GluD1, linked to schizophrenia, controls the burst firing of dopamine neurons.

Human mutations of the GRID1 gene encoding the orphan delta1 glutamate receptor-channel (GluD1) are associated with schizophrenia but the explicit role of GluD1 in brain circuits is unknown. Based on the known function of its paralog GluD2 in cerebellum, we searched for a role of GluD1 in slow glutamatergic transmission mediated by metabotropic receptor mGlu1 in midbrain dopamine neurons, whose dysfunction is a hallmark of schizophrenia. We found that an mGlu1 agonist elicits a slow depolarizing current in HEK cells co-expressing mGlu1 and GluD1, but not in cells expressing mGlu1 or GluD1 alone. This current is abolished by additional co-expression of a dominant-negative GluD1 dead pore mutant. We then characterized mGlu1-dependent currents in dopamine neurons from midbrain slices. Both the agonist-evoked and the slow postsynaptic currents are abolished by expression of the dominant-negative GluD1 mutant, pointing to the involvement of native GluD1 channels in these currents. Likewise, both mGlu1-dependent currents are suppressed in GRID1 knockout mice, which reportedly display endophenotypes relevant for schizophrenia. It is known that mGlu1 activation triggers the transition from tonic to burst firing of dopamine neurons, which signals salient stimuli and encodes reward prediction. In vivo recordings of dopamine neurons showed that their spontaneous burst firing is abolished in GRID1 knockout mice or upon targeted expression of the dominant-negative GluD1 mutant in wild-type mice. Our results de-orphanize GluD1, unravel its key role in slow glutamatergic transmission and provide insights into how GRID1 gene alterations can lead to dopaminergic dysfunctions in schizophrenia.

ßV-tubulin expression is associated with outcome following taxane-based chemotherapy in non-small cell lung cancer.

BACKGROUND: Tubulin-binding agents (TBAs) are effective in non-small cell lung cancer (NSCLC) treatment. Both ßIII- and ßV-tubulins are expressed by cancer cells and may lead to resistance against TBAs. METHODS: Pre-treatment samples from 65 locally advanced or oligometastatic NSCLC patients, who underwent uniform induction chemotherapy with paclitaxel and platinum followed by radiochemotherapy with vinorelbine and platinum were retrospectively analysed by immunohistochemistry. Protein expression of ßIII- and ßV-tubulin was morphometrically quantified. RESULTS: Median pre-treatment H-score for ßIII-tubulin was 110 (range: 0-290), and 160 for ßV-tubulin (range: 0-290). Low ßIII-tubulin expression was associated with improved overall survival (OS) (P=0.0127, hazard ratio (HR): 0.328). An association between high ßV-tubulin expression and prolonged progression-free survival (PFS, median 19.2 vs 9.4 months in high vs low expressors; P=0.0315, HR: 1.899) was found. Further, high ßV-tubulin expression was associated with objective response (median H-score 172.5 for CR+PR vs 120 for SD+PD patients, P=0.0104) or disease control following induction chemotherapy (170 for CR+PR+SD vs 100 for PD patients, P=0.0081), but not radiochemotherapy. CONCLUSION: Expression of ßV-tubulin was associated with treatment response and PFS following paclitaxel-based chemotherapy of locally advanced and oligometastatic NSCLC patients. Prolonged OS was associated with low levels of ßIII-tubulin. Prospective evaluation of ßIII/ßV-tubulin expression in NSCLC is warranted.

Stimulation of corticosteroid biosynthesis by angiotensin I [des-asp1]angiotensin I, angiotensin II and [des-asp1]-angiotensin II in bovine adrenal fasciculata cells.

The effect of angiotensin I (AI), angiotensin II (AII), [des-asp1]AI, [des-asp1]AII and [des-asp1-arg2]AII on corticosteroid production in isolated fasciculata cells from bovine adrenals has been studied. AII and [des-asp1]AII in concentrations ranging from 10(-9)M to 10(-6)M had a potent stimulatory effect on steroid biosynthesis. The dose-response curves for both peptides were identical. AI was about 3 times less potent than AII and [des-asp1]AII. The effect of AI was not due to its conversion to AII. [Des-asp1]AI was as active as AI. No significant conversion to [des-asp1]AII was observed. [Des-asp1-arg2]AII had only a minimal effect on steroidogenesis. The structural analog [sar1,-ala8]AII inhibited all angiotensins specifically and competitively. The affinity of the cellular binding site was higher for AII and [des-asp1]AII than for [sar1,ala8]ALL, but lower for AI and [des-asp1]AI than for the inhibitor. Combination of submaximal doses of AI and AII resulted in an additive effect on steroid production. By contrast, combination of maximal doses of both peptides had the same effect as AII alone. These data demonstrate a potent steroidogenic activity for AII as well as AI, [des-asp1]AI and [des-asp1]AII in bovine adrenal fasciculata cells. A common receptor site for all four peptides is suggested.

SNAP-25 regulation during adrenal gland development: comparison with differentiation markers and other SNAREs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.

Effect of long-term treatment with high doses of guanethidine on sperm transport and fertility of rats.

1. Male rats treated with guanethidine 25 mg/kg daily for eight weeks had no demonstrable ejaculatory function immediately after cessation of treatment. Four weeks later, ejaculatory function had returned in two-thirds of the males, and after eight weeks, all males were able to deposit sperm in the vagina. However, fertility had returned at that time in only two of the nine males. On the basis of these findings it might be expected that fertility would return in the other males after a more prolonged observation period.2. At autopsy, 66 days after cessation of treatment, the vasa deferentia were congested and filled with viscous material, and along their course dilated regions and pseudocysts containing sperm debris and spermatozoa were found.3. The isolated ducts of the guanethidine-treated males showed a markedly increased response to noradrenaline if compared with ducts from control rats. Response to tyramine was similar in both treated and untreated rats. In view of the increased response to noradrenaline it may be concluded that the noradrenaline stores, which are sensitive to tyramine, were still reduced 66 days after guanethidine treatment had been stopped.

New targeted treatments in lung cancer--overview of clinical trials.

Combination chemotherapy has been established as the cornerstone of systemic treatment for advanced lung cancer in the last ten to fifteen years. However, improvements with new drug combinations in recent years have been rather small and a general outcome plateau has been reached with one-year survival rates of about 40% and two-year survival rates of less than 15%. Survival over three to four years is still a rare event in this disease, and more and more efforts are being made to develop innovative systemic treatment strategies with mechanisms of action different from conventional cytotoxic drugs. These molecular targeted agents have made a strong move forward in the management of this disease since Gefitinib--a small molecule EGF-receptor tyrosine kinase inhibitor--was registered in 2003 by the FDA and a number of further countries for the third-line treatment of non-small-cell lung cancer. Since then, every month findings have been reported about new cellular targets on lung-cancer cells and, consequently, new agents aiming at these molecular targets are being developed, preclinically. Some of these agents have already been tested in the clinics within phase-I, phase-II and some even within randomised phase-III trials. In this review we will try to summarise the current knowledge and data on the clinical activity of these new drugs in lung cancer and to give a perspective on the future for these new treatment principles. The most promising strategies have been aiming at the EGF-receptor family, serum-VEGF and the VEGF-receptor family (VEGF-1 and -2, respectively). Results from pivotal registration trials are expected within the next one or two years for a number of these new drugs, and the standards of care for advanced lung cancer may change dramatically, comparable to what we have seen in other solid tumours such as metastasised breast and colon cancer.

NGF enhances depolarization effects on SNAP-25 expression: induction of SNAP-25b isoform.

The 25 kDa synaptosomal associated protein (SNAP-25), which is implicated in neuronal plasticity and neurosecretion, exists as two isoforms generated by alternative splicing of exons 5a and 5b. The aim of the present study was to characterize factors influencing isoform expression. We report that chronic depolarization of PC12 cells alone or in the presence of NGF induces the expression of isoform-b, in addition to a 1.8- to 3-fold increase in SNAP-25 mRNA and protein as determined by immunoblotting and combined RT-PCR and Southern blot analysis. When cerebellar granule neurons were cultured in elevated K+, the predominant isoform switched from SNAP-25a to SNAP-25b. Taken together these results suggested that chronic depolarization regulates the transcription and processing of SNAP-25 mRNA.

Adrenal gland SNAP-25 expression is altered in thyroid hormone receptor knock-out mice.

SNAP-25 is a protein in neurons and neuroendocrine cells, which is involved, together with syntaxin and VAMP, in neurotransmitter release and neurite outgrowth. Since the thyroid hormone receptors TR alpha and TR beta are essential for nervous system development, their possible role in regulating the expression of these vesicle trafficking proteins was examined by analysing SNAP-25 levels in TR alpha and TR beta knock-out mice. Immunoblotting and RT-PCR showed that SNAP-25 levels are increased in the adrenal gland, but not in cerebellum, in knock-out mice, while syntaxin-1 and VAMP-2 are unaffected in either tissue. Treatment of the pheochromocytoma-derived cell line PC12 with the thyroid hormone L-3,5,3'-triiodothyronine (T3) decreased SNAP-25 expression. Together, these data suggest that thyroid hormones exert a negative regulatory effect on SNAP-25 in adrenal medullary neuroendocrine cells.

Restriction of indication for automated percutaneous lumbar discectomy based on computed tomographic discography.

This report details the authors' early experience using the automated percutaneous lumbar discectomy (APLD) procedure, developed by Onik et al., in 97 patients with a disc protrusion. In the evaluation of a herniated disc, we used computed tomography (CT) discography. According to the distribution of the dye inside the disc, five different disc types can be differentiated. With a follow-up after 3-7 months, the short-term outcomes of the first 40 APLD-treated patients varied, depending on the shape of the protruded nuclear material. Patients with a broad dye base on CT discography had better short-term outcomes than patients with a narrow dye base. In the next 57 patients we treated with APLD, this tendency was confirmed. The success rate of a consecutive group of patients with a disc protrusion with a broad dye base, treated with APLD, was 80%. In comparison, the patients with a disc protrusion with a narrow dye base had an overall success rate of only 53%. The difference is statistically significant (P < 0.05). The message of this report is that APLD is a useful invasive treatment for patients with a disc protrusion. The outcome depends, however, on the shape of the protruded nuclear material as shown by CT discography, which makes this examination as a conditio sine qua non before treating patients with a disc protrusion with APLD.

An unfused acromial epiphysis. A reason for chronic shoulder pain.

We report on a case of a 34 year-old female patient who had been suffering from chronic shoulder pain for 2 years although she received conservative treatment. The X-ray revealed a prominent distal clavicle and was interpreted as an old AC dislocation. An axillary view showed an os acromiale. The bone scintigraphy confirmed the clinical suspicion of reaction at the epiphysis. Local corticoid injections improved the patient's condition so that she refused further surgical treatment.

Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons.

The ubiquitous second messenger cyclic GMP (cGMP) is synthesized by soluble guanylate cyclases in response to nitric oxide (NO) and degraded by phosphodiesterases (PDE). We studied the homeostasis of cGMP in living thalamic neurons by using the genetically encoded fluorescence resonance energy transfer sensor Cygnet, expressed in brain slices through viral gene transfer. Natriuretic peptides had no effect on cGMP. Basal cGMP levels decreased upon inhibition of NO synthases or soluble guanylate cyclases and increased when PDEs were inhibited. Single cell RT-PCR analysis showed that thalamic neurons express PDE1, PDE2, PDE9, and PDE10. Basal cGMP levels were increased by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and BAY60-7550 but were unaffected by PDE1 or PDE10 inhibitors. We conclude that PDE2 regulates the basal cGMP concentration in thalamic neurons. In addition, in the presence of 3-isobutyl-1-methylxanthine (IBMX), cGMP still decreased after application of a NO donor. Probenecid, a blocker of cGMP transporters, had no effect on this decrease, leaving PDE9 as a possible candidate for decreasing cGMP concentration. Basal cGMP level is poised at an intermediate level from which it can be up or down-regulated according to the cyclase and PDE activities.

SNAREs during development.

Soluble N-ethylmaleimide-sensitive factor attached protein receptor (SNARE) molecules are implicated in many fundamental cellular processes that require membrane fusion, and the interactions of the SNARE proteins, SNAP-25, syntaxin and VAMP/synaptobrevin, have been extensively studied. This review documents recent data on their role at different stages of development. SNARE proteins are expressed very early and play important roles in fertilization and in cell division during early embryogenesis. In the developing nervous system, they are important for neurite outgrowth and transformation of the growth cone into the mature synapse. In the neuroendocrine system, in addition to neurosecretion, they are involved in processes related to morphological plasticity. Although few data exist on regulation of SNARE proteins during development, growth factors, intracellular messengers and depolarization are known to modify their cellular expression. The putative importance of these factors during development is discussed.

[Joint infections following intra-articular injections]. / Gelenkinfektionen nach intraartikulären Injektionen.

The results of treatment after joint infections caused by articular injection or puncture, were examined in 198 patients. The state of arthrosis and the range of joint motion were chosen as objective criteria; the patient's personal opinion was also assessed. The duration of hospital stay--average 12.8 weeks--was longer in conservative treatment than in patients who received operative therapy. When patients were operated early during their hospitalisation, they had better radiological results and postoperative range of motion. However, in patients who received early operative treatment due to severe infections, range of postoperative motion was most restricted. The importance of limited indications and thorough information of the patient must be stressed considering 15 deaths during therapy.

[Conservative treatment of ductal carcinoma in situ of the breast]. / Tratamiento conservador del carcinoma ductal in situ de la mama.

BACKGROUND: Since the widespread use of mammography, the incidence of ductal carcinoma in situ of the breast has increased. Until few years ago the standard treatment was mastectomy, however, from the analysis of conservative treatment trials for invasive carcinoma, it was evident that ductal carcinoma in situ could also be treated conservatively. This was confirmed later by randomized trials. AIM: To analyze the experience of our Institution with conservative treatment of ductal carcinoma in situ of the breast. PATIENTS AND METHODS: A search through the data base of our Institution found 69 patients treated with lumpectomy and radiotherapy between the years 1976 and 1997. RESULTS: Twenty three of 69 patients (33%) were diagnosed because of a palpable mass. Eleven of twelve were diagnosed prior to 1990 and 12 of 57 after 1990. With a median follow-up of 48 months local control and overall survival is 97%. None of the patients underwent mastectomy. CONCLUSIONS: Conservative treatment of ductal carcinoma in situ of the breast is a reasonable alternative, mainly if we realize that with increasing frequency--the diagnosis is made through mammography and with non-palpable lesions. The results reported in this study are similar to those reported by other centers.

Stimulating effects of angiotensin I, angiotensin II and des-Asp1-angiotensin II on steroid production in vitro and its inhibition by Sar1-Ala8-angiotensin II.

Two of the agents known to block the renin-angiotensin-aldosterone system, namely Sar1-Ala8-angiotensin II and the nonapeptide SQ 20881, have been used to clarify the role of angiotensin II (AII) and its cogeners upon the steroidogenesis in isolated fasciculata cells from bovine adrenal tissue. It could be concluded that: (1) des-Asp1-angiotensin II is as active as AII on steroidogenesis from bovine fasciculata cells; (2) angiotensin I, although less potent, stimulates steroid production without being converted to AII or des-Asp1-AII, and (3) Sar1-Ala8-AII inhibits all three peptides in a competitive manner. The presence of a common receptor for all these three peptides is suggested.

Effects of varying sodium intake on blood pressure and renin-angiotensin system in subtotally nephrectomized rats.

In rats in which the renal mass had been reduced by 70 per cent, the effects of varying sodium intake on blood pressure, serum electrolytes, renin-angiotensin system, and some other parameters that were modified simultaneously were studied. Within 4 weeks, a high sodium diet (750 mEa. per kilogram) resulted in marked hypertension, whereas a standard sodium diet (150 mEq. per kilogram) elevated the blood pressure only slightly. A low sodium diet (less than 0.2 mEq. per kilogram) prevented the rise in blood pressure. In the hypertensive group, the hematocrit values were markedly decreased, indicating the expansion of extracellular and intravascular spaces. The compensatory renal hypertrophy was accelerated by the high sodium diet and retarded during restriction. During low sodium intake, the serum concentration of sodium was diminished and that of potassium elevated. During the high sodium diet, the sodium concentration was unchanged, but the potassium concentration was decreased. Subtotal nephrectomy diminished the plasma angiotensin II concentration, and the renin content of the kidney remnant was lower than that of the kidneys from control animals. Sodium restriction stimulated the renin angiotensin system markedly, whereas high sodium intake suppressed it. After subtotal nephrectomy, elevation of blood pressure, renal hypertrophy, and suppression of the renin-angiotensin system are closely related to sodium intake.

Differential expression of SNAP-25 isoforms and SNAP-23 in the adrenal gland.

In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.

Pharmacokinetic study of methotrexate, folinic acid and their serum metabolites in children treated with high-dose methotrexate and leucovorin rescue.

The pharmacokinetics of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diaminomethylpteroic acid (APA), folinic acid, and 5-methyltetrahydrofolate (5-MTHF) have been studied during 21 high-dose MTX (HDMTX) infusions (5 g.m-2 in 24 h) with leucovorin (LCV) rescue, a component of the therapy of 5 children with acute lymphoblastic leukemia (ALL). The median steady-state concentration of MTX was 66 mumol.l-1. Three elimination half-lifes were determined for MTX: 1.8 h, 6.4 h and a terminal 15 h. The median systemic MTX clearance was 110 mg.m-2.min-1. The 7-OHMTX level increased during each infusion and a Cmax of 19 mumol.l-1 was achieved at the end. Its initial half-life was 5 h and the terminal half-life was 12 h. Thus, the peak serum concentration ratio of 7-OHMTX to MTX was reached 24 h after the end of the infusion at a median ratio of 8. The MTX metabolite APA was detected in concentrations less than 0.06 mumol.l-1. The median folinic acid level during rescue, 48 h after starting the infusion, was 7.0 mumol.l-1 and 18 h following the last dose of LCV it was 0.44 mumol.l-1, leading to ratios of folinic acid to MTX of 31 and 6, respectively. The median 5-MTHF level during rescue was 0.44 mumol.l-1 with a median ratio of 5-MTHF to MTX of 2. Twenty infusions with 48 h MTX levels of less than 0.5 mumol.l-1 were without marked toxicity. Only one patient with a 48 h MTX concentration of 5.5 mumol.l-1 and a ratio of 5-MTHF to MTX of 0.08 suffered from ulcerating mucositis and septicaemia despite increased and prolonged LCV rescue.