Leucomycin U

The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction. Whole-cell assays using Escherichia coli cells expressing RosC mutants with single and double amino acid substitutions derived from RM30 indicated that P107S/L176Q, P107S/V277A, P107S/I319N, L176Q/V277A, L176Q/I319N, and S254N/V277A significantly reduced the catalytic activity of the second reaction, which is alcohol oxidation. Of the previously mentioned mutants, double mutants containing L176Q, which was presumed to occur in the FG loop region, lost the total catalytic activity of the third reaction (aldehyde oxidation). Additionally, an engineered M. rosaria strain with rosC disruption, which introduced the gene encoding the RosC mutants P107S/L176Q and P107S/V277A preferentially produced 20-dihydrorosamicin, which is formed after the first oxidation reaction of RosC.

The case report presents the case od 66-year-old woman with schock, severe general and grave local symptoms after a snake bite into the left limb. The species of attacking snake was not clearly specific, it should not have been clearly seen, but it was propably a common viper, a snake of viper family, which commonly occurs in our latitude. Due to the unclear origin of the bite, no specific antiserum and symptomatic treatment were given.

Objective: To report on a unique combination of cerebral toxoplasmosis and ocular toxoplasmosis in an HIV-positive patient in Slovakia. Methods: A 35-year-old heterosexual patient who presented with headache and major seizures underwent computed tomography (CT) and magnetic resonance imaging (MRI). Based on clinical findings, serological tests for toxoplasmosis were performed on serum and ocular fluid specimens. PCR was also used to detect Toxoplasma gondii and cytomegalovirus DNA. Goldmann and Witmer coefficient calculation was applied to demonstrate the synthesis of intraocular IgG antibodies. Results: CT and MRI revealed cystic lesions suspected of metastasis in the occipital and temporal regions, and we searched for the primary tumor. After vision loss in the left eye, which rapidly progressed to complete blindness, an eye examination detected macular edema. Anti-edema treatment was initiated. HIV positivity with a very low CD4 T-cell count (20/μL) was found, and the viral load was 100 400 HIV-RNA copies/ml. The serum was positive for anti-Toxoplasma IgG antibodies (> 200 IU/mL), IgM negative, and IgA borderline. As toxoplasmic encephalitis and retinitis were suspected, antitoxoplasmic therapy with pyrimethamine, spiramycin, and folinic acid was started. The ophthalmologist considered cytomegalovirus retinitis, which was not confirmed by serology or PCR. In contrast, the presence of IgG antibodies in ocular fluid and serum with the calculation of the Goldmann-Witmer coefficient (GW = 32) as well as PCR DNA positivity pointed to Toxoplasma gondii as the etiological agent. Follow-up MRI scan confirmed regression of the pathological lesions, neurological deficit also improved, CD4 T-lymphocytes increased above 200/μL, but blindness of the left eye persisted. Conclusion: CT and MRI scans offered no clue as to Toxoplasma etiology of the brain and eye involvement in an HIV-positive patient, which was only confirmed by laboratory tests. Due to the delay in the diagnosis of toxoplasmosis, time from the epileptic seizure to treatment initiation was 16 days, which assumedly caused irreversible blindness in the patient.