A simple and inexpensive method to control oxygen concentrations within physiological and neoplastic ranges.

Traditional methods for regulating oxygen concentration ([O2]) in in vitro experiments over the range found in normal and tumor tissues require the use of expensive equipment to generate controlled gas atmospheres or the purchase of a range of gas cylinders with certified O2 percentages. Here we describe a simple and inexpensive enzymatic method for generating low, precise steady-state [O2] levels that are stable for several hours. This method is particularly applicable to the in vitro study of some classes of hypoxia-targeted antitumor prodrugs and bioreductively activated agents.

pH-dependent bending in and out of purple membranes comprising BR-D85T.

The light-driven proton pump bacteriorhodopsin (BR) embedded in a purple membrane (PM) from Halobacterium salinarum undergoes a series of conformational changes while transporting a proton from the cytoplasmic to the extracellular side over the course of the so-called photocycle. Wild-type BR variant D85T, where aspartic acid 85 is replaced by threonine, allows for the study of structural intermediates of this photocycle that are formed in a light-dependent manner in the wild-type and in thermal equilibrium by tuning the pH of the D85T purple membrane suspension. Especially the last and least studied O-intermediate of the photocycle of bacteriorhodopsin has caught recent attention. First AFM images of D85T under acidic conditions resembling wild-type BR under physiological conditions in the O-photocycle-intermediate are presented. Bacteriorhodopsins embedded in the strongly bent purple membranes were analyzed by single molecule force spectroscopy (SMFS) providing the first single molecule force spectra of BR in the O-intermediate. SMFS was further employed to determine the absolute sign of membrane curvature. Complementary electrostatic force microscopy (EFM) was performed to support PM side discrimination and determination of the bending direction. Bending of PM-D85T was analyzed in more detail providing further insight into the structure-function relationship of the bacteriorhodopsin proton pump as well as PM behaviour at the solid-liquid junction. Findings reported here are of general interest to the field of chemomechanical transducers.

Bending of purple membranes in dependence on the pH analyzed by AFM and single molecule force spectroscopy.

The first AFM images of strongly bent purple membranes as well as the first single molecule force spectra of bacteriorhodopsins embedded therein are presented. AFM images of purple membranes to date always showed a flat membrane topology. Bacteriorhodopsin variants like BR-D85N and BR-D85T resemble an intermediate state of wild-type BR which is slightly 'wedge'-shaped. Due to the strong interaction within the 2-D crystalline lattice of the purple membrane, the geometrical anisotropy of the individual bacteriorhodopsins adds up to a macroscopic change in the geometry of the purple membranes. Instead of being flat they appear like domes at physiological conditions. Single molecule force spectroscopy was employed to determine the absolute sign of the membrane curvature. As the bacteriorhodopsins in the center of the dome-like purple membranes are not supported by any solid state substrate, the presented force spectra are the first of non-supported bacteriorhodopsin, resembling the natural occurrence in the halobacterial cell.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): I. Direct inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic species generated by decomposition.

PURPOSE: To investigate the interaction of the electrophilic species generated by the decomposition of the antineoplastic prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M) on the ability of O(6)-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine DNA lesions. MATERIALS AND METHODS: The contributions of inhibitory electrophilic species generated from VNP40101M towards AGT was assessed using analogues that selectively generated either the chloroethylating or the carbamoylating components of VNP40101M. The activity of AGT was determined from the inhibition of crosslink formation from O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine lesions. The half-lives of sulfonylhydrazine derivatives and isocyanates were measured using an acidification assay which gives a change in absorbance proportional to the release or consumption of small quantities of protons. RESULTS: Both of the reactive components produced by VNP40101M directly inactivated cloned human AGT; the carbamoylating moiety (IC(50) about 13 micro M) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC(50) about 100 micro M). These inhibitory actions were moderated by the addition of naked T5 bacteriophage DNA. Thus, AGT bound to DNA was markedly more resistant than free AGT to these electrophilic species. DNA also blocked the spontaneous loss of AGT activity which occurred upon incubation of this protein under mild conditions. CONCLUSIONS: The reaction of AGT with the methyl isocyanate generated from the decomposition of VNP40101M increased the net number of crosslinks generated by VNP40101M compared to a sulfonylhydrazine prodrug that formed the equivalent alkylating species in the absence of the cogeneration of methyl isocyanate. These actions may be of significance to the antineoplastic activity of VNP40101M.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): II. Role of O6-alkylguanine-DNA alkyltransferase in cytotoxicity.

PURPOSE: VNP40101M (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy in murine models. VNP40101M activation generates chloroethylating species that alkylate DNA at the O(6)-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O(6)-alkylguanine-DNA alkyltransferase (AGT) in model systems. We determined whether expression of AGT in Chinese hamster ovary (CHO) cells decreased sensitivity to VNP40101M and explored the mechanism of VNP40101M cytotoxicity by employing analogs of VNP40101M that generate reactive intermediates with either carbamoylating or chloroethylating activity. METHODS: AGT was overexpressed in CHO cells by transfection with an expression vector containing the human AGT gene. Cell lines expressing AGT were employed in clonogenic assays to determine the cytotoxicity of VNP40101M and its analogs. RESULTS: VNP40101M was more active against AGT-expressing CHO cells than 90CE (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine), a chloroethylating generator devoid of carbamoylating activity. Furthermore, the greater the degree of AGT expression the more resistance to VNP40101M cytotoxicity. Combination chemotherapy experiments support the conclusions that methyl isocyanate and the chloroethylating species generated from the activation of VNP40101M function synergistically to kill cells. CONCLUSIONS: The findings support the concept that alkylation of the O(6)-position of guanine residues in DNA is the predominant lesion created by VNP40101M, and that methyl isocyanate resulting from the base-catalyzed activation of VNP40101M inhibits AGT and presumably other enzymes involved in DNA repair, thereby enhancing the yield of the DNA G-C interstrand crosslinks responsible for the antitumor activity of this agent.

Reversal of mitomycin C resistance by overexpression of bioreductive enzymes in Chinese hamster ovary cells.

The clinical utility of antineoplastic agents is limited by the development of drug resistance by tumors. Mitomycin C (MC) is a bacterial product that must be enzymatically reduced to exert anticancer activity. We have demonstrated that expression of the bacterial MC resistance-associated (MCRA) protein in Chinese hamster ovary (CHO) cells confers profound resistance to this antibiotic under aerobic conditions, but not under hypoxia. MCRA produces resistance to MC by redox cycling of the activated hydroquinone intermediate back to the prodrug form. A CHO cell line developed by stepwise exposure to increasing concentrations of MC likewise expressed high level resistance to MC in air, but not under hypoxia. The overexpression of DT-diaphorase and NADPH:cytochrome c (P-450) reductase, two enzymes known to activate MC, restored sensitivity to MC in both MCRA-transfected and drug-selected cell lines. The level of sensitization was proportional to the quantity of enzyme activity expressed, supporting the concept that the levels of these two activating enzymes are important for sensitivity to MC. The findings of resistance to MC in air but not under hypoxic conditions and of restoration of sensitivity to MC by increasing levels of DT-diaphorase activity, properties not adequately explained by other resistance mechanisms (i.e., decreases in MC activation, repair of DNA lesions, and/or drug efflux), support the hypothesis that a functional mammalian homologue of MCRA may be involved in producing resistance to MC.

[Carpal tunnel syndrome with an unusual cause: a malignant nerve sheath tumor of the median nerve]. / Syndrome du canal carpien de cause rare: une tumeur maligne des gaines nerveuses du nerf médian.

We report on the follow-up of a patient who developed symptoms suggestive of carpal tunnel syndrome. Symptoms were however atypical with involvement of the nondominant hand and with selective, fascicular, electroneurographic changes. During the surgical decompression of the median nerve at the wrist a tumor was found, corresponding to an isolated malignant peripheral nerve sheath tumor (MPNST) of mild type. A course of local radiation therapy was completed, with no sign of recurrence, and a normalization of the serum level of neurone specific enolase.

Cardiac vein thrombosis and haemorrhagic myocardial necrosis; report of a case with review of the literature.

A 29-year-old woman, addicted to heroin since the age of 15 years, presented with a 4-day history of acute inspiratory chest pain, dyspnoea and vomiting associated with hypoventilation. She died 3 h after admission to the intensive care unit in spite of active resuscitative measures. The main autopsy findings were limited to the heart, which showed widespread cardiac vein thrombosis, and both ventricles and the atria were associated with multiple areas of haemorrhagic myocardial necrosis. We review the literature of this uncommon pathological entity and discuss its possible pathogenesis.

[Medical data in pathology--evaluation of a large collection. (530,000 diagnoses coded in SNOMED II)]. / Données médicales en anatomie pathologique--évaluation d'un grand collectif (530,000 diagnostics codés en SNOMED II).

The paper is describing the design and the performance of the computerized system, from its introduction in 1982 until the present day. The first device, using the MUMPS language on a mini-computer, followed by a VAX computer with terminals have been replaced in 1996 by an application program, using ORACLE, based on the client-server concept. The content and the particularities of the different data groups are discussed, concerning the functional components of the data bank: 'PATIENTS', 'SPECIMEN', 'SENDERS', 'REPORT' and 'DIAGNOSES'. By means of examples, we demonstrate the chronological evolution of the registration of persons, the distribution of the diagnoses according to the organ systems, the possibilities to combine various lesions and an algorithm to assure that a given lesion is registered only once per patient. In first place, the efficiency and the reliability of manual coding by a pathologist using the Systematized Nomenclature of Medicine (SNOMED; 2nd edition [1979/1982]) is discussed. The data bank currently contains 530,000 diagnoses, distributed among on SNOMED's five main modules, obtained from 1500 autopsies, 140,000 surgical and 180,000 cytological specimens. Concluding the article, an analysis is made of desirable developments in the future with the aim of a better integration of the acquired information in routine work and an enhanced use of the medical content for epidemiological research or statistical analysis.

[Cardiac fibrosarcoma discovered by cerebral metastases]. / Fibrosarcome cardiaque découvert par métastases cérébrales.

Primary heart tumors are rare, and the fibrosarcomas are exceptional. We present the findings in a 78 year old female who has been hospitalized in April 1997 with a history of 4 months of malaise, headaches and weight loss. Clinical examination showed a right lateral homonymous hemi-anopsia. Computed tomography (CT-Scan) of the brain showed two lesions in the occipital and temporal lobe, consistent with metastasis. Thoracic and abdominal-pelvic CT-Scan revealed a voluminous mass of the left auricle that has been interpreted as a probable sarcoma. Biopsy specimen of one of the cerebral lesions, performed at the Centre hospitalier universitaire vaudois (CHUV) on April 29th 1997, revealed fragments of a poorly differentiated sarcoma. After referral back to Neuchâtel, the patient suffered suddenly three weeks later from a cerebral vascular insult with instant onset of deep coma. She deceased on May 27. At autopsy we found a neoplastic lesion of the left auricle with invasion of the pulmonary veins. The histological appearance is similar to the appearance of the cerebral metastasis, showing a proliferation of spindled cells with a partial positivity for Vimentine. It was classified as a fibrosarcoma. Metastasis were found only in the brain with postoperative ischemic left occipitotemporal necrosis and neoplastic emboli.--We are discussing the various incidences of cardiac neoplasms in autopsies or biopsies.

[Weighted kappa statistics for measuring the divergence of rapid section and definitive diagnosis]. / Gewichtete Kappa-Statistik zur Messung der Divergenz von Schnellschnitt- und definitiver Diagnose.

Weighted Kappa statistics measure or quantify the disagreement between diagnoses given by different observers or which were established in different time periods. The study reports on 2226 frozen-section examinations performed in the period of 1986 to 1995 at the Pathology Institute of Neuchâtel, Switzerland. The two cohorts formed by the "first diagnosis" and the "definitive diagnosis" are classified in four SNOMED Morphology categories and, in case of disagreement, the weight of the error is computed according to an arbitrary scheme of 2 to 20 points. Of the diagnoses 2093 (= 94.0%) were completely identical. The kappa of the cohort is 0.91 (+/- 0.008), the weighted kappa 0.95 (+/- 0.005). Analyses of different parameters show a constantly higher value for weighted kappa than for nonweighted kappa. The 133 cases with disagreements reveal, among others, that frozen-section examination of the thyroid gland has a high risk of error, whereas biopsies of the mammary gland give the best results (rare and practically no significant disagreement). Subsequent analyses investigate diagnostic quality according to the chronological evolution and the performance of the responsible pathologists.

[Primary cardiac lymphoma with obstructive cardiac insufficiency]. / Lymphome cardiaque primaire avec insuffisance cardiaque obstructive.

A 80 year old man, in general good health, presented with a rapidly progressive congestive heart failure, without response to treatment. At echocardiography, there were masses in the right atrium and ventricle. The patient deceased two months after the first clinical manifestations. At autopsy, we found a massive right-sided atrio-ventricular infiltration by a malignant non-Hodgkin's lymphoma of centroblastic type, causing a subtotal obstruction of the tricuspid and pulmonary valves. We observed also four intestinal metastases without other manifestations of lymphoma.

[25 years of pathology at Neuchâtel]. / Vingt-cinq ans de pathologie à Neuchâtel.

The article introduces the Neuchâtel Institute of Pathology (INAP) which celebrated its 25th anniversary in 1991. The institute is organised according to the statutes of a private foundation, the State of Neuchâtel covering the excess running costs. The task of INAP is fulfilled by its services of clinical pathology which have evolved differently according to each sector. The autopsies reached their peak in 1979, a progressive decline in number is noted since then. The biopsies have increased regularly, especially since 1985. The service of cytopathology has had an irregular development and is actually in full expansion. The article then shows the solutions that INAP has found in the technical and medical fields. The role of medical informatics, introduced in 1983, is pointed out. In conclusion, we discuss the current and future questions of our discipline and their possible solutions in an institution such as ours.

Viral proteins associated with the Epstein-Barr virus transactivator, ZEBRA.

The BamHI Z Epstein-Barr replication activator (ZEBRA) mediates disruption of latency and induction of Epstein-Barr virus (EBV) early gene expression in latently infected lymphocytes. Polyclonal rabbit sera raised against ZEBRA were used to immunoprecipitate ZEBRA-associated proteins (ZAPs). ZAPs of 19, 21, 23, and 42 kDa were coimmunoprecipitated with ZEBRA from extracts of EBV-producing lymphoid cell lines. ZAPs were not recognized directly by the rabbit sera, but they were antigenic for EBV+ human sera. Immunoprecipitation of ZAPs by ZEBRA-specific antisera required the presence of ZEBRA. ZAPs were not coprecipitated with ZEBRA from mouse cells expressing only ZEBRA, from Raji (a cell line in which EBV is unable to complete lytic replication), or from cells treated with inhibitors of viral DNA synthesis. Thus, ZAPs are late EBV-encoded proteins. ZEBRA and ZAPs colocalized to a salt-insoluble nuclear fraction, and both were found extracellularly in crude preparations of virions. ZAPs might function to affect the cellular localization of ZEBRA, to alter its capacity to transactivate, or to influence its target gene specificity.

ZEBRA and a Fos-GCN4 chimeric protein differ in their DNA-binding specificities for sites in the Epstein-Barr virus BZLF1 promoter.

Epstein-Barr virus (EBV) encodes a protein, ZEBRA, which enables the virus to switch from a latent to a lytic life cycle. The basic domain of ZEBRA is homologous to the Fos/Jun oncogene family, and both proteins bind the canonical AP-1 site (TGAGTCA). However, ZEBRA does not contain a leucine zipper dimerization domain which has been shown to be necessary for DNA binding of Fos/Jun proteins. Additionally, ZEBRA binds to sites which deviate from the AP-1 consensus sequence. Thus, it was of interest to define the domain of the ZEBRA protein required for DNA binding. We have determined by mutagenesis that ZEBRA residues 172 to 227, representing the basic domain and a putative dimerization domain, are required for specific binding to AP-1 and divergent sites. Mutagenesis of the basic amino acids 178 to 180 or 187 to 189 abrogates ZEBRA binding to all DNA target sequences. These residues are conserved in Fos and are also necessary for Fos DNA-binding activity. We have found that a Fos-GCN4 chimera and ZEBRA have different cognate binding specificities. The autoregulated BZLF1 promoter contains three divergent AP-1 sequences, ZIIIA (TGAGCCA), ZIIIB (TTAGCAA), and Z-AP-1-octamer (TGACATCA). ZEBRA binds with high specificity to ZIIIA and ZIIIB but weakly to the Z-AP-1 octamer. Conversely, the Fos-GCN4 chimera recognizes only the Z-AP-1 octamer. ZEBRA binds the ZIIIA and ZIIIB sites together in a noncooperative fashion, while Fos-GCN4 binds these sites as a higher-order complex. Additionally, we have found that flanking sequences influence binding of Fos-GCN4 to a degenerate AP-1 site (TGAGCAA). The characteristic binding specificities of ZEBRA and cellular AP-1 proteins suggest that they differentially affect viral and cellular transcription.

[Comparison of 2 series of autopsies observed at Johns-Hopkins Medical Center, Baltimore (JHMI) and at the Neuchâtel Institute of Pathology (INAP)]. / Comparaison de deux séries d'autopsies, observées au Johns Hopkins Medical Institutions, Baltimore (JHMI) et à l'Institut neuchâtelois d'anatomie pathologique (INAP).

We are reporting the first results of a comparative study of 100 consecutive autopsies and their clinical diagnoses, observed at the Johns Hopkins Medical Institutions (JHMI) and at the Institut neuchâtelois d'anatomie pathologique (INAP). The diagnoses of the two series were coded according to the two different systems used currently at the two institutions. The data from Baltimore were automatically classified by a special "key word method" using the categories of the Index Medicus (MeSH = Medical subject headings). We proceeded then to a second recording by SNOMED codes, introduced into the computer system in the same way as we document the autopsy diagnoses in Neuchâtel. The two series could be compared in detail according to topographical, morphological and aetiological parameters. The over-all repartition of the examined cases shows a higher incidence of newborns in Baltimore (23), in Neuchâtel we observed only 7 newborn autopsies. The mean age was inferior in Baltimore (males: 53.5 years for JHMI, 73.1 years for INAP; females: 58.4 years for JHMI, 66.2 years for INAP). The number of diagnoses per autopsy was 31.9 at JHMI, and 50.1 in Neuchâtel. The topographical distribution of clinical and autopsy diagnoses showed a higher frequency of central nervous system lesions in JHMI which might be explained by the activity of a neuropathological division. Findings concerning the morphological categories revealed a higher frequency in JHMI for traumatic abnormalities (7.2% vs 2.5%), malformations (4.3% vs 0.8%), whereas inflammation and fibrosis and degenerative lesions were more often encountered in Neuchâtel. The differences in morphological observations could be attributed to a higher proportion of newborn cases in JHMI with complex malformation syndromes.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the site of recombination in the generation of the genome of DI particles of equine herpesvirus type 1.

Defective interfering particles (DIPs) are generated by serial, undiluted propagation of equine herpesvirus type 1 (EHV-1). DIP-rich preparations of EHV-1 mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. The defective genomes consist of reiterations of sequences from the left terminus (0.00 to 0.04 map units) of the long (L) region covalently linked to sequences from the inverted repeats (0.78 to 0.79, 0.83 to 0.87, 0.91 to 0.95, and 0.99 to 1.00 map units) of the short (S) region of the standard genome. We have identified and determined the nucleotide sequences of these segments of the standard genome as well as the component of the defective DNA that contains the site at which these two viral sequences recombined. Comparison of these sequences revealed that there is an 8-nucleotide sequence that is common to both the left terminus sequences and the inverted repeat sequences. These 8-nucleotide identical sequences are located at 3.25 kbp from the left terminus and at 9 kbp downstream of the L-S junction. The recombination between the left terminus and the inverted repeat sequences occurred at the site of homology and resulted in the generation of a novel open reading frame. The last 97 amino acids of an open reading frame of 469 amino acids encoded by sequences within the inverted repeats were replaced by a sequence of 68 amino acids encoded by a 204-bp sequence mapping at 0.023 map units. It will be of interest to determine whether this altered open reading frame, generated by recombination of sequences separated by more than 110,000 bp in the standard genome, plays a role in the varied outcomes of infection mediated by EHV-1 DIPs.