Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance.

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.

The use and advantages of a multichannel vaginal cylinder in high-dose-rate brachytherapy.

PURPOSE: This paper describes California Endocurietherapy's (CET) high-dose-rate (HDR) multichannel cylinder, the rationale for its design, procedure for its insertion, and the dosimetry involved in its use. A study was done that compared the doses achieved using the CET multichannel cylinder to the same cylinder if it only had a central channel. METHODS AND MATERIALS: The CET multichannel vaginal cylinder was inexpensively constructed, using parts from various suppliers. After insertion in the patient, the cylinder is affixed to a base plate to prevent displacement. Two sets of orthogonal films (without and with rectal barium) are taken in preparation for digitization of the catheters, bladder, rectal, and pelvic sidewall points. Using HDR brachytherapy planning software, the dose distribution is adjusted to achieve the prescribed dose (5 Gy HDR) 5 mm lateral to the cylinder surface, 5 mm lateral at the proximal parametrial tissue, and 4 mm superior to the vaginal apex. Doses to the bladder and rectum are limited to approximately 85% and 75%, respectively, of the prescribed dose. The plan is optimized on geometric parameters. For dose comparison to treatment using a central channel cylinder, the lateral channels are de-activated, leaving only the central channel activated. Dose points are placed 5 mm laterally and superiorly from the cylinder surface, and the plan is optimized to deliver a uniform dose to the defined dose points. The doses and treatment volumes are statistically compared. RESULTS: The CET multichannel cylinder allows much better dose control than the central channel cylinder. The multichannel cylinder achieves lower bladder and rectal doses by 14% and 15%, respectively, when compared to the central channel cylinder. By increasing the dwell times of certain dwell positions, the prescription dose is achieved in the vaginal apex and proximal parametrial tissues and along the length of the cylinder. CONCLUSION: The multichannel cylinder enables more flexibility in isodose shaping and dose control to various points and structures when compared to the conventional central channel cylinder.

Hydronephrosis in pregnancy: simultaneous depiction of fetal and maternal hydronephrosis by magnetic resonance urography.

Magnetic resonance urographic (MRU) techniques possess image quality and diagnostic capability that are improving with increasingly sophisticated imaging sequences and shorter scanning times. We describe the application of a fast breath-hold MR sequence (HASTE) in the assessment of ureteric obstruction in pregnancy. In the patient presented, HASTE MRU was successful in depicting ureteral anatomy and demonstrated dilation of both ureters below the level of the pelvic brim. This observation suggested distal ureteral obstruction rather than simple hydronephrosis of pregnancy. As a result, bilateral nephrostomies were performed and neonatal prematurity was avoided. Interestingly, in this patient, HASTE MR imaging also showed evidence of concurrent fetal hydronephrosis.

Tirapazamine with cisplatin in patients with advanced non-small-cell lung cancer: a phase II study.

PURPOSE: A phase II study was conducted to evaluate the safety and efficacy of tirapazamine combined with cisplatin for the treatment of patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Forty-four patients with stage IIIB/IV NSCLC were treated with a combination of tirapazamine and cisplatin. Patients received tirapazamine 260 mg/m2 administered intravenously over 2 hours, followed 1 hour later by cisplatin 75 mg/m2 administered over an additional hour, repeated every 21 days. The duration of therapy was meant to be limited to four cycles for nonresponders and eight cycles for responders. RESULTS: Ten of 44 patients (23%) showed a partial response. The estimated median survival for all patients was 37 weeks. Toxicities were treatable and included grade 3 nausea or vomiting (25%), fatigue (27.3%), and muscle cramps (4.5%). No dose reductions were necessary. CONCLUSION: The results show that tirapazamine can safely be added to cisplatin. Both the median survival and response rate observed strongly suggest that tirapazamine with cisplatin is more active than cisplatin alone.

p53 and PCNA expression in advanced colorectal cancer: response to chemotherapy and long-term prognosis.

In a series of 71 patients with advanced colorectal cancer treated with biochemically modulated 5-fluorouracil (5-FU) and methotrexate (MTX), we investigated the relationship between the proliferating-cell nuclear antigen (PCNA) (PC10) and p53 (Pab1801) primary-tumor immunohistochemical expression with respect to clinical response and long-term prognosis. Nuclear p53 expression was demonstrated in 44% of samples (any number of positive tumor cells) while all tumors showed a certain degree of PCNA immunostaining. PCNA immunostaining was correlated with histopathologic grade and p53 expression, while p53 was not correlated with any of the parameters considered. The probability of clinical response to biochemically modulated 5-FU was independent of p53 and PCNA expression. p53 expression (all cut-off values) was not associated with short- or long-term clinical prognosis, whereas patients with higher PCNA primary-tumor expression showed longer survival from treatment and survival from diagnosis, according to univariate and multivariate analysis, particularly in the sub-set of colon-cancer patients. We conclude that the clinical response of advanced-colorectal-cancer patients to biochemically modulated 5-FU and MTX cannot be predicted by PCNA and p53 primary-tumor expression, but high PCNA expression appears to be independently related to long-term prognosis.

Phase II trial of docetaxel (Taxotere) in patients with metastatic melanoma previously untreated with cytotoxic chemotherapy.

A phase II study was undertaken to evaluate the clinical efficacy and safety of docetaxel in patients with malignant melanoma. Between April 1992 and February 1996, 37 patients with metastatic malignant melanoma and no prior chemotherapy were treated with docetaxel 100 mg m-2 administered intravenously over 1 hour every 21 days. Patients were premedicated prior to each course with dexamethasone and diphenhydramine. Toxicity and follow-up were provided. Objective responses were seen in two out of 35 patients evaluable for response, one complete response and one partial response. These two responses were of a duration of greater than two years. The most common toxicity was grade 4 neutropenia, which occurred in 92% of patients; 49% required hospitalization for an episode of neutropenic fever. Additional patients had reversible grade 3-4 toxicities including nausea, vomiting, diarrhea, stomatitis, arthralgias, myalgias, peripheral neuropathy and fatigue. Eighteen patients had hypersensitivity reactions, two were grade 3-4. Fluid retention, grade 1-3 was observed in seven patients. Alopecia occurred in most patients. Docetaxel has definite but low-level activity against malignant melanoma. Further investigation of this drug should be considered in multidrug combination programs.

Effect of clitoral stimulation after artificial insemination on conception in Zebu-crossbred heifers in the tropics.

In order to increase conception rates to AI, an experiment was conducted to determine the effect of clitoral stimulation on pregnancy rate. Seven hundred and thirty-five Zebu-cross heifers were assigned to either of the following treatments: 1) control without clitoral stimulation (n=351) or 2) clitoral stimulation after IA (n=384). The data were analyzed using a linear model procedure. Clitoral stimulation increased pregnancy rates (57 vs 45 % for treated and control groups, respectively; P<0.02). Holstein x Zebu and Brown Swiss x Zebu heifers attained similar rates (P>0.05). There was a significant treatment-by-season interaction (P<0.01), with pregnancy rates being higher using clitoral massage during the rainy season (54.7 and 25.5% for the treated and control group respectively). In this study, manual clitoral stimulation increased conception rates in Zebu cross heifers under tropical conditions.

Endoscopic management of cholangitis: critical review of an alternative technique and report of a large series.

OBJECTIVE: To assess the outcome of endoscopic techniques as the solitary treatment modality for the complete management of ascending, bacterial cholangitis, compared with results of radiological and surgical methods as historical controls. METHODS: Endoscopic techniques were used to decompress bile ducts obstructed by stones (898 patients) or stenosis (49 patients). Endoscopic sphincterotomy (ES) was performed in 839 patients, and either 7-Fr straight stents (79), or nasobiliary tubes (29), were utilized as initial therapy in 108 patients. Of these latter patients, 68 subsequently underwent ES and stone removal, 17 had ES, lithotripsy, and stone removal, 18 were left with stents in place, and 5 were lost to follow-up. Follow-up was conducted by direct patient contact, by telephone, or through the referring physicians. RESULTS: All patients were managed by endoscopic techniques. There were four deaths (0.42%) in the first 30 days (none before 2 wk); no deaths were related to the procedures but were attributed to intercurrent medical problems. Two patients underwent surgery: one pancreatitis, one perforation. Complications were infrequent, occurring in 6% of patients. Bleeding occurred in 3%, pancreatitis in 2.8%, and perforation 0.2%. CONCLUSIONS: Endoscopic management of cholangitis is as effective as surgical or radiological methods for managing bacterial cholangitis, a potentially fatal syndrome, but ERCP and ES have been shown to be safer. Endoscopy is the preferred index technique both for establishing a definitive diagnosis and providing therapy.

[Carcinoma of Bellini's duct]. / Carcinoma del conducto de Bellini.

First case in the Spanish literature of a carcinoma of Bellini's duct. Differentiation of this tumor from the classic renal adenocarcinoma, mainly the papillary type, is based on the different embryological origin, microscopic characteristics and immunohistochemical studies. Prognosis is worse than for the other renal adenocarcinomas.

[Multiple drug resistance: a problem in cancer chemotherapy]. / Resistencia múltiple a drogas: un problema en la quimioterapia de cáncer.

One of the main problems in clinical oncology is an acquired cellular drug resistance. Special attention deserves the multidrug resistance phenomenon (MDR) involving tumors which become resistant to a wide spectrum of non-related drugs to which they have never been exposed. Several mechanisms responsible for this phenomenon have been described. Among them is the increased expression of the MDR1 gene which encodes the plasma membrane glycoprotein P-gp. This glycoprotein is an energy-dependant multidrug efflux pump of wide specificity. It seems to have a normal physiological function but in some tumors resistant to chemotherapy its expression is increased. In cell lines the increased expression of P-gp is correlated with a decreased accumulation and retention of drugs inside the cells. In addition to P-gp, at least two other mechanisms of multidrug resistance have been described: a decreased expression and changes in the catalytic activity of topoisomerase II enzyme, and changes in glutathione transferase levels. Through biochemical and molecular methods researchers continue to look for a correlation between non-responding tumors and changes in the known drug-resistance mechanisms. These studies suggest that several factors are involved in the cellular drug resistance observed in human tumors, and probably are interacting between them. In clinical practice, the need of controlling MDR phenomena has led to the creation of alternate therapeutic strategies.

A DNA polymerase from maize axes: its purification and possible role.

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100-120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30-37 degrees C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28-40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase alpha. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase alpha-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.

Meso-diencephalic infarction: a not so rare form of stroke.

26 patients admitted with clinical pictures consistent with meso-diencephalic infarction were studied using computerized axial tomography, magnetic resonance imaging and angiography. All of the patients presented symptoms in two of three major categories which comprise the "top of the basilar" syndrome. Lesions in the territory of the basilar-communicating artery were demonstrated in 22 of them. The remaining patients died before adequate neuroimaging documentation was obtained. Overall prognosis appeared relatively good since 80% of the patients recovered completely or incompletely from the ictus. Meso-diencephalic infarction is a very important and not uncommon subtype of cerebrovascular disorder.

Reconstruction of the medial canthus.

The keystone for successful reconstruction of the medial canthal area is adequate positioning of the medial canthal complex to maintain proper intercanthal distance and apposition of the lids to the globe. This requires an understanding of the dynamics of the tripartite insertion of the MCT and its relationship to the medial orbital wall. We have previously described a technique for transnasal wiring based on anatomic studies that is anatomically and physiologically precise and that is applicable to a variety of clinical situations. Soft-tissue problems need to be dealt with on an individual basis with grafts, flaps, or a combination of these modalities.

The behavioural effects of an N-methylaspartate receptor antagonist following application to the lumbar spinal cord of conscious rats.

The selective N-methylaspartate (NMA) receptor antagonist 2-amino-5-phosphonopentanoate (AP5) has been applied to the lumbar region of the spinal cord in conscious rats through chronically implanted intrathecal cannulae. Administration of AP5 was compared with administration of 2-amino-4-phosphonobutyrate (AP4), morphine and control injections. Intrathecal injection of AP5 (250 nmol) produced extensor paralysis in the hindquarters and depression of aversive responses to tail heat (54 degrees C), paw heat (hotplate 56.5 degrees C) and paw pressure. Depression of AP5 of the vocal response to aversive electrical stimuli, applied to the tail, suggested that AP5 produced analgesia independently of locomotor paralysis. Intrathecal injection of morphine (20-30 nmol) produced no observed locomotor effects, but depressed responses to all the aversive stimuli. Intrathecal injection of AP4 had no significant effects. When tritiated AP5 was administered intrathecally, the proportion of radioactivity recovered from the lumbar spinal cord suggested that the concentration of AP5, which produced locomotor depression and analgesia, was likely to have been in the range (15-75 microM) known to depress excitatory transmission in isolated spinal cord preparations. It is concluded that synaptic activation of n-methylaspartate receptors is important in spinal pathways which control locomotion, posture and central transmission of nociceptive information.

Two cases of death involving dicyclomine in infants. Measurement of therapeutic and toxic concentrations in blood.

In two deaths of infants investigated by the Medical Examiner, high levels of dicyclomine were detected in blood. In one of the cases, a level of 0.505 micrograms/ml was found, nearly 10 times reported adult therapeutic blood concentrations, and death was ascribed to an overdose of dicyclomine. Gas chromatography/mass spectrometry was used for the analysis of biological specimens.

Growth factor requirements of Chinese hamster lung fibroblasts in serum free media: high mitogenic action of thrombin.

We have defined a chemical medium to grow an anchorage and highly serum-dependent Chinese Hamster Lung fibroblast cell line (CCl 39) in the absence of serum. This serum-free medium referred to as DM4, contains Transferrin (5 microgram/ml), Insulin (10 microgram/ml), Epidermal Growth Factor (10 ng/ml) and Thrombin (0.05 U/ml). Thrombin appeared to be a very potent mitogen for Chinese Hamster Lung cells. DM4 allows exponential growth of CCl 39 cells plated in serum-coated dishes without any lag and with a generation time of 15 hours. Continuous subculturing and clonal growth can be obtained by passaging the cells with Ca++, Mg++ free phosphate-buffered saline in fibronectin coated dishes. The entire definition of growth factor requirements for this anchorage dependent cell line is of great value to approach the mechanisms of escape of growth control by these cells.

[Histochemical study of glutamate dehydrogenase activity in the medulla, bulb and in the central cerebellar nuclei of various vertebrates]. / Etude histochimique de l'activité glutamique déshydrogénase dans la moëlle, dans le bulbe et dans les noyaux cérébelleux centraux de quelques vertebrés.

In this work we research into the GDH location of the spinal cord, in the medullar oblongata and in the central cerebellar nuclei of the rat, cat, dog, chicken, lizard (Lacerta lepida), and Toad (Bufo calamita). The GDH is located at the cytoplasm and at the neuronal prolongations, at oligodendrocytes cells, at Lecerta ependimary glial cells, and at the capillary walls.