Metabotropic glutamate receptor 1 internalization induced by muscarinic acetylcholine receptor activation: differential dependency of internalization of splice variants on nonvisual arrestins

In this study, we characterized the glutamate- or second-messenger kinase-dependent internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M(1) muscarinic receptors in human embryonic kidney 293 cells, induced the internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca(2+) calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.     

Aging and intestinal motility: a review of factors that affect intestinal motility in the aged

Normal aging is associated with significant changes in the function of most organs and tissues. In this regard, the gastrointestinal tract is no exception. The purpose of this review is to detail the important age-related changes in motor function of the various parts of the gastrointestinal tract and to highlight some of the important motility changes that may occur, either in relation to common age-related disorders, or as a result of certain drugs commonly prescribed in the aged. A major confounding factor in the interpretation of motor phenomena throughout the gastrointestinal tract in this age group is the frequent coexistence of neurological, endocrinological and other disease states, which may be independently associated with dysmotility. Overall, current data are insufficient to implicate normal aging as a cause of dysmotility in the elderly. Normal aging is associated with various changes in gastrointestinal motility, but the clinical significance of such changes remains unclear. More important is the impact of various age-related diseases on gastrointestinal motility in the elderly: for example, long-standing diabetes mellitus may reduce gastric emptying in up to 50% of patients; depression significantly prolongs whole-gut transit time; hypothyroidism may prolong oro-caecal transit time; and chronic renal failure is associated with impaired gastric emptying. In addition, various, frequently used drugs in the elderly cause disordered gastrointestinal motility. These drugs include anticholinergics, especially antidepressants with an anticholinergic effect, opioid analgesics and calcium antagonists.     

Tumour specific promoter region methylation of the human homologue of the Drosophila Roundabout gene DUTT1 (ROBO1) in human cancers

The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RT-PCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for the tumour suppressor gene (TSG) at 3p12, a region defined by hemi and homozygous deletions and functional analysis.     

Irinotecan,cisplatin and mitomycin in inoperable gastro-oesophageal and pancreatic cancers - a new active regimen

Irinotecan, mitomycin and cisplatin all demonstrate activity in gastro-oesophageal cancers. This novel combination was administered to outpatients with previously untreated inoperable gastro-oesophageal or pancreatic cancer, in a 28-day cycle. A total of 26 out of 31 patients with gastro-oesophageal cancer and 12 out of 14 patients with pancreatic cancer have been treated with this combination, and were evaluable for response. The overall response rates for patients with gastro-oesophageal cancer was 42%, with a median survival of 9.5 months. In patients with pancreatic cancer, the overall response rate was 42% with a median survival of 8 months. There was a statistically significant increase in survival between those patients who achieved a stable disease response and those who achieved either a partial response or complete response. The toxicity profiles for both cancers were virtually identical. There were five treatment-related deaths, and a high admission rate (42%). Thus irinotecan, mitomycin and cisplatin is a new combination with activity in inoperable upper gastro-oesophageal cancers, but with a high toxicity profile. Future developments include reducing the dose of irinotecan and number of cycles of therapy to four.     

Altering the NICU and measuring infants' responses

The aim of the study was to measure the impact of a designated Quiet period on the NICU environment and its influence on the infants' physiological and movement responses. The study group comprised 10 preterm infants on assisted ventilation (mean gestational age 28.7 wk (range 24-32 wk), mean birthweight 1,322 g (range 600-2,060 g), mean age 5.2 d). The environment in which the infants were nursed was altered in terms of reduced light, noise, staff activity and infant handling. The infants' heart rate, blood pressure, oxygen saturation and movement responses were recorded during this Quiet period and compared with a period of Normal activity. When the Quiet period was compared with the Normal period (median values), the NICU environment had significantly altered in terms of Light: Quiet period 3.0 Lux, Normal period 254.5 Lux (p < 0.01); Noise: Quiet period 54.0 dB, Normal period 58.0 dB (p < 0.01); Alarm events: Quiet period 491.5 sec, Normal period 1,180.5 sec (p < 0.01); Staff conversation: Quiet period 16.0 occasions per hour, Normal period 60.0 occasions per hour (p < 0.01); Staff activity: Quiet period 25.5 occasions per hour, Normal period 59.0 occasions per hour (p <0.01); Infant handling: Quiet period 0.0 events per hour, Normal period 4.5 events per hour (p < 0.01). Infants' diastolic blood pressure and mean arterial pressure: median reduction of 2 mmHg for both during the Quiet period (p < 0.05). Infants' movements: Quiet period 14.5 movements per hour, Normal period 84.0 movements per hour (p < 0.05). DISCUSSION: This study demonstrates that Quiet periods are feasible for infants undergoing neonatal intensive care. The NICU environment was altered significantly for light, noise, infant handling and staff activity for a specified time period. These changes were associated with a reduced median diastolic blood pressure and mean arterial pressure and a decrease in infant movements.     

Continuous infusional topotecan in advanced breast and non-small-cell lung cancer: no evidence of increased efficacy.

Two open, phase II studies were performed to evaluate the activity and toxicity of infusional topotecan in patients with advanced non-small-cell lung carcinoma (NSCLC) and advanced breast cancer who had not received previous chemotherapy for metastatic disease. Twenty-five patients with an ECOG performance score < 2 were treated with infusional topotecan administered as a daily, continuous intravenous infusion starting at 0.6 mg m(-2) day(-1) (NSCLC) and 0.5 mg m(-2) day(-1) (breast cancer) for 21 days every 4 weeks. Three patients achieved a partial response as defined by WHO criteria: one with NSCLC (8%; 95% CI 0-39%) and two with advanced breast cancer (15%; 95% CI 2-45%). The major toxicities were neutropenia and thrombocytopenia, with one episode of neutropenic sepsis. These data suggest that topotecan delivered as a continuous intravenous infusion over 21 days as single-agent therapy does not appear to offer a clinical advantage over conventional 5-day schedules against advanced NSCLC and advanced breast cancer.     

Pupillary dysfunction in myasthenia gravis.

The constriction-dilation cycles of pupils exposed to a stationary, discrete slit-lamp beam were significantly prolonged in 25 myasthenic patients (1,060.4 +/- 45.8 msec) undergoing therapy with steroids, anticholinesterases, or both, compared to normal controls (801.9 +/- 8.6 msec) or subjects receiving steroids for nonneurological disease (860.9 +/- 14.9 msec). The duration of myasthenia correlated with the slowing of the cycle time. Myasthenia gravis may affect ectodermally derived smooth muscle or the autonomic neuromuscular junction or both, and not be restricted to the well-demonstrated alterations of neuromuscular junction in striated muscle of mesodermal origin. Alternatively, prolonged pupillary cycles could be attributed to dysfunction of central pathways of the pupillary light reflex.