemotherapy is also the most important treatment modality in extensive-stage disease, but its role is only palliative. Radiotherapy is reserved primarily for disease-related complications in patients in whom chemotherapy has failed.


[Preoperative chemotherapy of nephroblastoma. Preliminary results of the SIOP-9/GPO therapy study]. The therapeutic strategy of the SIOP-9 study includes pre-operative chemotherapy for all patients with nephroblastoma diagnosed by imaging methods aged between 0.5 and 16 years. By pre-operative chemotherapy the rate of radical resectable tumors should be increased and thereby the intensity of postoperative therapy, in particular of radiotherapy, diminished. Patients with nephroblastoma stage I-III were in case of tumorresponse randomised in either a 4 weeks or 8 weeks arm of pre-operative treatment with ACT D and VCR. The question was, if a prolongation of pre-operative chemotherapy could increase the relative part of stage I. Between 1/1/89 and 6/30/91 from 49 oncologic centres of former western Germany 188 patients were registered in the SIOP-9/GPO. From the stage I-III patients between 0.5 and 16 years 80.1% were pretreated with cytostatic agents. Only 53.9% of the patients with tumorresponse were randomised. The relative frequency of intraoperative ruptures was with 3% lower in the group of pretreated patients than in the primary operated (15.3%). The stage distribution for all Wilms' tumor patients showed a prevalence of stage I with 43.3% (after pre-operative treatment 59%; after primary operation 28%). Abdominal radiotherapy was performed in 22.4%. The event-free survival rate of all nephroblastoma lay at 85% 3 years after diagnosis (stage I standard 96%; unfavorable histology all stages 45%). 7.3% of the patients developed a hepatopathy under treatment and 7.8% even a VOD according to the criteria of McDonald.(ABSTRACT TRUNCATED AT 250 WORDS)


Epstein-Barr virus genotypes in AIDS-associated lymphomas are similar to those in endemic Burkitt's lymphomas. PCR was used to screen EBV-positive lymphomas from endemic and sporadic Burkitt's lymphoma patients, including EBV-positive lymphomas derived from patients with HIV infection. Only 10% of sporadic lymphomas from either North America (1/15) or South America (2/14) were associated with the type 2 EBV strain, whereas 50% (8/16) of lymphomas from equatorial Africa and 46% (10/22) of HIV-associated lymphomas were positive for the type 2 strain. These data, in conjunction with previous reports, suggest that the proportions of strain types in Burkitt's lymphoma reflect the proportions of strain types in peripheral lymphocytes, and not simply the prevailing regional strain. The increased association of the type 2 strain in lymphocytes and lymphomas from HIV-infected individuals and from Africa may be a result of intermittent (malaria) or continuous (HIU) compromise of immune function in these populations.


Characterization of Philadelphia-chromosome-positive acute leukemia by clinical, immunocytochemical, and gene analysis. Philadelphia chromosome (Ph') was detected at presentation in 10 out of 110 patients with acute lymphoblastic leukemia (ALL) and five of 168 patients with acute myelogenous leukemia (AML). Two other ALL patients who had studies at relapse were also included in the analyses. One of the 12 Ph'-positive (Ph+) ALL patients had simultaneous expression of myeloid-associated antigen on the leukemic blasts, while all the five AML patients coexpressed markers of lymphoid cells. Double labeling of the cells with myeloperoxidase and CD10 on three Ph+ AML cases showed that most leukemic blasts expressed either myeloperoxidase activity or CD10 but not both. Cross-lineage gene rearrangements of T-cell receptor (TCR) beta-chain gene were detected in three of the eight Ph+ ALL patients tested. All the four Ph+ AML cases studied showed immunoglobulin heavy chain gene rearrangements, and three of them also had simultaneous rearrangements of TCR beta-chain gene. The results revealed that Ph+ acute leukemia in this study belonged either to ALL or mixed lineage leukemia, and none was pure AML. This finding is contrary to that of acute blast crisis of chronic myelogenous leukemia in which the majority of patients had myeloid transformation. Rearrangements of bcr were detected in four of the 10 Ph+ ALL and three of the four Ph+ AML patients tested. No significant difference was noted in the clinical or hematologic manifestations among Ph+ leukemia with or without bcr rearrangements.


Detection of clonal Epstein-Barr virus in malignant proliferation of peripheral blood CD3+ CD8+ T cells. Epstein Barr virus (EBV) DNA was detected in a monoclonal proliferation of T cells in a three-year-old girl who presented with a history of fever, hepatosplenomegaly, and generalised lymphadenopathy. The disease ran a rapid, fulminant course and the patient died 11 days after presentation. Examination of the blood showed a lymphocytosis of 50 x 10(9)/l with all the cells showing the morphology of large granular lymphocytes. These cells were CD2+3+8+25+. Cytogenetic studies showed the presence of a 6q- clone. Southern blotting and hybridisation with a constant region probe for the T-cell receptor (TCR) beta chain gene showed clonal rearrangement of the TCR beta gene. Hybridisation of the Southern blot to the EBV XhoI probe revealed a clonal pattern of episomal EBV DNA. Our results establish the association between clonal EBV infection to a malignant proliferation of peripheral blood CD8+ T cells.


Human papillomavirus identified by nucleic acid hybridization in concomitant nasal and genital papillomas. Presence of human papillomavirus (HPV) as the etiologic agent in nearly all upper respiratory tract recurrent papillomas is well-established. The technique of nucleic acid hybridization now allows specific typing of HPV with a high degree of accuracy. This article reports a series of nine consecutive patients treated for nasal papillomas over the past 9 years. Eight of these patients had a personal history of genital papillomas (seven patients) or exposure (one patient). With the use of in situ hybridization and autoradiographic technique on paraffin-embedded tissue sections, HPV RNA type 6/11 was expressed in eight of nine nasal papillomas, and corresponding HPV types were also found in the two cases with which concurrent anogenital papilloma tissue was also available for analysis. Human papillomavirus RNA types 16 and 18 were not detected in any of the specimens. Signals of HPV messenger RNA type 6/11 were stronger in the fungiform areas than in the inverted areas of papillomas.


Human papillomavirus in sinonasal papillomas and squamous cell carcinoma. The diagnostic and prognostic relevance of human papillomavirus (HPV) types 6, 11, 16, and 18 in squamous papilloma, inverted papilloma, and squamous carcinoma of the sinonasal epithelium was examined using the polymerase chain reaction (PCR) technique. Four (15%) of 26 squamous papillomas, 7 (24%) of 29 inverted papillomas, and 1 (4%) of 24 squamous carcinomas were positive for HPV when examined using the PCR amplification technique. Human papillomavirus 6 was present in 5 specimens (3 squamous and 2 inverted papillomas); HPV-11 was present in 6 specimens (1 squamous and 5 inverted papillomas); and HPV-18 was present in 1 of 24 squamous carcinomas. HPV-16 was not identified in any specimen. The proportion of tissue samples showing HPV presence, and the association of HPV types 6 and 11 with benign lesions and HPV-18 with malignant lesions, are both in accord with findings from prior investigations. Two major questions regarding nasal papilloma are the probability for lesion recurrence after surgical excision and the risk for malignant transformation. There is no unanimity of opinion regarding the prognostic value of histopathologic dysplasia to forecast these outcomes. HPV is etiologically related to a subset of sinonasal papillomas and squamous carcinoma, and those with benign and malignant clinical course are separable on basis of HPV type. Because of the paucity of these nasal lesions, a multi-institutional prospective collaborative study is the ideal way to address these questions.


Neuroendocrine responses to cocaine do not exhibit sensitization following repeated cocaine exposure. Cocaine-induced enhancement of motor activity and extracellular dopamine concentrations exhibits sensitization upon repeated exposure. In this study, the neuroendocrine responses to cocaine were examined following cocaine pretreatment regimens which have been shown to produce behavioral sensitization. Adult male rats were injected with cocaine (15 mg/kg, IP) once daily for 14 days, followed by a dose-response challenge with cocaine (1-15 mg/kg, IP) either 18 hours or 7 days after the final pretreatment injection. Blood was collected 15 minutes following injections for radioimmunoassay of ACTH, corticosterone, prolactin, and renin. Cocaine increased plasma ACTH and corticosterone, while it decreased prolactin and renin concentrations. Pretreatment with cocaine for 2 weeks did not alter any of these endocrine responses after either the 18 hour or 7 day interval between pretreatment and challenge injections. In contrast, sensitization to the locomotor stimulant effects of cocaine was observed on the final day of pretreatment injections, and 7 days later. These data suggest that these endocrine effects of cocaine do not exhibit sensitization following repeated cocaine exposure.


Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes. The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.


Localized magnetic resonance spectroscopy measurement of brain lactate during intravenous lactate infusion in healthy volunteers. Proton magnetic resonance spectroscopy (1H MRS) localized to the left temporal-parietal region in 8 healthy volunteers detected a 2.1-fold +/- 0.7-fold increase (all values +/-SD) in brain lactate during intravenous infusion of 0.5 molar (M) sodium lactate (5 meq/kg over 20 minutes). Significant increases in brain lactate occurred within 5-10 minutes after starting lactate infusion, progressively rose during the infusion, then decreased towards baseline levels during 30 minutes post-infusion. Venous lactate concentration increased from 0.8 +/- 0.2 mM to 10.9 +/- 4.1 mM or 13.6-fold during the infusion. Flow phantom findings in vitro suggest attenuation of 1H MRS blood lactate signal from arteries and veins as a result of flow velocity effects. Correlations between paired blood and brain lactate measurements at each sampling time indicate a non-linear relationship between compartments during lactate infusion.


Antagonism of kainic acid lesions in the mouse hippocampus by U-54494A and U-50488H. A morphometric study of kainic acid- (KA) induced lesions was designed for the study of the interaction of the diamines U-5449A and U-50488H with excitatory amino acids, and the dose-response relationship thereof. IC50S determined for binding at the kappa receptor and other opioid receptors demonstrated the lack of kappa activity of U-54494A, a structurally related analog of U-50488H. Both opiate kappa receptor related anticonvulsant diamines were tested for their ability to protect the mouse hippocampus from the cytopathological changes induced by KA in neurons and glia. The damage observed with i.c.v. KA in mouse was restricted to neurons of the CA3 pyramidal region and glia of the hippocampus. It involved massive cell loss and shrunken neurons with dark cytoplasm and nuclei. Groups treated with combinations of KA and U-54494A or U-50488H showed scarce damage, but patches of necrotic changes were still observed. Control animals treated with saline (i.c.v.) and U-54494A (s.c.) or U-50488H (s.c.) did not suffer any noticeable alterations of the polymorphic layers of the hippocampal formation. Image analysis of the CA3 area of the hippocampus was used to quantitate the vacuolization induced by KA lesions in the control and treated groups. By this method, both U-54494A and U-50488H were shown to protect this area in a dose-related fashion as evidenced by reduced vacuolization. The anticonvulsant properties of these compounds may result in the antagonism of the excitotoxic lesions. More specifically, the ability of these diamines to block depolarization-induced influxes of Ca++ may protect the CA3 cells from the cytotoxic effects of persistent depolarization.


L-dopa is protective against indomethacin-induced small intestinal ulceration in the rat: possible role of an alpha-2-adrenergic mechanism. Dopaminergic agents ameliorate experimentally induced gastroduodenal mucosal injury, but there is no information about their effect on small intestinal mucosa. We studied the effect of L-dopa and related substances on indomethacin-induced intestinal ulceration in the rat. Ulceration was produced by s.c. injection of 30 mg/kg indomethacin, 30 min after refeeding fasted rats. Total ulcer area was measured 24 hrs after indomethacin administration. L-dopa, 5 mg/kg given in two divided doses 5 h apart, starting 30 minutes before administration of indomethacin, was found to protect the small bowel mucosa against indomethacin- induced damage (ulcer area 122 +/- 5.5 vs 224.2 +/- 5.4 mm2, mean +/- SEM, p less than 0.006). Administration of 5 mg/kg haloperidol, a dopa antagonist, did not abolish the protective effect of L-dopa. On the other hand, yohimbine, an alpha-2-adrenoreceptor antagonist, almost completely abolished the protective effect (180.4 +/- 5.3 vs 122 +/- 5.5, p less than 0.004). Clonidine 20 micrograms/kg, an alpha-2-adrenoreceptor agonist, closely mimicked the protective effect of L-dopa (141.5 +/- 10.9 vs 224.2 +/- 5.4, p less than 0.006). All drugs were give i.p. in two divided doses, at the same schedule as described for L-dopa. The results demonstrate that L-dopa has a protective effect on indomethacin-induced small bowel injury in the rat. The protective effect is probably mediated through stimulation of alpha-2-adrenoreceptors.


Effect of protein kinase inhibitors on ACTH-stimulated aldosterone production in rat zona glomerulosa cells. In order to obtain further evidence for the involvement of protein kinases in the short-term ACTH-stimulated aldosterone synthesis in rat zona glomerulosa cells, the effects of three different compounds with protein kinase inhibitory properties were investigated. Staurosporine, H-7 and trifluoperazine inhibited ACTH-stimulated aldosterone release in a dose-dependent manner. While the inhibitory effect of H-7 was reversible upon washing of the cells with inhibitor-free medium, the inhibition was maintained in cells treated with staurosporine or trifluoperazine. In contrast to the stimulated production, basal release of aldosterone even at the highest drug concentrations tested was not completely inhibited. We thus conclude that protein kinases may play a crucial role in short-term ACTH-stimulated aldosterone production in rat glomerulosa cells.


Phospholipids in Trypanosoma cruzi: phosphoinositide composition and turnover. The polyphosphoinositides from Trypanosoma cruzi were isolated by preparative thin-layer chromatography (TLC) and identified. When myo-[3H]inositol was present in the culture medium for five days, analyses showed the presence of phosphatidylinositol (PI), lysophosphatidylinositol (lysoPI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). Short-term incubation with 32Pi led to higher percentages of incorporation into phosphatidylethanolamine (PE), lysophosphatidylethanolamine (lysoPE) and PI compared to the other glycerophospholipids. The phosphoinositides (PI, PIP and PIP2) contained a larger proportion of unsaturated than saturated fatty acids. High proportions of 18:2 were found in the three phosphoinositides analyzed, whereas the major saturated fatty acid was 18:0. Water-soluble inositol phosphates (IP, IP2 and IP3) were also identified.


Glycolipids of a human glioma cell line bearing receptors for platelet-derived growth factor (PDGF). Glycolipids of U-1242 MG were characterized because results of previous studies showed that exogenous gangliosides, especially GM3, inhibit PDGF-stimulated growth of this human glioma cell line. GM3 and GM2 are the major gangliosides; both separate as doublets with thin-layer chromatography. The major neutral glycolipid is glucocerebroside with nonhydroxy fatty acids, but paragloboside, ceramide dihexoside, globoside, and asialoGM2 (GA2) are also present. The coexistence in U-1242 MG of these gangliosides and the PDGF receptor, whose mitogenic signal is modulated by GM3 in these cells, suggests a possible functional relationship among them with respect to growth regulation.


The influence of gender on developing copper deficiency and on free radical generation of rats fed a fructose diet. The present investigation was conducted to determine whether differences in copper and iron status between male and female rats can be detected during the development of copper deficiency. These differences may explain the protection of the female against the severity of copper deficiency. In addition, the livers of all rats were exposed to electron-spin resonance (ESR) spectroscopy for the presence of free radicals. Male and female rats were fed from weaning either copper-deficient or -adequate diets containing fructose for 31 days. Rats were killed at day 0, 8, 16, 24, and 31 of the study. Throughout the study, copper-deficient males exhibited the same organ copper concentrations as copper-deficient female rats. However, only in the male did copper deficiency cause a reduction in body weight and an increase in liver and heart sizes but a decrease in pancreas size. In contrast, organ iron concentrations were different between males and females. Only copper-deficient males were anemic. Only the livers of copper-deficient males showed the presence of free radicals. Although the livers of copper-deficient female rats exhibited higher concentrations of hepatic iron than their male counterparts, their livers did not show the presence of free radicals. The data of the present study suggest that changes in organ sizes and the severity of copper deficiency are not solely due to the total concentrations of iron and/or copper. The type of iron compound and the presence of free radicals may be involved in the pathology of copper deficiency of the male.


Effects of fasting, refeeding, and fasting with T3 administration on Na-K,ATPase in rat skeletal muscle. It is known that Na-K,adenosine triphosphatase (ATPase) in cell membranes represents an important consumer of cellular energy, eg, adenosine triphosphate (ATP), and that the concentration and activity of this enzyme change in a dose-dependent manner with serum thyroid hormone levels. To examine the hypothesis that low triiodothyronine (T3) syndrome represents a cellular adaptation in generalized severe illnesses that saves tissue energy expenditure, we measured the muscle Na-K,ATPase concentration and its activity in rats that led to low T3 syndrome induced by fasting. The Na-K,ATPase concentration was measured by 3H-ouabain binding to soleus muscle, and its activity was measured by 42K uptake in the contralateral soleus muscle. The effects of refeeding or T3 administration on Na-K,ATPase in soleus muscle in fasted rats were also examined. Na-K,ATPase concentration and activity were both increased in hyperthyroid rats and decreased in hypothyroid rats. In the fasting state, they were decreased to as low as the levels seen in hypothyroidism. Furthermore, with fasting + refeeding or fasting + T3 administration, Na-K,ATPase in soleus muscle returned to the normal level. These results suggest that tissue energy expenditure, as assessed by Na-K,ATPase, in skeletal muscles of fasted rats with low T3 syndrome is actually decreased to levels seen in hypothyroidism, due at least partly to the decrease in serum T3 concentrations, and that there exist some adaptation mechanisms in the peripheral tissues for the accommodation of energy metabolism in the body through decreased thyroxine (T4) to T3 conversion.


The role of octreotide (Sandostatin) in non-growth hormone-, non-thyroid-stimulating hormone-, and non-prolactin-secreting adenomas. Octreotide may act on non-growth hormone-, non-thyroid-stimulating hormone, and non-prolactin-secreting adenomas. Its efficacy was reported in some corticotropin-secreting adenomas from Nelson's syndrome and from Cushing's disease. In gonadotropin-secreting adenomas, octreotide was shown to be effective in two of eight cases. In nonfunctioning adenomas, visual improvement was observed with octreotide in 14 of 23 cases in a French multicenter study. Among the 33 patients whose tumor volume was checked, shrinkage occurred in seven, but an increase in tumor volume was observed in another seven patients. Mechanism(s) and prediction of efficacy of octreotide remain to be documented.


Preclinical studies on the anticancer activity of the somatostatin analogue octreotide (SMS 201-995). The antiproliferative effect of somatostatin-14 and its analogue, octreotide, on in vitro pancreatic and breast tumor cells has led to the suggestion that octreotide may have further oncological indications in addition to its use in the treatment of gastroentero-pancreatic (GEP) tumors. To extend these in vitro observations, we evaluated the effect of octreotide in rodent models of pancreatic and breast tumors. Octreotide at a dose of 5 micrograms or 50 micrograms twice a day in nude mice bearing solid MiaPaCa pancreatic tumors (subline 21) or ZR-75-1 breast tumors induced a significant inhibition of tumor growth from week 2 until the end of treatment at week 5. After 5 weeks, the mean volume of ZR-75-1 tumors in animals treated with the 50-micrograms regimen was 48% of that in controls. Autoradiographic studies showed that a high percentage (71%) of ZR-75-1 tumors were somatostatin receptor-positive. In addition, the growth of ZR-75-1 cells in vitro was significantly inhibited by octreotide. The drug was also tested in a second breast cancer model, 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in rats, and continuous administration of 10 micrograms/kg/h over 6 weeks led to an approximate 50% reduction in the number of tumors arising in the rat mammary gland. These data suggest that pancreatic and breast cancer may be among the malignant diseases clinically susceptible to octreotide.


Gene-environment interactions in atherosclerosis. The importance of environment and genetics working together to shape an individual's risk for atherosclerosis seems intuitively obvious. However, it is only recently that research strategies have begun to evolve that attempt to answer questions related to apportionment of risk that is due to specific environmental and genetic factors. These factors may impact upon risk either singly or, more likely, through a complex interaction that affects the metabolic history of the whole organism. Because the genetic bases of lipid and lipoprotein metabolism have been well-studied, and because of the epidemiologic and pathobiochemical associations between genetic disorders of lipid metabolism and atherosclerosis, researchers have searched for gene-environment interactions within animal and human systems in which the phenotype is defined by some index of lipoprotein metabolism. From work done in the lipoprotein area to this point a clear case can be made for: 1) the genetic influence over the phenotypic response to an environmental stimulus; 2) the environmental modulation of the phenotypic expression of severe genetic defects. In the realm of gene-environment interactions that affect lipoprotein phenotype, diet is the best-studied environmental factor.


Gastric inhibitory polypeptide-dependent cortisol hypersecretion--a new cause of Cushing's syndrome. Corticotropin-independent nodular adrenal hyperplasia is a rare cause of Cushing's syndrome, and the factors responsible for the adrenal hyperplasia are not known. We studied a 48-year-old woman with Cushing's syndrome, nodular adrenal hyperplasia, and undetectable plasma corticotropin concentrations in whom food stimulated cortisol secretion. Cortisol secretion had an inverse diurnal rhythm in this patient, with low-to-normal fasting plasma cortisol concentrations and elevated postprandial cortisol concentrations that could not be suppressed with dexamethasone. The cortisol concentrations increased in response to oral glucose (4-fold increase) and a lipid-rich meal (4.8-fold increase) or a protein-rich meal (2.6-fold increase), but not intravenous glucose. The infusion of somatostatin blunted the plasma cortisol response to oral glucose. Intravenous infusion of gastric inhibitory polypeptide (GIP) for one hour increased the plasma cortisol concentration in the patient but not in four normal subjects. Fasting plasma GIP concentrations in the patient were similar to those in the normal subjects; feeding the patient test meals induced increases in plasma GIP concentrations that paralleled those in plasma cortisol concentrations. Cell suspensions of adrenal tissue from the patient produced more cortisol when stimulated by GIP than when stimulated by corticotropin. In contrast, adrenal cells from normal adults and fetuses or patients with cortisol-producting or aldosterone-producing adenomas responded to corticotropin but not to GIP. Nodular adrenal hyperplasia and Cushing's syndrome may be food-dependent as a result of abnormal responsiveness of adrenal cells to physiologic secretion of GIP. "Illicit" (ectopic) expression of GIP receptors on adrenal cells presumably underlies this disorder.


Food-dependent Cushing's syndrome mediated by aberrant adrenal sensitivity to gastric inhibitory polypeptide. Some patients with Cushing's syndrome have nodular adrenal hyperplasia. In most the disease is corticotropin-dependent, but in others it is corticotropin-independent. The cause of the adrenal hyperplasia in the latter patients is not known. We studied a 49-year-old woman with Cushing's syndrome and nodular adrenal hyperplasia in whom food stimulated cortisol secretion. Plasma cortisol concentrations were measured in response to the ingestion of mixed meals, glucose, protein, and fat and after the administration of various gastrointestinal and other types of hormones. We also studied the ability of the long-acting somatostatin analogue octreotide to prevent the food-induced increase in plasma cortisol concentrations and to ameliorate the clinical manifestations of Cushing's syndrome in this patient. The patient's fasting plasma cortisol concentrations were subnormal, ranging from 3.0 to 7.5 micrograms per deciliter (83 to 207 nmol per liter), and they increased to as high as 16.5 micrograms per deciliter (455 nmol per liter) after a mixed meal. Her urinary cortisol excretion ranged from 164 to 250 micrograms per day (453 to 690 nmol per day) and could not be suppressed by a large dose of dexamethasone. Plasma corticotropin concentrations were virtually undetectable at all times. The ingestion of glucose, protein, and fat increased plasma cortisol concentrations to 3.6, 2.2, and 4 times the base-line value, respectively; the meal-induced and glucose-induced increases were inhibited by octreotide. The infusion of gastric inhibitory polypeptide (GIP) increased the patient's plasma cortisol concentration to 3.7 times the base-line value, but had no effect in normal subjects. The patient's fasting plasma GIP concentrations were normal both before and after a meal, and there was a close correlation between her plasma cortisol and GIP concentrations. Treatment with octreotide decreased urinary cortisol excretion and ameliorated the clinical manifestations of Cushing's syndrome. The development of aberrant adrenal sensitivity to GIP can result in food-dependent adrenal hyperplasia and therefore in Cushing's syndrome.


Cerebral hypermetabolism produced by intraventricular endothelin-1 in rats: inhibition by nimodipine. Injection of endothelin-1 (9 pmol) into the lateral cerebral ventricle of rats produced barrel-rotations, convulsions, tonic hindlimb extensions, facial clonus, and kinetic ataxia for up to 45 min. Quantitative metabolic autoradiographs produced from the [14C]deoxyglucose method and analyzed over 60 individual brain structures or subregions revealed intense hypermetabolism in periventricular tissues close to the injection site and in many of their efferent projection sites. Histological examination of these areas proved that this dose of endothelin was without toxic or ischemic effects on neurons or glial cells. Structures metabolically affected ipsilateral to injection were caudate nucleus (+164%), lateral septal nucleus (+270%), and two white matter tracts--corpus callosum (+236%) and hippocampal fimbria (+318%). Distant stimulated structures included cerebellar cortical layers, but not cerebellar nuclei or white matter. Increased rates of glucose metabolism among many other nuclei, particularly distinct subunits of the hippocampal formation and structures in contact with the ventricular system, signify that endothelin induced widespread metabolic stimulation over much of the neuraxis. Furthermore, although the 9 pmol concentration of endothelin produced convulsive movements and diverse metabolic stimulation, it did not evoke detectable electroencephalographic seizure activity assessed by intra- or extracerebral electroencephalography. Both the convulsions and hypermetabolic activation were inhibited by intraventricular pretreatment with the dihydropyridine calcium-channel antagonist, nimodipine. The results identify endothelin-1 as a calcium-mediated 'convulsive' peptide with selective stimulatory effects on cerebral glucose metabolism.


Endothelial cell antigens recognized by IgA autoantibodies in patients with IgA nephropathy: partial characterization. Antibodies directed against endothelial cells (AECA) have been described in IgA nephropathy. We have previously measured AECA of the IgA subclass (AECA-IgA) in the sera of patients with glomerulonephritis by an ELISA method, and have shown that patients with IgA nephritis (IgA N) and lupus nephritis (LN) had significantly greater AECA-IgA activity compared with normal controls [1]. In the current study, on Western blotting of the EC membrane components, 6 of 18 (33%) IgA N sera positive for AECA-IgA bound to molecules of 135 and 116 kDa and 10 of 18 (56%) to a molecule of 205 kDa. These EC membrane components recognized by AECA-IgA may be of importance in the pathogenesis of IgA nephropathy.


Erythropoietin for anaemia in haemodialysis patients: results of a maintenance study (the Canadian Erythropoietin Study Group). Most published studies of recombinant human erythropoietin (rHuEpo) have been of limited duration and in small patient populations. The present study examines the long-term effects of rHuEpo in 98 haemodialysis patients treated for up to 18 months. All patients had completed a 6 month placebo-controlled study of rHuEpo. Patients previously on placebo (group 1; n = 31) received rHuEpo at an initial dose of 50 U/kg thrice weekly with subsequent dose adjustments to maintain haemoglobin (Hb) in the range 105-120 g/l. Patients previously on rHuEpo (group 2; n = 67) continued on their usual dose with adjustments made to maintain Hb at 105-120 g/l. Haematological parameters were measured every 2 weeks. Quality of life, assessed by a disease-specific kidney disease questionnaire (KDQ), was measured every 6 months. Mean Hb in group 1 increased from 74.2 +/- 11.4 g/l at baseline to 112.9 +/- 12.6 g/l after 12 months of rHuEpo therapy. After 12 weeks of rHuEpo therapy Hb in groups 1 and 2 was indistinguishable. Hb remained constant in both groups throughout the period of follow-up. Mean rHuEpo dose requirements were similar in both groups. At the end of the study the mean intravenous rHuEpo dose in group 1 patients was 176.6 +/- 154.4 and in group 2 patients was 210.0 +/- 144.0 U/kg per week. Access failure was increased during rHuEpo therapy in patients with synthetic grafts (46% versus 7% failures compared to fistulae; P less than 0.001). Group 1 patients receiving rHuEpo had a significant increase in diastolic but not systolic blood pressure despite a 32% increase in overall antihypertensive prescriptions.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of recombinant human erythropoietin on erythropoiesis in homozygous sickle-cell anaemia and renal failure. The development of end-stage renal disease (ESRD) in patients with sickle-cell anaemia results in increased transfusion dependence, increasing the risk of iron overload. Correction of anaemia with recombinant human erythropoietin (rHuEpo) in dialysis patients might also result in stimulation of haemoglobin F production, which protects against sickling, although very high doses were required to achieve this effect in non-uraemic animals. rHuEpo was administered to three transfusion-dependent patients with ESRD and homozygous sickle-cell disease (initial dose 100 U/kg twice weekly, increasing to 125 U/kg at 6 weeks, and to 150 U/kg at 9 weeks in two patients). This resulted in reticulocytosis and increased circulating erythroid blast-forming units. Total haemoglobin was predominantly HbA (i.e. transfused blood) at the start of the study, reflecting transfusion dependence, but after 3 months' treatment was between 60 and 94% HbS. No sickling crises occurred. Haemoglobin F remained at less than 3% of total haemoglobin. One patient was withdrawn at 10 weeks with CAPD peritonitis. The other two patients completed 12 weeks' treatment without transfusion but final Hb concentrations were 4.5 and 5.5 g/dl. Whether larger doses of rHuEpo will be more successful in managing such patients remains unclear. No effect on HbF production can be expected.


Effect of chronic intravenous calcitriol on parathyroid function and set point of calcium in dialysis patients with refractory secondary hyperparathyroidism. This study evaluates the effect of intravenous calcitriol on parathyroid function and ionized calcium-PTH sigmoidal curve obtained during low- and high-calcium haemodialysis in 10 patients with osteitis fibrosa whose secondary hyperparathyroidism was refractory to conventional therapy. After 4 months of intravenous calcitriol, serum ionized calcium increased from 1.28 +/- 0.08 to 1.37 +/- 0.11 mmol/l (P less than 0.001), serum phosphate from 1.54 +/- 0.18 to 1.79 +/- 0.4 mmol/l (P NS), serum calcitriol from 16.7 +/- 9.9 to 34.3 +/- 6.4 pg/ml (P less than 0.001), while alkaline phosphatase decreased from 366 +/- 340 to 226 +/- 180 IU/l (P less than 0.05), osteocalcin from 46.4 +/- 20 to 34.5 +/- 15.3 ng/ml (P less than 0.05), and basal intact PTH from 1069 +/- 700 to 305 +/- 270 (P less than 0.01). Basal PTH started to decrease after 1 month of treatment prior to the increase in the ionized calcium. Because of hypercalcaemia the dialysate calcium was decreased from 1.75 to 1.5 mmol/l in three of five patients on haemodialysis, and calcium-containing solutions were replaced by calcium-free fluids in four of five patients on haemodiafiltration. Calcitriol dose, at the first month of therapy was 5.6 +/- 0.8 micrograms/week, but it was successively decreased because of hypercalcaemia to a final dose of 3.6 +/- 1.3 micrograms/week. After intravenous calcitriol the ionized calcium-PTH sigmoidal curve shifted to the left and downward. Maximally stimulated PTH and maximally inhibited PTH obtained during low- and high-calcium dialysis significantly decreased, as well as the ratio of basal PTH/PTHmax and the set point of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)


'Pulse' oral calcitriol in uraemic patients: rapid modification of parathyroid response to calcium. We performed dynamic parathyroid function tests, with generation of parathyroid hormone/ionized calcium (PTH/iCa) response curves, obtained during sequential hypercalcaemic and hypocalcaemic dialysis, on six haemodialysis patients before and after 4-weekly 'pulse' doses of oral calcitriol. There were no significant changes in the pretreatment, 2-week and 4-week values of blood ionized calcium, serum total calcium, phosphate, and alkaline phosphatase. PTH decreased significantly after 4 weeks of treatment with calcitriol (P less than 0.03), and the post-treatment PTH/iCa curves were all displaced leftwards and downwards, indicating a marked inhibitory effect of calcitriol on parathyroid responsiveness across and beyond the physiological range of calcaemia. The mean shift of the PTH/iCa relationship was equivalent to a reduction of iCa of 0.2 mM for any given concentration of PTH, and conversely to a 70% reduction of PTH for any given blood iCa. The results suggest that wide spacing of oral 'pulse' doses of calcitriol can achieve favourable modification of the PTH/iCa relationship, and that dosing interval and dose size, rather than route of administration, may be the major determinants of the previously reported superiority of intravenous over daily oral calcitriol regimens.


Total and regional bone densities in dialysis patients. Total and regional bone mineral densities (BMD) of ten male haemodialysis (HD) patients and ten male patients on continuous ambulatory peritoneal dialysis (CAPD) were measured using dual-energy X-ray absorptiometry (DEXA), and compared with that of age- and sex-matched controls. Our data showed that patients with renal failure on dialysis had reduced bone densities as manifested by a reduction in total body BMD, femoral neck BMD, and Ward's triangle BMD. In addition, head BMD and femoral trochanter BMD were also reduced in HD patients. Among HD patients, the length of the period of dialysis correlated with serum level of parathyroid hormone and the reductions in total body BMD and head BMD. Furthermore, there was a strong negative correlation between bone density of the skull and serum parathyroid hormone. Our results demonstrated regional variations in the reduction of bone density in patients with asymptomatic renal bone disease. DEXA bone scan is a useful adjunct in the early assessment of renal osteodystrophy and bone density of the skull can be used as a monitor in hyperparathyroid bone disease.


Sonographic evaluation of gallbladder motility in patients with end-stage renal disease on haemodialysis. Thirty nine patients with end-stage renal disease on haemodialysis therapy with upper gastrointestinal symptoms were investigated for gallbladder motor function as the cause of their symptoms using cholecystosonography in fasting state, after a cholecystokinetic agent (BILOPTIN fatty meal) and after a smooth muscle relaxant (BUSCOPAN). Forty healthy individuals served as a control group. No significant difference was found between dialysis patients and healthy controls regarding the gallbladder area during fasting state, or the variation in the area of gallbladder during maximal contraction and dilatation. However, the patients on dialysis therapy of longer duration had stronger gallbladder contraction in response to a cholecystokinetic agent. Serum gastrin concentrations were increased in haemodialysis patients, but there was no consistent relationship between serum gastrin and gallbladder motility. The upper gastrointestinal symptoms of dialysis patients are unlikely to be due to disturbed gallbladder motility.


Low-molecular-weight protein-permeable cuprammonium rayon haemofilter. Searching for a dialyser membrane with a cut-off similar to that of the human glomerulus, a modified cuprammonium rayon (AM-75-UP) and a polyacrylonitrile (PAN-15-DX) haemofilter were tested in vivo for the ability to eliminate substances of a molecular weight (MW) of 10-65 kilodaltons (kDa). Endogenous marker substances of a defined MW (beta-2-microglobulin 11.8 kDa; retinol binding protein 21 kDa; alpha-1-microglobulin 26.7 kDa; alpha-1-glycoprotein 41 kDa; alpha-1-antitrypsin 54 kDa; albumin 66.3 kDa) were measured by laser nephelometry or immunosorbent assay; sieving coefficients (SC) and protein elimination (PE) per 20 l haemofiltration were calculated for each low-MW protein. The PAN haemofilter shows elimination characteristics comparable to those of earlier tested haemofilters (polysulphone, AN69, cellulose triacetate) with a sharp cut-off in the MW range of 10-15 kDa. The cuprammonium rayon haemofilter is permeable for proteins with a higher MW; especially for alpha-1-microglobulin a relevant removal (SC 0.2; PE 0.56 g/20 l) was established. This membrane has a cut-off more similar to that of the human glomerulus; but far from the demanded quality with a relevant removal of substances in the MW range up to 60 kDa. Calculated albumin loss (2.4 +/- 0.2 g/20 l) seems to be tolerable for stable haemodialysis patients.


Pharmacokinetics of ciprofloxacin and vancomycin in patients with acute renal failure treated by continuous haemodialysis. To determine appropriate doses of ciprofloxacin and vancomycin for septic patients with acute renal failure (ARF) treated by continuous arteriovenous and venovenous haemodialysis, (CAVHD/CVVHD), we performed pharmacokinetic studies in patients receiving these antibiotics. All patients were treated by CAVHD/CVVHD using Hospal AN69S 0.43 m2 filters and Fresenius 1.5% peritoneal dialysis fluid at dialysate flow rates (Qd) of 1 and 2 l/h. Patients received ciprofloxacin 200 mg i.v. 12-hourly (n = 6) or 8-hourly (n = 5); vancomycin 1 g i.v. was administered to 10 patients approximately every 48 h to maintain therapeutic plasma levels. For ciprofloxacin, volume of distribution (Vdarea) was 136.5 +/- 9.81, terminal elimination half-life (t1/2) 6.4 +/- 0.8 h, and total body clearance (TBC) 264.3 +/- 22.9 ml/min (mean +/- SEM). Mean sieving coefficient (S/C) was 0.76 +/- 0.05 and filter clearances at Qd 1 and 2 l/h were 16.2 +/- 1.9 and 19.9 +/- 1.1 ml/min respectively. For vancomycin, Vdarea was 60.7 +/- 5.11, t1/2 24.7 +/- 2.6 h and TBC 31.0 +/- 4.6 ml/min. Mean S/C was 0.66 +/- 0.08 and filter clearances at Qd 1 and 2 l/h 12.1 +/- 2.0 and 16.6 +/- 2.0 ml/min. These data suggest that patients with ARF treated by CAVHD/CVVHD should be given ciprofloxacin 200 mg i.v. 8-12-hourly and vancomycin every 48 h.


Endoscopic placement of CAPD catheters: a review of one hundred procedures. One hundred consecutive endoscopically placed peritoneal dialysis catheters inserted in 95 patients over an 18-month period have been reviewed. All catheters were placed for chronic dialysis (CAPD). Following insertion there were five early catheter failures (4 failed to drain, 1 perforated viscus) and 13 early complications (7 leaks, 3 tunnel bleeds, 2 scrotal oedema, 1 wound infection). In the long term six patients required transfer to haemodialysis (2 recurrent peritonitis, 2 pain on outflow, 1 unable to cope, 1 persistent vomiting). Overall probability of catheter survival as predicted by Kaplan-Meier analysis was 0.85 at 18 months. These results confirm that endoscopic placement of CAPD catheters is safe and reliable. In addition there is a low early failure rate and the long-term catheter survival figure is comparable with the best series reported. This procedure allows direct visualization of the peritoneal cavity, thus minimizing the risk of visceral damage. Furthermore, the procedure is well tolerated under local anaesthesia and allows early institution of dialysis because of the extremely low leakage rate (11%). Endoscopic placement of CAPD catheters is now the procedure of choice in our centre. General anaesthetic and mini-laparotomy are thus avoided in most of this high-risk group.


Impact of schistosomiasis on patient and graft outcome after kidney transplantation. In this work the impact of schistosomiasis on kidney transplantation was investigated by comparing two groups of patients, group 1 (Schistosoma-infected cases) and group 2 (control cases). In group 1, schistosomiasis was diagnosed in both donor and recipient in 63 cases, in recipient only in 65 cases, and in donor only in eight cases. Schistosomal infection among kidney transplant recipients was S. haematobium in 17 cases, S. mansoni in 58 cases, and mixed in 53 cases. Schistosomiasis was diagnosed by finding Schistosoma eggs in urine, stools, rectal mucosal biopsy, recipient bladder mucosal biopsy, or in the donor ureter obtained during surgery. Patients and donors with active lesions were treated at least 3 weeks before transplantation by the antischistosomal drugs praziquantel and oxamniquine. Follow-up after kidney transplantation showed no significant difference between the two groups regarding the incidence of acute and chronic rejection. Nevertheless, dose of cyclosporin, HBs antigenaemia, incidence of urinary tract infection, renal stones, ureteric stricture, and urinary leakage were significantly greater among schistosomal patients when compared to control cases. Schistosomal reinfection was observed in 23% of cases at high risk. Antischistosomal treatment did not affect the graft function. We have concluded that schistosomiasis may affect the outcome of kidney transplantation.


Renal allograft rupture: diagnostic role of ultrasound. To evaluate the diagnostic role of ultrasound in spontaneous renal allograft rupture we reviewed 18 cases observed in our centre in 10 years. Ultrasound studies were performed immediately before surgery in 15 cases. Renal allograft rupture occurred during the first 3 weeks after transplantation in 17 cases (94%). Clinical findings were consistent with previous reports. The diagnosis was confirmed by surgical exploration in 17 cases, and by necropsy in the remaining one. Nine patients were treated by corsetage and eight by graft nephrectomy, while one patient died before surgery. Acute rejection was present in nine cases, and severe acute tubular necrosis in five; no renal tissue was available for histological study in four patients. On ultrasound examination, perirenal haematoma was the most frequent finding, while subcapsular/intrarenal haematoma or findings suggesting rejection or urinary tract obstruction were less frequently observed. In six cases, disruption of the white linear echoes of the capsule of the graft could be seen; this finding has not been described previously. Ultrasound has a definite role in presurgical evaluation of suspected renal transplant rupture.


Efficacy of hormone replacement therapy in postmenopausal women with oral discomfort. The increase in incidence of oral discomfort among women in a menopause is probably due to hormone modifications. This study evaluated the efficacy of hormone replacement therapy in 27 postmenopausal patients, aged 48 to 58 years, with oral discomfort and no local irritants and in 47 postmenopausal women with no oral discomfort. Patients were treated with conjugated estrogens for 21 days and medroxyprogesterone acetate from day 12 through day 21. Hormone-replacement therapy had no effect on oral cytology in the 40 symptom-free postmenopausal women compared with a group of 47 postmenopausal women who had no oral symptoms and were not treated. Hormone-replacement therapy relieved symptoms and improved oral cytohormonal features in 15 of 27 patients with symptoms. Nuclear estrogen receptors were found by immunohistochemical assay in 8 of 10 randomly selected patients with symptoms who responded to hormone-replacement therapy, but not in 2 patients who did not benefit from hormone-replacement therapy. Estrogen receptors were also found in 6 of 10 fertile women with no oral disease. Our results suggest that oral discomfort may be related to steroid hormone withdrawal only in some postmenopausal women and that replacement therapy may improve the clinical picture and cytologic features in this group of patients. Immunohistochemical identification of estrogen receptors may help to identify patients for whom hormone-replacement therapy may be beneficial.


Oral leishmaniasis associated with kala-azar. A case report. Mucosal leishmaniasis as an oral disease in the form of chronic periodontitis with involvement of the oral mucosa is described. Leishmania parasites were isolated from the oral lesions, lymph nodes, and bone marrow. The patient had a low-grade fever and hepatosplenomegaly that regressed along with the oral lesions after treatment with stibogluconate sodium.


[Cortical dysplasia as an epileptogenic focus: reduced binding of 123I-iomezanil with barely perceptible 99mTc-HMPAO SPECT]. In cortical areas associated with an epileptic focus, there is, in the majority of cases, a correlation between decreased benzodiazepine (Bz) receptor binding, detectable with 123I-Iomazenil, and decreased blood flow. A patient with focal epilepsy due to a localized abnormality in cortical differentiation is presented who showed a marked focal decrease in Bz receptor binding and a normal 99mTc-HMPAO SPECT. Based on this case the relation between blood flow and Bz receptor binding in epileptic foci is discussed.


Treatment and prophylaxis of cytomegalovirus disease. The diseases caused by cytomegalovirus (CMV) may threaten the life or sight of immunocompromised individuals such as patients undergoing transplantation and those with the acquired immunodeficiency syndrome. The management of CMV disease can be difficult. The antiviral agents ganciclovir and foscarnet are effective against CMV retinitis and gastrointestinal diseases, although dose-limiting adverse effects and the need for long-term maintenance therapy may hinder their use in many patients. When used to treat CMV pneumonitis in bone marrow transplant recipients, ganciclovir alone is not as effective as when it is combined with immune globulin. Since CMV disease can be fatal, several protocols have been developed for the transplant patient population, including administration of acyclovir, ganciclovir, screened blood products, and immune globulins.


Neuropeptide Y interaction with the adrenergic transmission line: a study of its effect on alpha-2 adrenergic receptors. Neuropeptide Y (NPY), first isolated in 1982, is widely distributed among the neurons of the central and peripheral nervous systems, often in close association with catecholamines. Because of its wide distribution and concentrations in selected areas of the brain, NPY is considered a putative neurotransmitter with several possible physiological effects including modulation of blood pressure, food intake and pituitary hormone release at a central level. Peripherally, the peptide seems to be involved, via direct and indirect mechanisms, in noradrenaline (NA)-mediated vasoconstriction. The ability of NPY to interact with the catecholamine transmission line may underly a possible modulatory influence of NPY on catecholamine receptor characteristics. We recently observed interaction between alpha-2 adrenergic receptors and those for NPY at the presynaptic level. Additional data from our studies in spontaneously hypertensive rats suggest that impairment of these interactions may contribute to the hypertension in this strain.


The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution. The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.


Tn10 insertion specificity is strongly dependent upon sequences immediately adjacent to the target-site consensus sequence. Transposon Tn10 inserts preferentially into particular "hotspots" that have been shown by sequence analysis to contain the symmetrical consensus sequence 5'-GCTNAGC-3'. This consensus is necessary but not sufficient to determine insertion specificity. We have mutagenized a known hotspot to identify other determinants for insertion into this site. This genetic dissection of the sequence context of a protein binding site shows that a second major determinant for Tn10 insertion specificity is contributed by the 6-9 base pairs that flank each end of the consensus sequence. Variations in these context base pairs can confer variations of at least 1000-fold in insertion frequency. There is no discernible consensus sequence for the context determinant, suggesting that sequence-specific protein-DNA contacts are not playing a major role. Taken together with previous work, the observations presented suggest a model for the interaction of transposase with the insertion site: symmetrically disposed subunits bind with specific contacts to the major groove of consensus-sequence base pairs, while flanking sequences influence the interaction through effects on DNA helix structure. We also show that the determinants important for insertion into a site are not important for transposition out of that site.


Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme. Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli. These cDNAs differed in size but exhibited a common overlapping DNA sequence. Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa. The amino acid sequence of this protein exhibits 35% identity with the E. coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme. The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E. coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes. A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases. Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species. All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues. Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems. We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.


A region of herpes simplex virus VP16 can substitute for a transforming domain of Epstein-Barr virus nuclear protein 2. Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for EBV-induced B-cell transformation in vitro. EBNA-2 contains a 14-amino acid domain that directly activates transcription and is required for transformation. To determine whether another transcriptional activator can substitute for this function, a chimeric virus was constructed that contained a portion of the transcriptional activation domain from the herpes simplex virus VP16 protein inserted in place of the 14-amino acid domain of EBNA-2. The chimeric virus was able to transform B cells efficiently and transactivate expression of EBV and B-cell genes. Randomization of the 14-amino acid sequence in the domain markedly reduced its transcriptional activating activity and the transforming efficiency of the recombinant EBV. Mutation of a tryptophan within the 14-amino acid domain of EBNA-2 completely abolished transcriptional activation and B-cell transformation. These experiments indicate that EBNA-2 and VP16 activate transcription by similar mechanisms and that transcriptional activation is required for EBV-induced B-cell transformation.


Expression of a family of complementary-strand transcripts in Epstein-Barr virus-infected cells. A major family of polyadenylylated cytoplasmic transcripts are expressed from the BamHI A-I region of the Epstein-Barr virus genome, off the strand complementary to that encoding several functions associated with viral replication and the lytic cycle, including the DNA polymerase (BALF-5). These complementary-strand transcripts (the main one is about 4.8 kilobases long), expressed in all cell types associated with Epstein-Barr virus, are present at high levels in nasopharyngeal carcinoma tumors. Sequence analysis of clones that correspond to spliced transcripts in a cDNA library from such a tumor, C15, generates a profile of the main complementary mRNA. It contains at least three AUG-initiated open reading frames, the largest of which could be translated to give a polypeptide of about 20 kDa. Evidence from several types of experiments suggests that conditions which support the up (or down) regulation of transcriptional expression from one viral DNA strand within the relevant region of the genome produce the opposite effect on transcripts from the other strand. The capacity for interference between complementary Epstein-Barr viral transcripts offers a mechanism for control of gene expression that may be related to maintenance of viral latency.


Single amino acid substitutions in "low-risk" human papillomavirus (HPV) type 6 E7 protein enhance features characteristic of the "high-risk" HPV E7 oncoproteins. HPV types associated with genital disease are termed "high-risk" or "low-risk" viruses according to their prevalence in cancers. Two viral genes, E6 and E7, are invariably expressed in cervical carcinomas. The E7 gene product has been found to bind the retinoblastoma tumor suppressor protein and to be phosphorylated by casein kinase II. Although present in both high- and low-risk E7 proteins, these activities are diminished in the low-risk HPV-6 E7 polypeptide. To better understand the oncogenic potential of the HPV-6 E7 protein, we replaced four of its amino acids with HPV-16 E7 residues present in the analogous region of the N-terminal half of the protein. Replacement of the arginine at position 4 of the HPV-6 E7 protein with an aspartate present in HPV-16 E7 slowed the mobility of the protein when expressed in vivo. Replacement of the glycine at position 22 with an aspartate resulted in higher affinity for retinoblastoma protein binding. Replacement of valine residues at positions 30 and 37 with asparagine and aspartate, respectively, resulted in higher levels of casein kinase II phosphorylation. The substitution at position 22 was the only mutation that exhibited increased transforming activity, suggesting a correlation between the HPV E7 protein affinity for the retinoblastoma tumor suppressor protein and its ability to transform established cells. Our results show that subtle changes in sequence may result in marked differences in biological activity of HPV oncogenes.


S-nitrosylation of tissue-type plasminogen activator confers vasodilatory and antiplatelet properties on the enzyme. Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing factor (EDRF) by way of the enzyme's single free sulfhydryl (Cys-83) to form a stable S-nitrosothiol protein adduct. S-nitrosylation endows t-PA with potent vasodilatory and antiplatelet properties that are accompanied by elevations in intracellular cyclic GMP analogous to those induced by low molecular weight (e.g., S-nitroso amino acid) S-nitrosothiols. Moreover, this chemical modification does not adversely affect the catalytic efficiency of t-PA, the fibrin stimulation of this activity, the binding of t-PA to fibrinogen, or the interaction of the enzyme with its physiologic serine protease inhibitor, plasminogen-activator inhibitor type I. The coupling of vasodilatory, antiplatelet, and fibrinolytic properties in one molecule makes the S-nitrosylated t-PA a unique molecular species and may provide insight into the mechanisms by which the endothelium maintains vessel patency. These data also suggest a pharmacologic approach to treatment of thromboocclusive disorders.


Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], plays an important role in the regulation of mineral ion homeostasis. As well as being the major steroid hormone that regulates calcium metabolism, 1,25(OH)2D3 suppresses transcription of the gene encoding parathyroid hormone, a peptide that plays a dominant role in regulating extracellular calcium levels. To identify DNA sequences that may mediate this transcriptional repression, nuclear extracts containing the 1,25(OH)2D3 receptor were examined for binding to sequences in the 5'-flanking region of the human parathyroid hormone gene. A 25-base-pair (bp) oligonucleotide containing the sequences from -125 to -101 from the start of exon I binds nuclear proteins recognized by monoclonal antibodies against the 1,25(OH)2D3 receptor. The sequences in this region contain a single copy of a motif (AGGTTCA) homologous to the motifs repeated in the up-regulatory 1,25(OH)2D3-response elements. When placed upstream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediate transcriptional repression in response to 1,25(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. This down-regulatory element, therefore, differs from the up-regulatory 1,25(OH)2D3-response elements both in sequence composition and in the requirement for particular cellular factors other than the 1,25(OH)2D3 receptor for repressing transcription.


A mitogenic peptide amide encoded within the E peptide domain of the insulin-like growth factor IB prohormone. We have identified an amino acid sequence within the E peptide of the insulin-like growth factor IB (IGF-IB) precursor that is biologically active and designated this peptide insulin-like growth factor IB-(103-124) E1 amide (IBE1). Its existence was predicted by a flanking Gly-Lys-Lys-Lys, a signal sequence for sequential proteolytic cleavage and peptidyl C-terminal amidation. A synthetic analog of the predicted IBE1 peptide, designated Y-23-R-NH2, was generated with tyrosine added at position 0. This peptide at 2-20 nM had growth-promoting effects on both normal and malignant human bronchial epithelial cells. Y-23-R-NH2 bound to specific high-affinity receptors (Kd = 2.8 +/- 1.4 x 10(-11) M) present at 1-2 x 10(4) binding sites per cell. Ligand binding was not inhibited by recombinant insulin or recombinant IGF-I. Furthermore, a monoclonal antibody antagonist to the IGF-I receptor (alpha IR3) did not suppress the proliferative response induced by Y-23-R-NH2. In addition, C-terminal amidation was shown to be important in receptor recognition since the free-acid analog of IBE1 (Y-23-R-OH) did not effectively compete for binding and was not a potent agonist of proliferation. Immunoblot analysis of human lung tumor cell line extracts using an antibody raised against Y-23-R-NH2 detected a low molecular mass band of approximately 5 kDa, implying that a protein product is produced that has immunological similarity to IBE1. Extracts of human, mammalian, and avian livers analyzed on an immunoblot with the anti-Y-23-R-NH2 antibody contained proteins of approximately 21 kDa that were specifically recognized by the antiserum and presumably represent an IGF-I precursor molecule. This implies that in species where an IGF-I mRNA with homology to the human IGF-IB E domain has not yet been described, an alternate mRNA must be produced that contains a sequence similar to that of the midportion of the human IGF-IB E domain. Our findings demonstrate that IBE1 is a growth factor that mediates its effect through a specific high-affinity receptor and is most likely conserved in many species.


Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification. Gene amplification is characteristic of tumors and continuous cell lines but not of primary, normal, diploid, senescing cells. However, the rat cell line REF52, which resembles primary cells in requiring expression of cooperating oncogenes for transformation, is unusual among cell lines as it is not permissive for amplification. REF52 cells did not form colonies in N-(phosphonacetyl)-L-aspartate (PALA), a drug for which the only known mechanism of resistance is amplification of the carbamoylphosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) gene. Colonies did form in a low concentration of methotrexate but did not contain amplified dihydrofolate reductase genes. Expression of two cooperating oncogenes in REF52 cells converted them to a state permissive for amplification. Cells expressing only the 12S E1A mRNA of adenovirus 5 did not give rise to PALA-resistant colonies, but expression of an activated ras gene together with E1A readily allowed the cells to form resistant colonies in which the CAD gene was amplified. Cells expressing E1A plus ras were fully transformed, but expression of simian virus 40 large tumor antigen alone converted REF52 cells to a state permissive for amplification without transforming them fully. The ability to manipulate gene amplification in REF52 cells by expression of oncogenes should contribute to an understanding of the nature of the permissive state.


Brain vascular endothelial cells express JC virus large tumor antigen in immunocompetent and cyclophosphamide-treated hamsters. When injected intracerebrally into newborn hamsters, the human polyomavirus JC virus (JCV) establishes a nonproductive infection resulting in brain tumor formation. Using immunostaining methods to detect the JCV regulatory protein, large tumor antigen (T antigen), we have now demonstrated JCV infection of brain vascular endothelial cells (EC) in infected hamsters. JCV T antigen was detected in lectin-labeled EC as well as in von Willebrand factor-expressing EC in both cyclophosphamide-treated and nonimmunosuppressed hamster brains 16, 21, and 31 days after birth. Cyclophosphamide-treated hamsters exhibited a greater number of JCV-infected EC, whereas T-antigen expression in nonvascular cells was not affected. The influence of cyclophosphamide was most pronounced in the cerebellum where increased numbers of JCV-infected EC were located predominantly at the internal granular layer-white matter junction, also a prominent location for T-antigen-expressing neoplastic foci. The hamster model demonstrates in vivo infection of EC by a human polyomavirus and directs interest toward the role of these cells in human JCV infection.


Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. A cDNA encoding the murine Ah receptor (Ahb-1 allele for aromatic hydrocarbon responsiveness) has been isolated and characterized. Analysis of the deduced protein sequence revealed a region with similarity to the basic region/helix-loop-helix (BR/HLH) motif found in many transcription factors that undergo dimerization for function. In addition to the BR/HLH domain, the N-terminal domain of the Ah receptor has extensive sequence similarity to the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein and two regulatory proteins of Drosophila, Sim and Per. Photoaffinity labeling and peptide mapping studies indicate that the Ah receptor binds agonist at a domain that lies within this conserved N-terminal domain. The Ah receptor appears to be a ligand-activated transcription factor with a helix-loop-helix motif similar to those found in a variety of DNA-binding proteins, including Myc and MyoD.


Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain. A cDNA library derived from human cerebral cortex was screened for the presence of sodium channel alpha subunit-specific clones. Ligation of three overlapping clones generated a full-length cDNA clone, HBA, that provided the complete nucleotide sequence coding for a protein of 2005 amino acids. The predicted structure suggests four homologous repeats and exhibits greatest homology and structural similarity to the rat brain sodium channel II. A second cDNA clone, HBB, that encodes a different subtype of sodium channel was isolated. Hybridization of DNA fragments from the 3' untranslated region of HBA and PCR with primers derived from HBB with human-hamster somatic cell hybrids localized these clones to human chromosome 2. In situ hybridization to human metaphase chromosomes mapped the structural genes for both HBA and HBB sodium channels to chromosome 2q23-24.3. The sodium channel HBA gene product was expressed by transfection in CHO cells. Expressed HBA currents were voltage-dependent, sodium-selective, and tetrodotoxin-sensitive and, thus, exhibit the biophysical and pharmacological properties characteristic of sodium channels.


Regulation of PACE propeptide-processing activity: requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. PACE (paired basic amino acid cleaving enzyme) is a subtilisin-like serine protease involved in processing of propeptides in the constitutive secretory pathway. We here demonstrate that the transmembrane and cytoplasmic domains of PACE are required for retention in the secretory pathway but not for propeptide-cleaving activity. Addition of the endoplasmic reticulum retention signal Lys-Asp-Glu-Leu (KDEL) to the carboxyl terminus of the truncated molecule resulted in intracellular retention of the protein and loss of activity, indicating that the endoplasmic reticulum is an inappropriate environment for propeptide processing. In addition, mutation of a consensus PACE cleavage site within the amino-terminal region prevented processing of PACE to a mature form and destroyed activity. These data indicate that PACE is synthesized as a proprotein which requires autoproteolytic removal of an 81-residue pro sequence for optimal activity. A mutant form of PACE that lacked the pro sequence was nonfunctional, and addition of a pro sequence from a homologous subtilisin-like serine protease, PC2, did not restore activity. By analogy to the bacterial subtilisin family, the propeptide of PACE may guide the folding of PACE into an active enzyme.