Mechanism of action of and mechanism of reduced susceptibility to the novel anti-Clostridium difficile compound LFF571.

LFF571 is a novel semisynthetic thiopeptide and potent inhibitor of Gram-positive bacteria. We report that the antibacterial activity of LFF571 against Clostridium difficile is due to inhibition of translation. Single-step mutants of C. difficile with reduced susceptibility to LFF571 were selected at frequencies of <4.5 × 10(-11) to 1.2 × 10(-9). Sequencing revealed a G260E substitution in the thiopeptide-binding pocket of elongation factor Tu. Importantly, this mutation did not confer cross-resistance to clinically used antimicrobials. These results support the development of LFF571 as a treatment for C. difficile infection.

Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

Type III phosphatidylinositol-4-kinase beta (PI4KIIIß) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIß inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIß in lymphocyte proliferation. Previously proposed functions of PI4KIIIß in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIß inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

In vitro and in vivo activities of novel, semisynthetic thiopeptide inhibitors of bacterial elongation factor Tu.

Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the in vitro and in vivo activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC(90) ≤ 0.25 µg/ml) but weaker against the streptococci (MIC(90) ≥ 4 µg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the tuf gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal S. aureus infections with 50% effective doses (ED(50)) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic E. faecalis infections with ED(50) of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active in vitro and in vivo activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.

Antihyperglycaemic medication modifies factors of postprandial satiety in type 2 diabetes.

AIM: Type 2 diabetes is characterized by hyperglycaemia, delayed gastric emptying and a blunted response of gut hormones during feeding that may modulate satiety. We hypothesized that it is associated with more hunger when treated by medication. METHODS: We studied nine type 2 diabetic men (A1C: 6.7+/-0.3%, waist circumference: 104+/-4 cm) after an overnight fast, during 5 h in response to a 2.88 MJ breakfast, twice, in a crossover design, with or without antihyperglycaemic agents. Satiety ratings, thermic effect of meal, gastric emptying, plasma concentrations of gut peptides, leptin, insulin and substrates and intake from a subsequent buffet were determined. RESULTS: With medication, fasting and postprandial plasma glucose levels were lower but area under the curve (AUC) did not vary vs. without medication. Gastric emptying was shortened, branched chain amino acids (BCAA) AUC and thermic effect were lower, and postprandial glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY3-36) were maintained at higher levels beyond 4 h. Correlations were significant between duration of diabetes and fasting ghrelin (r=0.779, p=0.013) and peak insulin (r=-0.769, p=0.016), 5-h postmeal ghrelin and peak glucose (r=0.822, p=0.007), 5-h glucose and GLP-1 (r=-0.788, p=0.012), and 5-h hunger scores and energy intake at buffet (r=0.828, p=0.006). Without medication, fullness scores correlated with BCAA levels. Visual analogue scale scores, ghrelin and leptin levels did not differ between studies. CONCLUSIONS: The decrease in factors associated with postprandial satiety with treatment is counterbalanced by higher GLP-1 and PYY3-36. Medication may normalize the link between perception of hunger and subsequent food intake.

Identification of spermine as an inhibitor of erythropoiesis in patients with chronic renal failure.

Fetal mouse liver and normal human bone marrow cell cultures were used for studies on the inhibition of erythroid colony formation (CFU-E) by sera from anemic patients with end-stage renal failure and the polyamine spermine. Sera from each of eight predialysis uremic anemic patients with end-stage renal failure produced a significant (P < 0.001) inhibition of erythroid colony formation in the fetal mouse liver cell cultures when compared to sera from normal human volunteers. In vivo or in vitro dialysis of the uremic sera with a 3,500-dalton exclusion limit membrane removed the inhibitor from uremic sera. The uremic serum dialysate provided by the membrane fractionation was significantly inhibitory in the erythroid cell cultures. When this dialysate was applied to gel filtration chromatography (Bio-Gel P-2) the inhibitor was found to be in the same molecular weight range as [(14)C]spermine. The polyamine spermine produced a dose-related inhibition of erythroid colony formation (CFU-E) in fetal mouse liver and normal human bone marrow cultures. Thus, the following evidence is provided that the in vitro inhibitor of erythropoiesis found in chronic renal failure patients' sera is identical with the polyamine spermine: (a) the inhibitor and radiolabeled spermine appeared in identical Bio-Gel P-2 effluent fractions; (b) when spermine was added to normal human sera at concentrations reported in sera of uremic patients, and studied in both the fetal mouse liver cell culture and normal human bone marrow cultures, a dose-related inhibition of erythroid colony (CFU-E) formation was noted; and (c) the inhibitory effects of crude uremic serum, uremic serum dialysate, and fractions of uremic serum dialysate from a Bio-Gel column, on erythroid colony formation were completely abolished by the addition of a specific rabbit antiserum to spermine.

The relationship between Taxol and (+)-discodermolide: synthetic analogs and modeling studies.

BACKGROUND: During the past decade, Taxol has assumed an important role in cancer chemotherapy. The search for novel compounds with a mechanism of action similar to that of Taxol, but with greater efficacy particularly in Taxol-resistant cells, has led to the isolation of new natural products. One such compound, (+)-discodermolide, although structurally distinct from Taxol, has a similar ability to stabilize microtubules. In addition, (+)-discodermolide is active in Taxol-resistant cell lines that overexpress P-glycoprotein, the multidrug-resistant transporter. Interestingly, (+)-discodermolide demonstrates a profound enhancement of the initiation process of microtubule polymerization compared to Taxol. RESULTS: The synthesis of (+)-discodermolide analogs exploiting our highly efficient, triply convergent approach has permitted structure-activity relationship (SAR) studies. Small changes to the (+)-discodermolide structure resulted in a dramatic decrease in the ability of all four discodermolide analogs to initiate tubulin polymerization. Two of the analogs also demonstrated a decrease in total tubulin polymerization, while a change in the olefin geometry at the C8 position produced a significant decrease in cytotoxic activity. CONCLUSIONS: The availability of (+)-discodermolide and the analogs, and the resultant SAR analysis, have permitted an exploration of the similarities and differences between (+)-discodermolide and Taxol. Docking of the X-ray/solution structure of (+)-discodermolide into the Taxol binding site of beta-tubulin revealed two possible binding modes (models I and II). The preferred pharmacophore model (I), in which the C19 side chain of (+)-discodermolide matches with the C2 benzoyl group of Taxol and the delta-lactone ring of (+)-discodermolide overlays with the C13 side chain of Taxol, concurred with the results of the SAR analysis.

Solution structure of (+)-discodermolide.

[structure: see text]. The solution structure of (+)-discodermolide (1) has been determined via 1- and 2-D NMR techniques in conjunction with Monte Carlo conformational analysis. Taken together, the results demonstrate that in solution (+)-discodermolide occupies a helical conformation remarkably similar to the solid state conformation.

Gram-scale synthesis of (+)-discodermolide.

[formula: see text] A triply convergent, highly efficient second-generation synthesis of the potent antimitotic agent (+)-discodermolide (1) has been achieved on a 1-g scale.

The impact of a decision process upon scalp recorded premovement potential.

Twelve normal subjects were asked to select one finger after a warning signal and to move it. Using different modes of averaging the EEG responses, it is shown that: (i) the selection of a finger is accompanied by a distinct negative slow potential consisting of a rapid set-up and a following plateau which is the most prominent under the Fz electrode, and (ii) the decision process, when dispersed within a large interval in repeated trials, gives rise to a slow progressive increase of negativity resembling the early component of premovement slow cortical potential from the classical self-paced paradigm.

The early component of the premovement readiness potential and its behavioral determinants.

Fifteen healthy subjects were asked to randomly select a tick of a mechanical clock which was operating throughout the experiment and to flex their wrist at the chosen tick. The individual averages of EEG recordings collected during the period of the putative decision to begin the task exhibited steep slopes of negativity followed by plateaus. These slopes preceded the selection of the signal sound at varying intervals, the longest interval was 3 s. The grand average of individual averages depicted a waveform whose shape was identical to the early component of the classical readiness potential. Another interesting finding came from experiments with mental counting of clock sounds, which demonstrated that the generation of the first mentally-counted number is preceded by a distinct negative potential shift.

A reappraisal of rat motor cortex organization by intracortical microstimulation.

The organization of the motor cortex was reinvestigated with intracortical microstimulation, in light-anaesthetized (ketamine) rats. A posterolateral (PL) vibrissae area was found in addition to the rostral one, and blinks of the contralateral eyelids were elicited from a part of this PL area. Several cortical representations such as neck or tail were largely overlapping with neighbouring areas. Vegetative effects (mainly myosis and swallowing) were obtained from a medial cortical strip. Within the PL vibrissae area, a topical arrangement related to the vibrissal rows was observed, whereas in the leg areas, no individual representation of muscles could be evidenced. These results are compared with the maps previously published, and discussed in terms of specificity, musculotopy and overlapping of motor areas.

Steep early negative slopes can be demonstrated in pre-movement bereitschaftspotential.

OBJECTIVES: The study presents data suggesting that the classic bereitschaftspotential hides in its early component (BP1) steep increases of negativity which precede the movement at varying intervals in repeated trials. METHODS: In 12 volunteers, surface EEG from Fz, Cz, C3, C4, and Pz electrodes and EMG from the flexor digitorum communis were recorded during self-paced wrist flexions. Two hundred trials were collected from each subject. The single trials were grouped for averaging in two different ways. In the first one, single trials for each subject were used to create individual averages. In the second, all single trials were divided into groups according to the point in time of small steep negative shift from the baseline detected on sweeps before the movement and time group averages were created. The identification of small shifts was based on the comparison of calculated mean amplitudes in the first and the second half of the 1 s time window moved along the time axis. RESULTS: The small negative shifts were identified in 97.2% of analyzed records. In each subject, their position on the time axis varied considerably. Individual averages exhibited the characteristics of classical readiness potential, i.e. slow early component, steep late component, laterality over motor cortices. On the other hand, all time group averages (26) displayed an early steep negative shift followed by plateau which, about 0.5 s before the movement, gave rise to the second, late steep negative shift. The slope values calculated in the definite segments of averaged curves were used as a measure of the amplitude of these shifts over various brain areas. MANOVA showed a significant effect of the electrode position both in the case of early slopes (F(4,115)=9.7; P<0.000) and in the case of late slopes (F(4,115)=22.5; P<0.000). In both cases, the largest value was demonstrated under Cz electrode. In contrast to the late slopes, the values of early slopes did not exhibit laterality and suggested greater importance of pre-frontal regions in their formation. CONCLUSION: We have suggested that the formation of steep early negative shifts from the baseline in time group averages was due to synchronization of a mental process which, under classical averaging procedure, was dispersed uniformly throughout the pre-movement period.

Parieto-temporal rhythms in the 6-9 Hz band recorded in epileptic patients with depth electrodes in a self-paced movement protocol.

OBJECTIVES: Description of 6-9 Hz rhythmic electrical activity observed on recordings from electrodes implanted in the cortex of epileptic patients undergoing presurgical evaluation. METHODS: Recordings were obtained from 74 patients with multilead electrodes in the frontal, parietal and temporal cortex. The motor task consisted of a self-paced fist clenching movement at approximately 10 s intervals. Events within a window extending from 4 s before to 1 s after movement EMG onset were analyzed. RESULTS: (i) Spindle-like rhythmic activity at 6-9 Hz was observed in 29 patients. (ii) This activity was located in the inferior parietal and superior temporal areas. (iii) Enhancement of rhythmic activity occurred when patients were asked to perform the motor task. (iv) A striking tendency to phase-locking of rhythmic oscillations on consecutive trials was noted during the 3-2 s epoch before movement EMG onset. CONCLUSION: Whether this intracerebrally recorded 6-9 Hz rhythmic activity belongs to the mu-alpha class or is a special type of theta, and if it is related to the epileptic process or to drug treatment remain open to discussion. Motor-task related enhancement and phase-locking suggest that this activity may be one more indicator of movement preparation.

Presence of very early events preceding self-paced movements in epileptic patients. An intracerebral exploration.

In epileptic patients explored with depth electrodes kept within the brain for several days ("chronic conditions") to localize their focus, recordings were taken from the less perturbed deep brain areas, before and during the execution of self-paced movements. A few deep leads (some of which were located in areas which did not display a classical readiness potential) showed that phasic paroxystic events very often occurred 2,000 ms and, sometimes, even about 3,000 ms before the movement (movement-related events, MREs). This finding suggests that the preparation of a "voluntary" self-paced movement may begin well before the appearance of the readiness potential or, alternatively, that intracerebral paroxystic spikes delay the decision making. This type of phasic process may be undetectable in scalp recording from normal subjects and only be revealed when some brain structures have become hyper-reactive due to the epileptic process.

Suppression of motor seizures after specific thalamotomy in chronic epileptic monkeys.

In the monkey with a chronic motor epileptogenic focus, thalamic lesions restricted to the anterior part of the ventro-postero-lateral nucleus produced a long-lasting improvement, leading in most cases to almost complete seizure suppression. This improvement was attributed to the interruption of a long-loop recurrent excitatory mechanism, involving muscular afferents which are known to relay in the thalamic region where lesions were produced. The specific nature of this effect was suggested by the preliminary results of lesions performed in thalamic regions with non-specific cortical projections. Compared with the pre-lesional situation in which focal activity resulted in permanent motor troubles, the post-lesional state did not show any additional motor deficit. It is concluded that such lesions could be a new approach to controlling intractable motor epilepsies in man.

Can epileptologists without access to intracranial EEG use reliably the International League Against Epilepsy classification of the localization-related epileptic syndromes? A stereo-electroencephalographic study.

The goal of the present study was to investigate the reliability of clinical and electroencephalographic (EEG) criteria for the classification of localization-related epileptic syndromes as listed in the Proposals of Revised Classification of Epilepsies and Epileptic Syndromes 1989 (ICE). ICE distinguishes between multiple syndromes within epilepsies of a given lobe. Intracranial recordings were the main element in the development of the revised ICE. Considering that most epilepsy centers have no access to such invasive techniques for precise anatomic localization, it was of interest to assess how accurately the seizure origin could be determined from the scalp EEG and clinical data as reported in ICE. In this retrospective study, we compared the accuracy of the topographic diagnosis made by two groups of physicians evaluating the same patients-one group with and the other without access to results of stereo-EEG (SEEG). Medical files of 87 patients with intractable localization-related epilepsy were analyzed: 38 with frontal, 37 with temporal, 10 with parietal, and 2 with occipital lobe epilepsy were included in the study. All patients underwent previous SEEG and successful cortectomy. Minimum follow-up was 5 years. In most cases, noninvasive techniques and criteria suggested by ICE allowed topographic diagnosis of focal epilepsies according to brain lobe involvement. More detailed diagnosis, localizing the origin of critical activity within a lobe, was often unreliable. Further data are required for a definition of the epileptogenic zone. A spatiotemporal evaluation of critical events, including the intracranial EEG recording, remains the best method for topographic diagnosis of localization-related epilepsy.

Synchronizing effect of clock rhythm on the 'when to move' decision in repeated voluntary movements.

In 13 volunteers, surface EEGs from F(z), C(z), C(3), C(4), and P(z) electrodes and EMG from the flexor digitorum communis were recorded during an experimental task in which the performance of wrist flexion was linked with the selection of a clock sound. In the epoch from 4 to 1 s before movement, more than 95% of the records exhibited a small negative shift from the baseline. The position of these shifts on the time axis made it possible to create groups of time trials. The average EEG curves from these groups showed one prominent negative shift from the baseline, which could be related to the position of particular clock sounds. The slope values of these shifts were highest under the F(z) and C(z) electrodes. We suggested that these negative shifts coincided with 'when to move' decisions, which preceded the execution of the movement at varying intervals.

Instability of immunoglobulins and complement (C3) as predictor of renal allograft rejection.

Ten renal allograft recipients were studied for a period of three months with weekly measurements of serum complement C3 including a baseline value before transplantation. Immunoglobulins (IgG, IgM, IgA) were studied in nine of these patients. Fifteen rejection episodes occurred, three patients losing their graft because of irreversible rejections. The patients were followed for 2.5 years. The degree of of change in serial C3 and immunoglobulin levels from baseline was determined by variability index s. Fourteen out of 15 rejections were preceded by si (C3) greater than .1, one to several weeks in advance. The si (Ig) was useful in week 1 and predicted the rejection missed by si (C3). We conclude that instability of immunoglobulins and complement as measured by s is not only associated with rejections but appears to be a precursor to rejection events. We suggest the following prognostic two-step procedure: (1) Week 1: If either si (C3) or si (Ig) greater than .1, predict rejection. (2) Cases that pass step 1 are monitored by C3 at weekly intervals. As soon as si (C3) greater than .1, predict rejection.

Event-related potentials, CNV, readiness potential, and movement accompanying potential recorded from posterior thalamus in human subjects. A SEEG study.

Intracranial recordings were obtained from three patients with intractable chronic pain who underwent analgesic electrical stimulation of the contralateral thalamus. Multilead electrode made it possible to record from several thalamic nuclei. The electrode was targeted into the ventroposterolateral (VPL) nucleus of the thalamus. During separate recording sessions, the following tests were performed: somatosensory evoked potentials (SEP) of the median or posterior tibial nerve, event-related cognitive potentials (auditory oddball P3 wave), readiness potential (RP) and contingent negative variation (CNV) using auditory warning (S1) and visual imperative (S2) stimuli. The movement accompanying potential (MAP), which was present in the VPL in all but one of the recordings, behaved as a far-field potential. Recordings obtained from the VPL confirmed its established role as a relay nucleus, processing somatosensory information to the primary somatosensory cortex. The VPL generated the 'thalamic' SEP, which was the only potential regularly recorded in this nucleus. In the recordings from one patient (No. 3), auditory and visual evoked potentials of the CNV protocol, peaking at approximately 300 ms, were obtained from the VPL and appeared to be generated in situ. Neither RP, CNV nor 'oddball' ERPs appeared in the VPL. From the pulvinar, only a visually evoked potential was recorded. Oddball P3, RP, CNV, and middle and long latency auditory and visual potentials (evoked in the CNV paradigm) appeared to be generated 'dorsally' to the VPL, probably in the nucleus posterolateralis (PL). This structure may therefore be involved in both the processing of afferent information and in cognitive operations.

Readiness potentials related to self-initiated movement and to movement preceded by time estimation: a comparison.

Two procedures for eliciting premovement potentials were compared: (1) the estimation of a 3 s interval elapsed after a warning auditory signal, and (2) classical "self-pacing". Eleven healthy right-handed subjects participated in the experiment, EEG records from scalp electrodes placed at CZ, C3+ and C4+ were analyzed. It has been shown that both procedures induced similar premovement potentials except that in the first procedure the early component of the potential was longer. The time estimation itself induced a negative slow potential consisting of a rapid set-up and a subsequent plateau.