Deciphering Tacrolimus-Induced Toxicity in Pancreatic ß Cells.

ß Cell transcription factors such as forkhead box protein O1 (FoxO1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), pancreatic and duodenal homeobox 1, and neuronal differentiation 1, are dysfunctional in type 2 diabetes mellitus (T2DM). Posttransplant diabetes mellitus resembles T2DM and reflects interaction between pretransplant insulin resistance and immunosuppressants, mainly calcineurin inhibitors (CNIs). We evaluated the effect of tacrolimus (TAC), cyclosporine A (CsA), and metabolic stressors (glucose plus palmitate) on insulinoma ß cells in vitro and in pancreata of obese and lean Zucker rats. Cells were cultured for 5 days with 100 µM palmitate and 22 mM glucose; CsA (250 ng/mL) or TAC (15 ng/mL) were added in the last 48 h. Glucose plus palmitate increased nuclear FoxO1 and decreased nuclear MafA. TAC in addition to glucose plus palmitate magnified these changes in nuclear factors, whereas CsA did not. In addition to glucose plus palmitate, both drugs reduced insulin content, and TAC also affected insulin secretion. TAC withdrawal or conversion to CsA restored these changes. Similar results were observed in pancreata of obese animals on CNIs. TAC and CsA, in addition to glucose plus palmitate, induced comparable inhibition of calcineurin and nuclear factor of activated T cells (NFAT); therefore, TAC potentiates glucolipotoxicity in ß cells, possibly by sharing common pathways of ß cell dysfunction. TAC-induced ß cell dysfunction is potentially reversible. Inhibition of the calcineurin-NFAT pathway may contribute to the diabetogenic effect of CNIs but does not explain the stronger effect of TAC compared with CsA.

Creutzfeldt-Jakob disease and non-convulsive status epilepticus: a clinical and electroencephalographic follow-up study.

OBJECTIVE: To describe the clinical and electroencephalographic findings from a confused elderly woman with Creutzfeldt-Jakob disease (CJD) that initially were compatible with the diagnosis of non-convulsive status epilepticus (NCSE). METHODS AND RESULTS: A 75-year-old right-handed woman was admitted to our hospital because of confusion and alteration of mental status. The two first electroencephalograms (EEGs) showed continuous diffuse spikes, rhythmic sharp waves and sharp-and-slow wave complexes which were completely abolished after the administration of 10 mg of intravenous diazepam. Over the following days, the clinical state of the patient was unmodified despite aggressive antiepileptic therapy. A third EEG revealed pseudo-periodic negative or positive-negative slow waves localised in the right frontal region. Subsequently, two consecutive EEGs showed continuous periodic generalised bi-triphasic complexes at a rate of 1 Hz, compatible with the diagnosis of CJD. Finally, the patient died, and postmortem examination was diagnostic of the sporadic form of CJD. CONCLUSIONS: Clinical and electroencephalographic features in the early stages of CJD may resemble NCSE. The administration of intravenous benzodiazepines and its clinical and electroencephalographic correlation, response to the antiepileptic therapy, and monitoring with serial EEG recordings may be helpful considerations in the differential diagnosis.

Increasing the number of synapses modifies olfactory perception in Drosophila.

The Drosophila mutant gigas produces an enlargement of postmitotic cells caused by additional rounds of DNA replication. In neurons, the mutant cell establishes more synapses than normal. We have taken advantage of this feature to study the effect of synapse number on odorant perception. Mosaic adults were generated in which one antenna was homozygous for gigas, whereas the contralateral side served as an internal control. Morphological analysis indicates that the number and type of sensory afferents forming the mutant antenna, as well as their projection to the olfactory glomeruli, are normal. In contrast, the volume of identified glomeruli increases to a variable extent, and mutant sensory neurons branch profusely. The number of synapses, estimated in the ventral (V) glomerulus that receives ipsilateral afferents only, is increased twofold to threefold. Large-dense-core vesicle-containing terminals that probably modulate olfactory centers are identified in the V glomerulus. Their number and size are not modified by the mutant input. Sensory transduction, measured by electroantennograms, is normal in amplitude and kinetics. In odorant tests, however, the profile of the behavioral response to ethyl acetate shows attractive responses to concentrations to which sibling controls remain indifferent (10(-)8 and 10(-)7 v/v). In addition, the intensity of the response is augmented both at attractive and repulsive odorant concentrations with respect to that of controls. These results demonstrate that increased synapse number in the sensory neurons can modify the behavior of the organism, allowing a higher sensitivity of perception.

Odor exposure causes central adaptation and morphological changes in selected olfactory glomeruli in Drosophila.

In an attempt to correlate behavioral and neuronal changes, we examined the structural and functional effects of odor exposure in Drosophila. Young adult flies were exposed to a high concentration of the selected odor, usually benzaldehyde or isoamyl acetate, for 4 d and subsequently tested for their olfactory response to a variety of odorants and concentrations. The behavioral response showed specific adaptation to the exposed odor. By contrast, olfactory transduction, as measured in electroantennograms, remained normal. In vivo volume measurements were performed on olfactory glomeruli, the anatomical and functional units involved in odor processing. Pre-exposed flies exhibited volume reduction of certain glomeruli, in an odor-selective manner. Of a sample of eight glomeruli measured, dorsal medial (DM) 2 and ventral (V) were affected by benzaldehyde exposure, whereas DM6 was affected by isoamyl acetate. Estimation of the number of synapses indicates that volume reduction involves synapse loss that can reach 30% in the V glomerulus of flies adapted to benzaldehyde. Additional features of odorant-induced adaptation, including concentration dependence and perdurance, also show correlation, because both effects are elicited by high odor concentrations and are long-lasting (>1 week). Finally, the dunce mutant fails to develop behavioral adaptation as well as morphological changes in the olfactory glomeruli after exposure. These neural changes thus appear to require the cAMP signaling pathway.

Cellular and molecular features of axon collaterals and dendrites.

Neural geometry is the major factor that determines connectivity and, possibly, functional output from a nervous system. Recently some of the proteins and pathways involved in specific modes of branch formation or maintenance, or both, have been described. To a variable extent, dendrites and axon collaterals can be viewed as dynamic structures subject to fine modulation that can result either in further growth or retraction. Each form of branching results from specific molecular mechanisms. Cell-internal, substrate-derived factors and functional activity, however, can often differ in their effect according to cell type and physiological context at the site of branch formation. Neural branching is not a linear process but an integrative one that takes place in a microenvironment where we have only a limited experimental access. To attain a coherent mechanism for this phenomenon, quantitative in situ data on the proteins involved and their interactions will be required.

Chiral photogeneration using perfluorocyclopentene-derived photochromes.

A new type of perfluorocyclopentenes which contain an optically active group at the 2-position of the thiophene ring were synthesized. Irradiation with UV light afforded the cyclized diastereoisomers in ratios dependent on solvent polarity and temperature.

A genetic approach to detect muscle protein interactions in vivo.

An organism is required to identify biologically relevant protein interactions. We propose Drosophila and its indirect flight muscles as a suitable experimental system for genetic screenings for muscle protein interactions. The first attempt focused on troponin I (TnI) in view of the key role in thin filament regulation that this protein performs. Suppressors of a defined Tn I allele have been isolated as mutations in the heavy chain of myosin (MhC). This unsuspected functional interaction between TnI and MhC serves to illustrate one of the benefits of the approach. Four of the suppressors identified to date reside in the MhC head, around the actin-binding site and near the lips of the pocket where ATP is hydrolyzed. Two other suppressors correspond to a second site mutation in TnI and a mutation in the conserved region of Tropomyosin II (TmII), respectively. All the identified suppressors are mutations in constituents of the sarcomere, and most of them are structurally similar to human mutations causing familial hypertrophic cardiomyopathy (FHC). At least seven sarcomere proteins can lead to FHC and, consequently, the disease is heterogeneous and difficult to diagnose. In addition, putative natural suppressors may help obscure the origin of FHC. The genetic procedure, used here for muscle proteins, could help diagnose FHC and other myopathies, and extend to proteins of clinical interest in other tissues, including the nervous and circulatory systems.

Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles.

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.

Single neuron mosaics of the drosophila gigas mutant project beyond normal targets and modify behavior.

gigas is a lethal mutant that differentiates enlarged cells, including the nucleus. This trait manifests only after the completion of the mitotic program. We have taken advantage of this phenotype to test in vivo the capacity of normal target cells to arrest the growth of mutant sensory axons. Single neuron connectivity changes have been analyzed in mosaics after horseradish peroxidase retrograde tracings. A mutant mechanoreceptor neuron, growing over a genetically normal substrate, contacts its normal target, and in addition projects to novel areas of the CNS. The mutant axon does terminate its growth eventually, and the new additional targets that are reached correspond to mechanoreceptor domains in other ganglia, indicating that this territorial constraint is operational in the mutant. gigas neurons maintain their stereotyped profile and represent an expanded version of the normal branching pattern. The ultrastructure of the invading projections does not reveal gliotic or necrotic reactions from the new cell contacts. The functional consequences of the connectivity changes produced by the mutant mechanoreceptors have been studied in grooming behavior. Mosaic flies carrying a single gigas mechanoreceptor show modified, albeit context-coherent, grooming responses after stimulation of the mutant bristle, whereas the response from neighboring normal sensory neurons remains unchanged. All of these experiments indicate that target recognition and growth arrest are two dissectible processes of neural development, and they highlight the autonomous features of the growth cone during pathfinding.

Disappearance of bromopropylate residues in artichokes, strawberries and beans.

Residues of Bromopropylate were determine in artichokes, strawberries and beans after foliar spray of acaricide at two rates. The rates used were 1 g/l formulated product (normal recommended) and 1.5 g/l. The residue levels of bromopropylate in the three crops after 14 days were lower than 0.7 ppm and did not exceed the Maximum Residual Level (MRL) recommended by FAO. In the artichokes and strawberries, the total concentration of residues decreased by 50% of the initial level after 2-3 days. Only trace levels of the bromopropylate residues (less than 0.01 ppm) were detected in the "hearts" of the artichokes. Bromopropylate residues in the green beans were also less than 0.8 ppm after the first day of foliar spraying. The kinetic of degradation occurred in two different steps. In the first step (4-6 days) the dissipation of bromopropylate was faster whereas in the second step (7-14 days) the loss of residues was much slower.

[Epilepsy with myoclonic absences]. / La epilepsia con ausencias mioclónicas.

Within generalized epilepsy, the syndrome of epilepsy with myoclonic absences is considered as intermediate between idiopathic and symptomatic forms. This syndrome is characterized by developing in childhood with a male predominance. Critical EEG shows paroxysms of PO at 3 Hz, and in the polygraphic recording rhythmic 3 Hz myoclonus is observed with a strict correspondence between EEG spike and myoclonus. The response to therapy is generally poor, and 18% develop generalized symptomatic epilepsy. We report 3 patients with epilepsy with myoclonic absences and good outcome, to emphasize the importance of a precise diagnosis by means of the polygraphic recording of the attack, the fact the association of sodium valproate and ethosuximide is the most useful therapy and, finally, the possibility that some patients with epilepsy with myoclonic absence may develop Janz's juvenile myoclonic epilepsy.