Induction of proliferation of basal epidermal keratinocytes by cold atmospheric-pressure plasma.

BACKGROUND: Over the past few decades, new cold plasma sources have been developed that have the great advantage of operating at atmospheric pressure and at temperatures tolerable by biological material. New applications for these have emerged, especially in the field of dermatology. Recently it was demonstrated that cold atmospheric-pressure plasma positively influences healing of chronic wounds. The potential of cold plasma lies in its capacity to reduce bacterial load in the wound while at the same time stimulating skin cells and therefore promoting wound closure. In recent years, there have been great advances in the understanding of the molecular mechanisms triggered by cold plasma involving signalling pathways and gene regulation in cell culture. AIM: To investigate cold plasma-induced effects in ex vivo treated human skin biopsies. METHODS: Human skin tissue was exposed to cold plasma for different lengths of time, and analysed by immunofluorescence with respect to DNA damage, apoptosis, proliferation and differentiation markers. RESULTS: After cold plasma treatment, the epidermal integrity and keratin expression pattern remained unchanged. As expected, the results revealed an increase in apoptotic cells after 3 and 5 min of treatment. Strikingly, an induction of proliferating basal keratinocytes was detected after cold plasma exposure for 1 and 3 min. As these are the cells that regenerate the epidermis, this could indeed be beneficial for wound closure. CONCLUSION: We investigated the effect of cold plasma on human skin by detecting molecules for growth and apoptosis, and found that both processes are dependent on treatment time. Therefore, this approach offers promising results for further applications of cold plasma in clinical dermatology.

Synaptic proteins and the assembly of synaptic junctions.

Synapses are highly specialized contact sites between neurons and their target cells where information in the form of chemical substances travels from a pre- to a postsynaptic cell. In the central nervous system of mammals, most nerve cells are innervated by functionally distinct types of synapses, each requiring a specific set of molecular constituents for proper function. Various molecular players that may be involved in the assembly of synaptic junctions have been identified recently.

Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals.

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

[Wound management-biology and wound healing disorders]. / Wundmanagement ­ Biologie und Störung der Wundheilung.

Wound healing is one of the most complex biological processes in an organism. It proceeds in three consecutive stages: the exudative, the proliferative, and the reparative phase. For a better understanding of new treatment possibilities, knowledge of the fundamental principles of these phases is required. Depending on the extent, location, bacterial colonization, and stage of a wound, it is important to find the appropriate treatment modality. In the present article, the basic principles of wound healing and disruptive factors are described in preparation for the next part on modern treatment modalities.

SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo.

Synapse-associated proteins (SAPs) are constituents of the pre- and postsynaptic submembraneous cytomatrix. Here, we present SAP102, a novel 102kDa SAP detected in dendritic shafts and spines of asymmetric type 1 synapses. SAP102 is enriched in preparations of synaptic junctions, where it biochemically behaves as a component of the cortical cytoskeleton. Antibodies directed against NMDA receptors coimmunoprecipitate SAP102 from rat brain synaptosomes. Recombinant proteins containing the carboxy-terminal tail of NMDA receptor subunit NR2B interact with SAP102 from rat brain homogenates. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. These data represent direct evidence that in vivo SAP102 is involved in linking NMDA receptors to the submembraneous cytomatrix associated with postsynaptic densities at excitatory synapses.

Piccolo, a presynaptic zinc finger protein structurally related to bassoon.

Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle-associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.

Molecular determinants of presynaptic active zones.

The presynaptic cytoskeletal matrix (cytomatrix) assembled at active zones has been implicated in defining neurotransmitter release sites. Munc13, Rim, Bassoon and Piccolo/Aczonin are recently identified presynaptic cytomatrix proteins. These multidomain proteins are thought to organize the exocytotic and endocytotic machinery precisely at active zones.

Identification of a cis-acting dendritic targeting element in MAP2 mRNAs.

In neurons, a limited number of mRNAs have been identified in dendritic processes, whereas other transcripts are restricted to the cell soma. Here we have investigated the molecular mechanisms underlying extrasomatic localization of mRNAs encoding microtubule-associated protein 2 (MAP2) in primary neuronal cultures. Vectors expressing recombinant mRNAs were introduced into hippocampal and sympathetic neurons using DNA transfection and microinjection protocols, respectively. Chimeric mRNAs containing the entire 3' untranslated region of MAP2 transcripts fused to a nondendritic reporter mRNA are detected in dendrites. In contrast, RNAs containing MAP2 coding and 5' untranslated regions or tubulin sequences are restricted to the cell soma. Moreover, 640 nucleotides from the MAP2 3' untranslated region (UTR) are both sufficient and essential for extrasomatic localization of chimeric mRNAs in hippocampal and sympathetic neurons. Thus, a cis-acting dendritic targeting element that is effective in two distinct neuronal cell types is contained in the 3' UTR of MAP2 transcripts. The observation of RNA granules in dendrites implies that extrasomatic transcripts seem to assemble into multimolecular complexes that may function as transport units.

Dispersion of epinephrine sensitive and insensitive adenylate cyclase from the ciliate Tetrahymena pyriformis.

The membrane bound adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of the ciliate Tetrahymena pyriformis could be extracted by washing the membrane fraction with 0.25 M sucrose. The enzyme dissociated in this way did not sediment after centrifugation at 105 000 times g for 2 h and was still responsive to stimulation by epinephrine. Dispersion of the membranes with Triton-X 100 led to purified preparation of the cyclase, which was no longer stimulated by epinephrine but retained fully the activation by fluoride and serotonin.

Aspartokinase isoenzymes of the fruiting myxobacterium Myxoccus xanthus.

Two isoenzymes of aspartokinase can be found in extracts of the differentiating bacterium Myxococcus xanthus. Aspartokinase I is repressed by L-lysine and feedback is inhibited by meso-diaminopimelate and by low concentrations of L-lysine. However, the inhibition by L-lysine is no longer observed at high concentration of this amino acid. Aspartokinase II is repressed and feedback inhibited specifically by L-threonine. Both enzymes are stimulated significantly by L-methionine and L-isoleucine; the effect is greater with aspartokinase I. The role of these enzymes in relation to growth conditions of the organism is discussed and a correlation with life cycle activity is indicated.

Economic evaluation of the effects of planting date and application rate of imidacloprid for management of cereal aphids and barley yellow dwarf in winter wheat.

The effects of planting date and application rate of imidacloprid for control of Schizaphis graminum Rondani, Rhopalosiphum padi L. (Homoptera: Aphididae), and barley yellow dwarf virus (BYDV) in hard red winter wheat were studied. The first experiment was conducted from 1997 to 1999 at two locations and consisted of three planting dates and four rates of imidacloprid-treated seed. The second experiment was conducted from 2001 to 2002 in Stillwater, OK, and consisted of two varieties of hard red winter wheat seed and four rates of imidacloprid. Aphid densities, occurrence of BYDV, yield components, and final grain yield were measured, and yield differences were used to estimate the economic return obtained from using imidacloprid. In the first study, aphid populations responded to insecticide rate in the early and middle plantings, but the response was reduced in the late planting. Yields increased as insecticide rate increased but did not always result in a positive economic return. In the second study, imidacloprid seed treatments reduced aphid numbers and BYD occurrence, protected yield, and resulted in a positive economic return. The presence of aphids and BYDV lowered yield by reducing fertile head density, total kernel weight, and test weight. Whereas the application of imidacloprid seed treatments often provided positive yield protection, it did not did not consistently provide a positive economic return. A positive economic return was consistently obtained if the cereal aphid was carrying and transmitting BYDV and was more likely to occur if wheat was treated with a low rate if imidacloprid and planted in a "dual purpose" planting date window.

Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix.

In this study, we describe a novel 420 kDa protein, called Piccolo, found at a wide variety of adult rat brain synapses. High protein levels in the cerebellum, the olfactory bulb and the hippocampus were frequently observed to be associated with asymmetric type 1 synapses. Piccolo is selectively enriched in presynaptic terminals, but is not a component of synaptic vesicles (SVs). Immunogold electron microscopy revealed that Piccolo localizes to the amorphous material among SVs at the presynaptic plasma membrane. Biochemical studies showed that it is very tightly bound to this structure. Thus, we speculate that Piccolo is a structural component of the presynaptic cytomatrix which anchors SVs to the presynaptic plasmalemma.

Effect of hypophysectomy on rat preadipocyte replication and differentiation.

The effects of hypophysectomy, which results in decreased fat pad weight, fat cell number, and new fat cell formation, on preadipocyte replication were examined. Perirenal and epididymal preadipocytes were cultured from hypophysectomized, sham-operated and unoperated 3-month-old rats. In cloned preadipocytes, hypophysectomy resulted in a 19% decrease in cloning efficiency and a 60% reduction in cell number after 3 weeks in culture. Perirenal cells underwent more extensive replication than epididymal cells. The mechanism of reduced replication following hypophysectomy differed from that of donor site: hypophysectomy resulted in an increased percentage of cells which underwent 2 or fewer population doublings at the expense of cells capable of more than 2 doublings. However, donor site had little effect specifically on these slowly replicating preadipocytes; rather, perirenal preadipocytes underwent more extensive replication than epididymal cells because of the higher percentage of preadipocytes capable of 13 or more doublings in perirenal fat pads. Hypophysectomy did not result in decreased differentiation of preadipocytes. These observations are in accord with the hypothesis that hypophysectomy reduces fat cell number in maturing rats partly through an effect on preadipocyte replicative capacity. Additionally, it seems that more than one form of preadipocyte exists, the various forms having differing susceptibilities to factors such as pituitary function and anatomic site.

The formation of 5 alpha-reduced androgens in stromal cells from human breast adipose tissue.

The present study was designed to determine if stromal cells derived from human breast adipose tissue contain 5 alpha-reductase activity, and to study the effect of 5 alpha-reduced androgens on aromatase activity under basal and cortisol stimulated conditions. Stromal cells were prepared from breast adipose tissue obtained at the time of surgery from four patients. The cells were isolated after collagenase digestion and were cultured in alpha-minimum essential medium with 15% fetal calf serum. Studies were carried out between days 4 and 11 of the third subculture in the presence or absence of cortisol (10(-6) M). Metabolism of androstenedione (A) was studied over a period of 8 h after addition of medium containing 20 X 10(6) dpm (100 pM) [3H]A. The cells metabolized A to estrone (E1), testosterone (T), 5 alpha-androstane 3, 17-dione (5 alpha-A-dione), androsterone (AND), and dihydrotestosterone. On day 7 of culture, product formation expressed as percent conversion of A per 1 X 10(6) cells ranged as follows: E1, 0.02-0.13; T, 0.12-0.36; 5 alpha-A-dione, 2.05-9.91; and a fraction containing AND and dihydrotestosterone, 0.38-0.59. In the presence of cortisol the rate of cell growth was decreased by 25% to 50%. The formation of E1 increased 150- to 1500-fold and AND formation increased 2- to 8-fold. There was no consistent change in the formation of 5 alpha-A-dione and T. The addition of 5 alpha-A-dione (10(-6) M) to the culture medium at the time of assay resulted in greater than 90% inhibition of E1 formation under both basal and cortisol stimulated conditions. The studies indicate that adipose tissue is an important site for the formation of 5 alpha-reduced androgens.

Conditioned avoidance acquisition and extinction following repeated electroconvulsive shock: strain effect and response to vasopressin.

Male albino rats (Sabra strain) were exposed to electroconvulsive shock (ECS) once daily for periods ranging from 1 to 13 days, and proactive effects on conditioned avoidance response (CAR) acquisition and extinction were studied. CAR acquisition was intact following both single and repeated ECS, but extinction was accelerated by multiple ECS administration. These findings resembled the effect of repeated ECS on anterograde memory function in humans and confirmed previous observations based on a passive avoidance paradigm. However, extinction was not accelerated in a different rat strain (LC2). Parallel open field activity measures suggested that these findings were not related to ECS-induced alterations in locomotor activity. Administration of arginine vasopressin prior to each ECS, or following acquisition sessions, as well as 1-desamino-8-D-arginine vasopressin administration following acquisition sessions, did not ameliorate ECS-induced deficits in the Sabra rats. Differences between the present paradigm of ECS administration and those in which positive effects of vasopressin and other neuropeptides have been reported are discussed. The potential research applications of a rodent model of ECS-induced memory impairment that parallels deficits encountered in the clinical context are considered.

Olfactory sensitivity in major depressive disorder and obsessive compulsive disorder.

Olfactory sensitivity to two odorants, isoamyl acetate and androsterone, was assessed in 14 obsessive compulsive disorder (OCD) patients, nine major depressive disorder (MDD) patients, and 16 sex- and age-matched healthy controls. Tests were performed during a drug-free period, and 3 and 6 weeks after initiation of antidepressant drug therapy. No difference in olfactory sensitivity, to either odorant, was found between OCD patients and controls at any time. In MDD patients, a significant increase in the sensitivity to isoamyl acetate was observed 6 weeks after initiation of treatment, compared to controls.

Antidepressant and cognitive effects of twice- versus three-times-weekly ECT.

OBJECTIVE: The purpose of this study was to determine which of the two commonly used schedules of ECT administration, twice or three times weekly, is clinically optimal in terms of antidepressant efficacy and cognitive effects. METHOD: In this double-blind study, 52 consenting, medication-free patients with major depressive disorder, endogenous subtype (Research Diagnostic Criteria), were randomly assigned to bilateral, brief-pulse, constant-current ECT administered over 4 weeks at a rate of three times weekly or twice weekly with the addition of one simulated ECT (anesthesia and muscle relaxant only) per week. Outcome measures were the Hamilton Depression Rating Scale, Acute Cognitive Effects Battery, and Chronic Cognitive Effects Battery. RESULTS: Hamilton depression scale scores were significantly improved by both schedules, with no difference in outcome either 1 week or 1 month after the end of the ECT series. However, the rate of response to ECT three times a week was significantly faster and was related to the rate of real ECT administration. Cognitive effects were more prominent with ECT three times a week. CONCLUSIONS: ECT twice a week is an effective schedule for clinical practice and is potentially advantageous in view of a therapeutic outcome identical to that of ECT three times a week and less severe cognitive effects. ECT three times a week may be specifically indicated when early onset of clinical effect is of primary importance.

Purification of a pituitary polypeptide that stimulates the replication of adipocyte precursors in culture.

A bovine anterior pituitary polypeptide that stimulates the replication of rat and human adipocyte precursors has been purified. Its Mr is 44 000-53 000 and its isoelectric point is 9.8-10.3. While pituitary basic fibroblast growth factor is equally mitogenic on adipocyte precursors and skin fibroblasts, the polypeptide described here is selectively more active on the precursors. We postulate that this adipocyte growth factor plays a physiological role by modulating the number of adipocyte precursors.

Current concepts in the pharmacological treatment of obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) is a chronic and often disabling disease. OCD is characterised by intrusive, unwanted and persistently recurring mental events (obsessions) that usually evoke discomfort or anxiety, and/or repetitive ritualistic behaviours (compulsions) that are aimed at reducing discomfort and anxiety. However, the compulsions succeed only in achieving transient relief, followed by a growing sense of pressure. 10 years ago, OCD was considered a rare and treatment-refractory disorder. Recent well designed studies document a lifetime prevalence rate for OCD of more than 2% in the general population. The outlook for patients with OCD has changed in the last decade, with many well controlled studies showing that OCD patients respond to specific behavioural and pharmacological treatments. The specific form of behavioural therapy is in vivo exposure coupled with response prevention. Only serotonin reuptake inhibitors, such as clomipramine, fluoxetine and fluvoxamine, are effective in the treatment of both depressed and not depressed OCD patients. Fluoxetine and fluvoxamine lack the anticholinergic side effects of clomipramine and, thus, provide an alternative treatment for patients who cannot tolerate clomipramine. Other nonserotonergic antidepressants (tricyclics and monoamine oxidase inhibitors) and anxiolytic agents have not been found to be consistently effective in this disorder. Insufficient data on the efficacy of neuroleptics and their potentially irreversible side effects limit their use in OCD patients. Behavioural and the pharmacological treatment are complementary, and a combination of the 2 therapies is apparently more effective than either modality alone.

Four repeat MAP2 isoforms in human and rat brain.

In mammalian brain, variations in the primary structure of the characterized microtubule-associated protein 2 (MAP2) isoforms have only been observed in their projection domains whose length determines the spacing between neighboring microtubules. We now report that, as with MAP4 and tau, MAP2 isoforms containing four (4R) instead of three (3R) tandem repeats in their microtubule binding domains do exist in human and rat brain. The additional sequence, inserted between the first and second repeat of the 3R-MAP2 messages, appears on mRNAs encoding both high and low molecular weight (Hwt and Lwt) rat MAP2 variants. In contrast to the corresponding 3R-messages, 4R-Hwt MAP2 concentrations decrease during early postnatal rat brain development, while the amount of 4R-Lwt MAP2 messages remains constant. In general, 4R-/3R-MAP2 mRNA ratios appear to be low with the highest levels of 4R-messages found in the cerebellum.