Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway.

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose.

Imaging haemodynamic changes related to seizures: comparison of EEG-based general linear model, independent component analysis of fMRI and intracranial EEG.

BACKGROUND: Simultaneous EEG-fMRI can reveal haemodynamic changes associated with epileptic activity which may contribute to understanding seizure onset and propagation. METHODS: Nine of 83 patients with focal epilepsy undergoing pre-surgical evaluation had seizures during EEG-fMRI and analysed using three approaches, two based on the general linear model (GLM) and one using independent component analysis (ICA): The results were compared with intracranial EEG. RESULTS: The canonical GLM analysis revealed significant BOLD signal changes associated with seizures on EEG in 7/9 patients, concordant with the seizure onset zone in 4/7. The Fourier GLM analysis revealed changes in BOLD signal corresponding with the results of the canonical analysis in two patients. ICA revealed components spatially concordant with the seizure onset zone in all patients (8/9 confirmed by intracranial EEG). CONCLUSION: Ictal EEG-fMRI visualises plausible seizure related haemodynamic changes. The GLM approach to analysing EEG-fMRI data reveals localised BOLD changes concordant with the ictal onset zone when scalp EEG reflects seizure onset. ICA provides additional information when scalp EEG does not accurately reflect seizures and may give insight into ictal haemodynamics.

Differences in corpus callosum volume and diffusivity between temporal and frontal lobe epilepsy.

We analyzed volume and diffusivity measures of the corpus callosum (CC) in patients with temporal (TLE) and frontal (FLE) lobe epilepsy in comparison with healthy subjects. On high-resolution T1-weighted scans of 18 controls and 44 patients the volumes (cm(3)) of Witelson regions (WRs) and the entire CC were measured. The apparent diffusion coefficients (ADCs, 10(-5)mm(2)s(-1)) for the entire CC and three areas of interest were measured from co-registered ADC maps. The CC of patients with TLE and FLE, corrected for total brain volume, was smaller than that of controls. Patients' ADC values were higher than those of controls. Findings were significant for WR1, WR2, and WR6, the CC regions connecting the frontal and temporal lobes. Patients with FLE had smaller WR1 and higher ADC values; in patients with TLE, the findings were similar for WR6. Atrophy and increased diffusivity in subregions of the CC connecting homotopic contralateral cortical regions indicate anatomical abnormalities extending beyond the epileptogenic zone in FLE and TLE.

Anti-genotoxic and anti-mutagenic activity of Escherichia coli Nissle 1917 as assessed by in vitro tests.

Anti-genotoxic or anti-mutagenic activity has been described for a number of Gram-positive probiotic bacterial species. Here we present evidence that Gram-negative Escherichia coli Nissle 1917 (EcN) also displays anti-genotoxic/anti-mutagenic activity, as assessed in vitro by the Comet Assay and the Ames Test, respectively. This activity was demonstrated by use of the mutagens 4-nitroquinoline-1-oxide (NQO), hydrogen peroxide (H2O2) and benzo(a) pyrene (B[a]P). For both assays and all three test agents the anti-genotoxic/anti-mutagenic activity of EcN was shown to be concentration dependent. By the use of extracts of bacteria that were inactivated by various procedures (heat treatment, ultrasound sonication or ultraviolet light irradiation), mechanistic explanations could be put forward. The proposed mechanisms were enforced by treating the bacterial material with proteinase K prior to testing. The mutagen H2O2 is most likely inactivated by enzymic activity, with catalase a likely candidate, while several explanations can be put forward for inactivation of B[a]P. NQO is most likely inactivated by metabolising enzymes, since the formation of the metabolite 4-aminoquinoline could be demonstrated. In conclusion, the in vitro results presented here make a strong case for antimutagenic properties of EcN.

Quality assessment of unfractionated heparin using 1H nuclear magnetic resonance spectroscopy.

Due to problems, especially anaphylactoid reactions, raised by impure unfractionated heparin the quality assessment of heparin has to be reconsidered. Neither the USP nor the European Pharmacopoeia are able to guarantee the purity of heparin, i.e., the limitation of oversulfated chondroitin sulfate (OSCS) which was found to be the reason for the allergic adverse effects. In the first run the regulatory authorities ask for 1H NMR spectroscopic and capillary electrophoretic measurements in order to characterize the impurity profile of heparin. Using an optimized 1H NMR method the limit of detection for OSCS was found to be 0.1%. In addition, it is possible to reliably quantify both OSCS and dermatan sulfate (DS), the latter being an indicator of poor purification of the unfractionated heparin. Screening of more than 100 heparin samples collected from international markets revealed a high number of samples containing substantial amounts of DS and a number of samples containing OSCS in an amount higher than 0.1%.

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

Influence of buttermilk powder or buttermilk addition on phospholipid content, chemical and bio-chemical composition and bacterial viability in Cheddar style-cheese.

The effect of buttermilk powder addition post-curd formation or buttermilk addition to cheese milk on total and individual phospholipid content, chemical composition, enzyme activity, microbial populations and microstructure within Cheddar-style cheese was investigated. Buttermilk or buttermilk powder addition resulted in significant increases in total phospholipid content and their distribution throughout the cheese matrix. Addition of 10% buttermilk powder resulted in higher phospholipid content, moisture, pH and salt in moisture levels, and lower fat, fat in dry matter, L. helveticus and non-starter bacteria levels in cheeses. Buttermilk powder inclusion resulted in lower pH4.6/Soluble Nitrogen (SN) levels and significantly lower free amino acid levels in 10% buttermilk powder cheeses. Buttermilk addition provided a more porous cheese microstructure with greater fat globule coalescence and increased free fat pools, while also increasing moisture and decreasing protein, fat and pH levels. Addition of buttermilk in liquid or powdered form offers potential for new cheeses with associated health benefits.

Membrane lipids of Rhodopseudomonas viridis.

In search of the precyanobacterial origin of the typical thylakoid lipids found in cyanobacteria and chloroplasts, we analyzed the polar lipids of the anaerobic phototrophic bacterium Rhodopseudomonas viridis. Glycolipids (monogalactosyl-, digalactosyl- and glucuronosyl diacylglycerol), phospholipids (phosphatidyl choline, -ethanolamine, -glycerol and cardiolipin) and an ornithine lipid were isolated and identified by NMR (1H, 13C, 31P) and mass spectrometry. Positional distribution and pairing of fatty acids in molecular species show small, but significant differences between glyco- and phospholipids. In this context, a new enzymatic method is described for assigning the enantiomeric structure of the diacylglycerol moiety in glyco- and phospholipids. 14C-Labelling studies suggest that monogalactosyl diacylglycerol is formed by galactosylation of diacylglycerol as in chloroplasts and not by glucosylation followed by epimerization as in cyanobacteria. The two 1,6-linked galactopyranose residues of digalactosyl diacylglycerol are both in beta-linkage and thus differ from the corresponding chloroplast lipid with its alpha-beta-sequence. R. viridis does not contain the sulfolipid, and even phosphate starvation does not induce the synthesis of this most characteristic thylakoid lipid, which on the other hand is present in other anaerobic phototrophic bacteria.

Molecular analysis of Saccharomyces cerevisiae chromosome I: identification of additional transcribed regions and demonstration that some encode essential functions.

Saccharomyces cerevisiae chromosome I has provided a vivid example of the "gene-number paradox." Although molecular studies have suggested that there are greater than 100 transcribed regions on the chromosome, classical genetic studies have identified only about 15 genes, including just 6 identified in intensive studies using Ts- lethal mutations. To help elucidate the reasons for this disparity, we have undertaken a detailed molecular analysis of a 34-kb segment of the left arm of the chromosome. This segment contains the four known genes CDC24, WHI1, CYC3 and PYK1 plus at least seven transcribed regions of unknown function. The 11 identified transcripts have a total length of approximately 25.9 kb, suggesting that greater than or equal to 75% of the DNA in this region is transcribed. Of the transcribed regions of unknown function, three are essential for viability on rich medium and three appear to be nonessential, as judged by the lethality or nonlethality of deletions constructed using integrative transformation methods. No obvious phenotypes were associated with the deletions in the apparently nonessential genes. However, two of these genes may have homologs elsewhere in the genome, as judged from the appearance of additional bands when DNA-DNA blot hybridizations were performed at reduced stringency. Taken together, the results provide further evidence that the limitations of classical genetic studies of chromosome I cannot be explained solely by a lack of genes, or even a lack of essential genes, on the chromosome.

Worsening seizures after surgery for focal epilepsy due to emergence of primary generalized epilepsy.

Our patient underwent right anteromesial temporal resection at 17 years of age for intractable complex partial seizures due to hippocampal sclerosis, and then developed juvenile myoclonic epilepsy after a change in medication. Postoperative seizures ceased after a change to valproate monotherapy. Our patient reminds us to remain aware that generalized and focal epilepsy may coexist as an unusual cause for surgical failure. We feel that these patients may still be favorable candidates for epileptic surgery, as long as the focal epileptogenic zone is amenable to resection and the generalized epilepsy appears to be readily controllable.

Evidence of cingulate motor representation in humans.

A 44-year-old man with a right frontal lobe tumor and intractable seizures underwent subdural grid evaluation before resection. The electrode locations were identified on a three-dimensional surface-reconstructed image of the brain after subdural grid placement. Electrical stimulation of electrodes placed over the right cingulate gyrus revealed evidence of tonic posturing of the left forearm and wrist and tonic extension of the left leg. This finding provides further evidence of a motor area in the cingulate gyrus in humans.

Fluid-attenuated inversion recovery: correlations of hippocampal cell densities with signal abnormalities.

BACKGROUND: Hippocampal sclerosis (HS) is characterized by hippocampal atrophy and increased signal on T2-weighted images and on fluid-attenuated inversion recovery (FLAIR) images. OBJECTIVE: To quantitate cell loss and compare it with signal abnormalities on FLAIR images. METHODS: Thirty-one patients with temporal lobe resection, pathologically proven HS, and Engel class I and II outcome were included: 20 with HS only and 11 with HS associated with pathologically proven cortical dysplasia (dual pathology). The signal intensity on FLAIR was rated as present or absent in the hippocampus and correlated with the neuronal losses in the hippocampus. RESULTS: FLAIR signal increases were present in 77% (24/31) of all patients studied. In patients with isolated HS, 90% (18/20) had ipsilateral signal increases, but in patients with dual pathology, only 55% (6/11; p < 0.02) showed FLAIR signal increase. Hippocampal cell losses were significantly higher in the isolated HS group. The average cell loss in patients with FLAIR signal abnormalities was 64.8 +/- 8.0% as compared with only 32.7 +/- 5.1% in patients with no FLAIR signal abnormalities. There was a significant positive correlation between the presence of signal abnormality and average hippocampal cell loss in both pathologic groups. CONCLUSIONS: Ipsilateral FLAIR signal abnormalities occur in the majority of patients with isolated HS but are less frequent in those with dual pathology. The presence of increased FLAIR signal is correlated with higher hippocampal cell loss.

Disturbed benzodiazepine receptor function at the onset of temporal lobe epilepsy--lomanzenil-binding in de-novo TLE.

PURPOSE: Epileptogenic foci exhibit disturbed function at the level of the benzodiazepine receptor. The aim of our study was to investigate the incidence of focal reductions of temporal benzodiazepine receptor binding (BRB) as assessed by scintigraphy with 123I-iomazenil in patients with denovo temporal lobe epilepsy (TLE). METHODS: Forty adult patients (age: 34+/-12 years) with cryptogenic denovo TLE underwent scintigraphy with 123I-iomazenil. In all patients, symptomatic epilepsy was excluded by clinical investigation and MRI. The median duration of TLE was seven months, and the patients had a median of three documented seizures in their history of disease. BRB was quantified in four temporal regions covering the whole temporal lobe. Temporal asymmetry values (ASY) were compared with data determined in 13 age-matched controls yielding Z-scores for global and regional temporal BRB. RESULTS: A significant reduction of temporal BRB was found in 19 of the 40 patients (48 %), mainly in mesial temporal regions; temporal BRB asymmetries were also found in patients with a short history of seizures and low seizure frequency (< or = 1 year; n = 32, 13/32 (41 %)). Only in the entire cohort did the magnitude of temporal reduction of BRB correlate with the duration of TLE as well as with the number of previous partial seizures (r = 0.40 and r = 0.36; p < 0.03, respectively). CONCLUSIONS: Foci of decreased BRB can already be detected at the onset of TLE; their magnitude is related to ongoing epileptic activity.

Spontaneous oscillations in cerebral blood flow velocity in normal humans and in patients with carotid artery disease.

Spontaneous oscillations in cerebral blood flow velocity (CBFV) in normals and in patients with stenoses or occlusions of the internal cerebral artery were measured using transcranial Doppler sonography. In normal subjects, large oscillations of up to +/- 30% from the mean CBFV were found with low frequencies between 0.4 and 9 cycles/min. No correlations between CBFV oscillations and systemic circulatory parameters were detected. In patients with carotid artery obstructions the CBFV oscillations were significantly reduced in the middle cerebral artery ipsilaterally to a hemodynamically significant lesion, but not contralaterally. Our results support the hypothesis that spontaneous oscillations (B-waves) of small pial vessels are responsible for the CBFV fluctuations.

Persistent unihemispheric perceptual impairments in humans following focal seizures.

Perception has been linked to a highly coordinated activation of cortical regions whose functional organization and performance is subject to plastic changes. We tested whether chronic repetitive disturbances of the brain by focal epileptic activity have a long-standing detrimental effect on the perceptual performance in the affected hemisphere. Nine patients were examined who had a history of complex partial seizures but no structural cerebral damage on magnetic resonance imaging and no evidence of ongoing epileptic activity on scalp electroencephalography and who had clinically been without seizures for at least 3 days. The side of primary epileptic involvement was determined by seizure semiology (n = 2), focal electroencephalographic slowing (n = 3) or focal abnormality during single photon emission topography (SPECT) (n = 4). The computer controlled psychometric assessment of the somesthetic frequency discrimination revealed that the perception in the hand corresponding to the affected hemisphere was impaired relative to the contralateral hand (P < 0.01), and to the performance of a group of normal controls (P < 0.01). We conclude that mechanisms related to focal epileptic activity can result in regional perceptual decrements even when there is no clinical or surface-electroencephalographic evidence of epileptic discharges. This in turn suggests that somatosensory testing may be of help in localizing, or at least lateralizing an epileptic focus.

A double-blind comparison of amitriptylinoxide versus doxepine in the treatment of severe depression.

In a double-blind parallel group study the efficacy and safety of amitriptylinoxide were evaluated vs. doxepine in the treatment of in-patients with severe depression. Two groups of 22 patients each received amitriptylinoxide and doxepine respectively at a daily dosage of 120-360 mg for a period of 4 weeks. The total score on the Hamilton Depression Scale (HAMD) was reduced with amitriptylinoxide on an average from 28 +/- 5 before treatment to 12 +/- 8 at the end of treatment, with doxepine from 29 +/- 8 to 13 +/- 11. Of the amitriptylinoxide-treated patients, 12 showed a more than 50% reduction in this score compared with 15 under doxepine. The difference was not statistically significant. Twenty patients in each group experienced adverse drug reactions, the percentage of anticholinergic side effects being equal in the two groups.

PET and SPECT in medically non-refractory complex partial seizures. Temporal asymmetries of glucose consumption, benzodiazepine receptor density, and blood flow.

AIM: In contrast to medically refractory complex partial seizures (CPS), only limited knowledge exists on cerebral perfusion and metabolism in medically non-refractory CPS. The aim of this study was to investigate the frequency of temporal asymmetries in regional cerebral glucose consumption (rCMRGlc), regional cerebral blood flow (rCBF), and regional cerebral benzodiazepine receptor density (BRD) in this group of patients. METHODS: The study included 49 patients with medically non-refractory cryptogenic CPS (age: 36.0 +/- 16.1 years). rCMRGlc was studied with F-18-FDG-PET (FDG), rCBF with Tc-99m-ECD-SPECT (ECD), and BRD with l-123-iomazenil-SPECT (IMZ). All studies were performed interictally and within four weeks in each patient. Duration of epilepsy ranged from 0.1 to 42 years (median 4.0 years). SPECT was performed with the triple-headed SPECT camera Multispect 3, PET with the PET camera ECAT EXACT 47. Using linear profiles, glucose consumption, as well as uptake of ECD and IMZ, were measured in four temporal regions of interest (ROIs), and asymmetry indices were calculated (ASY). The results were compared to 95% confidence intervals determined in control subjects. RESULTS: Thirty-five of the 49 (71%) patients had at least one significantly elevated ASY; temporal rCMRGlc was asymmetrical in 41% of the patients, temporal BRD in 29%, and temporal rCBF in 24%. One patient had an asymmetry of all three variables, two of temporal rCMRGlc and BRD, three of temporal rCMRGlc and rCBF, and another four of rCBF and BRD. Fourteen patients had an isolated temporal asymmetry in rCMRGlc, seven in BRD, and four in rCBF. A discrepancy in lateralization between the three modalities was not observed. CONCLUSION: The majority of patients with medically non-refractory CPS have focal abnormalities of blood flow and metabolism in their temporal lobe. In this group of patients, FDG-PET demonstrates abnormalities with the highest frequency of the three modalities studied, followed by IMZ-SPECT, and ECD-SPECT.

Measurement of temporal asymmetries of glucose consumption using linear profiles: reproducibility and comparison with visual analysis.

AIM: One approach to regionally analyze temporal glucose consumption consists in drawing linear profiles over the maximal values measured in the temporal cortical ribbon. The aim of our study was to test the reproducibility of this method and to compare its diagnostic performance to that of visual analysis in patients with complex partial seizures (CPS). METHODS: Regional cerebral glucose consumption (rCMRGIc) was measured interictally in 25 CPS patients and 10 controls using F-18-deoxyglucose and the positron emission tomography (PET) camera ECAT EXACT 47. The PET scans were visually analyzed for the occurrence of unilateral temporal hypometabolism. Furthermore, rCMRGIc was quantified on six contiguous coronal planes by manually tracing maximal values of temporal glucose consumption, thus creating line profiles of temporal glucose consumption for each side. Indices of asymmetry (ASY) were then calculated from these line profiles in four temporal regions and compared to the corresponding 95% confidence intervals of the control data. All analyses were performed by two observers independently from each other and without knowledge of the clinical findings. RESULTS: The agreement between the two observers with regard to focus lateralization was 96% (kappa = 0.93) on visual analysis and 100% (kappa = 1) on quantitative analysis. There was an excellent agreement with regard to focus lateralization between visual and quantitative evaluation (kappa = 0.8). CONCLUSION: Quantitation of local temporal rCMRGIc by using linear profile analysis is highly reproducible; for the lateralization of epileptogenic foci, however, this method does not possess significant advantages over the visual evaluation of the scans.

NMR spectroscopy in pharmacy.

Since drugs in clinical use are mostly synthetic or natural products, NMR spectroscopy has been mainly used for the elucidation and confirmation of structures. For the last decade, NMR methods have been introduced to quantitative analysis in order to determine the impurity profile of a drug, to characteristic the composition of drug products, and to investigate metabolites of drugs in body fluids. For pharmaceutical technologists, solid state measurements can provide information about polymorphism of drug powders, conformation of drugs in tablets etc. Micro-imaging can be used to study the dissolution of tablets, and whole-body imaging is a powerful tool in clinical diagnostics. Taken together, this review covers applications of NMR spectroscopy in drugs analysis, in particular, methods of international pharmacopoeiae, pharmaceutics and pharmacokinetics. The authors have repeated many of the methods describe in their own laboratories.

Hamartomas and epilepsy: clinical and imaging characteristics.

PURPOSE: Cerebral hamartomas are lesions marked by a disorganized arrangement of mature neural elements and represent a rare cause of medically intractable focal epilepsy. We present the clinical presentation and imaging findings of this rare entity. METHODS: History and neurophysiological studies of 14 patients with pathologically confirmed hamartomas who had surgery for intractable focal epilepsy were reviewed. MRIs were available for review in 10 patients. RESULTS: The lesions were most commonly located in the temporal and frontal lobes. Seizure semiology was concordant with the anatomic location of the hamartoma in all patients. Nine of the thirteen patients (69%) with the hamartoma confined to one lobe had interictal spikes and sharp waves at the corresponding electrodes. The ictal pattern was confined to the same lobe of the hamartoma in five of nine patients with ictal recordings. Although imaging characteristics were variable, all patients had signal increase on T2-weighted images and 50% of them had mild mass effect. Neocortical involvement was present in the majority of patients (7/10), blurring of the gray/white matter interface was seen in seven patients. Five of those seven patients were found to have associated cortical dysplasia by pathology. CONCLUSION: Hamartomas represent a rare entity and may cause devastating epilepsy. Imaging characteristics are difficult to distinguish from those of some other developmental tumors. Hamartomas are frequently associated with microscopic cortical dysplasia (CD), thus underlining their malformative etiology.