Methyltransferase Contingencies in the Pathway of Everninomicin D Antibiotics and Analogues

Everninomicins are orthoester oligosaccharide antibiotics with potent activity against multidrug-resistant bacterial pathogens. Everninomicins act by disrupting ribosomal assembly in a distinct region in comparison to clinically prescribed drugs. We employed microporous intergeneric conjugation with Escherichia coli to manipulate Micromonospora for targeted gene-replacement studies of multiple putative methyltransferases across the octasaccharide scaffold of everninomicin effecting the A₁, C, F, and H rings. Analyses of gene-replacement and genetic complementation mutants established the mutability of the everninomicin scaffold through the generation of 12 previously unreported analogues and, together with previous results, permitted assignment of the ten methyltransferases required for everninomicin biosynthesis. The in vitro activity of A₁- and H-ring-modifying methyltransferases demonstrated the ability to catalyze late-stage modification of the scaffold on an A₁-ring phenol and H-ring C-4’ hydroxy moiety. Together these results establish the potential of the everninomicin scaffold for modification through mutagenesis and in vitro modification of advanced biosynthetic intermediates.     

High brachial artery bifurcation: a report of 2 cases

High division of the brachial artery was observed in two cadavers, during routine dissection of upper extremities. In the first case, the brachial artery of the right upper extremity was divided into its two terminal branches immediately after passing between the lateral and medial roots of the median nerve and just below the origin of the profunda brachii artery. The lateral branch was the radial artery, located in the space normally occupied by the brachial artery and the medial one was the ulnar artery. In the second case, the brachial artery was divided into its two terminal branches just below the origin of the profunda brachii artery. Accurate knowledge of the relationships and course of these major arterial conduits and particularly of their variational patterns, is of considerable practical importance in the conduct of reperative surgery in the arm, forearm and hand.     

Microsatellite markers in leukaemia and lymphoma: comments on a timely topic

Microsatellites are unique highly polymorphic and informative genetic markers dispersed in the human genome. Their detection by PCR is rapid and a wide variety of DNA sources including archival material are available for diagnostic purposes. Microsatellite typing of haematological neoplasms may be applied to the search for loss of heterozygosity at loci possibly harbouring tumour suppressor genes, for example in acute lymphoblastic leukaemia. The technique may detect submicroscopical chromosomal deletions which are not visible in the leukaemic karyotype. RER+ tumours exhibiting microsatellite instability appear to be rare among haematological cancers with the possible exception of lymphoid tumours in immunosuppressed patients and lymphomas derived from mucosa-associated lymphoid tissue. An X-chromosomal microsatellite near the human androgen receptor gene (HUMARA) may be used for clonal X-inactivation analysis. Microsatellites therefore represent a collection of powerful genetic markers suitable to tackle questions relevant to basic research and clinical problems in leukaemia and lymphoma.     

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.     

Diversity of pituitary cells in primary cell culture. An immunocytochemical study

A bioassay system for rapid detection of carcinogenic agents has been developed using male Fischer 344 rats to bridge the gap between long-term carcinogenicity tests and short-term screening assays. The system, called the medium-term liver bioassay, is fundamentally based on the 2-stage hypothesis of tumor production, employing initiation by diethylnitrosamine (200 mg/kg, i.p.) in the first stage and test chemical administration during the second, in combination with two-thirds partial hepatectomy. It requires only 8 wk for animal experimentation and a further few weeks for quantitative analysis of immunohistochemically demonstrated glutathione S-transferase placental form positive hepatic foci. A total of 291 chemicals/substances have already been analyzed in our laboratory. Among 63 chemicals that were proved to be carcinogenic in the liver of rat and/or mouse, 57 (90%) gave positive results irrespective of their mutagenicity. Negative compounds include peroxisome proliferators and tamoxifen. Even nonhepatocarcinogens were positive at a rate of 24%. Eighty-six percent (12/14) of mouse liver carcinogens were also positive. On the other hand, only 2 out of 45 noncarcinogens showed very weak positivity. Thus, the efficacy of the system for hepatocarcinogens has been well established. This bioassay is increasingly regarded as an appropriate alternative test for carcinogenicity risk assessment and is practically used for a rapid evaluation of hepatocarcinogenicity of chemicals.     

Elder mistreatment: its relevance to older women

Elder mistreatment, both abuse and neglect, is an important health care problem for women since they are involved as victims and as perpetrators. The incidence of both forms of such mistreatment is increasing, and both often occur within the context of long-term care. Elder abuse occurs in both unidirectional and dual directional forms, and includes parent abuse and spouse abuse. Elder neglect occurs in two forms; neglect by others and self-neglect. While elderly men and women are both neglected and/or abused, women may suffer greater physical and psychological consequences. To date, researchers have inadequately addressed the relevance of gender to both forms of elder mistreatment. This literature review addresses what is currently known about elder abuse and neglect that is of particular relevance to older women.     

Pulmonary function after one-lung ventilation in newborns: the basis for neonatal thoracoscopy

BACKGROUND: To maintain good exposure during major video-assisted thoracic surgery it is necessary to deflate completely the ipsilateral lung. However, little is known about the effects of one-lung ventilation (OLV) on pulmonary function in newborn patients. METHODS: Ten neonatal domestic pigs with a mean age of 6+/-0.6 days were intubated and ventilated in pressure-controlled mode (inspired oxygen fraction=1.0). One-lung ventilation was maintained for 120 minutes. Serial measurements of hemodynamics and gas exchange were done before, during, and until 90 minutes after OLV. Pulmonary function testing was performed before and after OLV for each lung separately. RESULTS: With the inspired oxygen fraction set at 1.0, arterial oxygen saturation remained stable at 100% during OLV. Venous admixture and alveolar-arterial oxygen tension gradient increased slightly from the baseline value of 2.6% +/-0.3% to 3.8%+/-0.3% during OLV (mean+/-standard error of the mean; p=0.02), and from 358+/-28 to 407+/-18 mm Hg (not significant), respectively. Both values returned to baseline during the subsequent ventilation of both lungs. Static compliance and resistance of the ventilated lung did not change. Compliance of the collapsed lung decreased after reexpansion from 0.42+/-0.07 to 0.29+/-0.06 mL x cm H2O(-1) x kg(-1), p=0.008). Resistance remained unchanged (0.22+/-0.02 versus 0.25+/-0.05 cm H2O x L(-1) x s(-1); not significant). CONCLUSIONS: There were only minor effects on pulmonary function during and after OLV in the neonatal piglet. Alterations in gas exchange during OLV were minimal. Prolonged collapse of the lung with subsequent reexpansion was associated with a slight decrease in compliance, indicating some mild lung injury.     

Pathology of Toxoplasma gondii infection in the nude rat. An experimental model of toxoplasmosis in the immunocompromised host?

Genetically athymic nu/nu (nude) rats, deficient in T cells, die from infection with various Toxoplasma gondii strains, including RH and Prugniaud strains. In contrast, these strains cause chronic infections without apparent symptoms in immunocompetent non-nude rats. We show here that nude rats die in two to three weeks after RH infection and three to four weeks after Prugniaud infection. Histological examination of brains from nude rats at different time points after infection, revealed an absence of lesions after RH strain infection and cysts with usually no inflammation after Prugniaud infection. Lungs from nude rats developed a fibrin alveolitis using either strain, whereas myocarditis with focal areas of necrosis were observed only after Prugniaud infection. Cysts and, in some cases, tachyzoites in the necrotic lesions were easily identifiable. The two strains of T. gondii elicited in nude rats a granulomatous hepatitis that only differed in intensity. Spleen and mesenteric lymph nodes appeared totally non reactive in both cases. This model allows immunological and parasitological studies by comparison with immunocompetent rat infection. Published data concerning toxoplasma pathology in AIDS, therefore, suggest that acute toxoplasma infection in nude rats may be a useful model for studying disseminated forms of toxoplasma infection found in AIDS patients.