Role of SNTA1 in Rac1 activation, modulation of ROS generation, and migratory potential of human breast cancer cells.

BACKGROUND: Alpha-1-syntrophin (SNTA1) has been implicated in the activation of Rac1. However, the underlying mechanism has not yet been explored. Here, we show that a novel complex, involving SNTA1, P66shc, and Grb2 proteins, is involved in Rac1 activation. METHODS: Co-immunoprecipitation assays were used to show the complex formation, while siRNAs and shRNAs were used to downregulate expression of these proteins. Various Rac1 activation assays and functional assays, such as migration assays, in vitro wound healing assays, cell proliferation assays, and ROS generation assays, were also performed. RESULTS: The results showed a significant increase in activation of Rac1 when SNTA1 and P66shc were overexpressed, whereas depletion of SNTA1 and P66shc expression effectively reduced the levels of active Rac1. The results indicated a significant displacement of Sos1 protein from Grb2 when SNTA1 and P66shc are overexpressed in breast cancer cell lines, resulting in Sos1 predominantly forming a complex with Eps8 and E3b1. In addition, the SNTA1/P66shc-mediated Rac1 activation resulted in an increase in reactive oxygen species (ROS) production and migratory potential in human breast cancer cells. CONCLUSION: Together, our results present a possible mechanism of Rac1 activation involving SNTA1 and emphasise its role in ROS generation, cell migration, and acquisition of malignancy.

Hepatopathy of Mauriac syndrome: a retrospective review from a tertiary liver centre.

BACKGROUND: Mauriac syndrome is characterised by growth failure, cushingoid appearance and hepatomegaly which occurs in patients with insulin dependent diabetes and was first described shortly after the introduction of insulin as a treatment for the condition. OBJECTIVE: To describe the clinical features, histological findings and outcome of young people with glycogenic hepatopathy, the hepatic manifestation of Mauriac syndrome (MS). DESIGN: Retrospective cohort study. PATIENTS: Young people with glycogenic hepatopathy. SETTING: Tertiary paediatric hepatology unit. RESULTS: Thirty-one young people (16 M), median age of 15.1 years (IQR 14-16.2) presented within the study period. Median age of diagnosis of diabetes was 10 years (IQR 8-11). Median insulin requirement was 1.33 units/kg/day; median HbA1c was 96.7 mmol/mol (IQR 84.7-112.0). Growth was impaired: median height z-score was -1.01 (-1.73 to 0.4) and median body mass index (BMI) z-score was 0.28 (-0.12 to 0.67). Hepatomegaly was universal with splenomegaly in 16%. Transaminases were abnormal with a median aspartate aminotransferase (AST) of 76 IU/L and gamma glutamyltransferase of 71 IU/L. Liver biopsy was undertaken in 19 (61%). All showed enlarged hepatocytes with clear cytoplasm with glycogenated nuclei in 17. Steatosis was present in the majority. Inflammation was present in 8 (42%). Fibrosis was seen in 14 (73%) and was generally mild though 2 had bridging fibrosis. Megamitochondria were described in 7. Presence of megamitochondria correlated with AST elevation (p=0.026) and fibrosis on biopsy (p=0.007). At follow-up 17 children had normal or improved transaminases, in 13 there was no change. Transaminases followed the trend of the child's HbA1c. CONCLUSIONS: Despite modern insulin regimens and monitoring in children with type 1 diabetes, MS still exists. Significant steatosis, inflammation and fibrosis were all seen in liver biopsies.

The anatomic variations of the circle of Willis in preterm-at-term and term-born infants: an MR angiography study at 3T.

BACKGROUND AND PURPOSE: It has been shown that the brain of a preterm infant develops differently from that of a term infant, but little is known about the neonatal cerebrovascular anatomy. Our aims were to establish reference data for the prevalence of the anatomic variations of the neonatal circle of Willis (CoW) and to explore the effect of prematurity, MR imaging abnormality, vascular-related abnormality, laterality, and sex on these findings. MATERIALS AND METHODS: We scanned 103 infants with an optimized MR angiography (MRA) protocol. Images were analyzed for different variations of the CoW, and results were compared for the following: 1) preterm-at-term and term-born infants, 2) infants with normal and abnormal MR imaging, 3) infants with and without a vascular-related abnormality, 4) boys and girls, and 5) left- and right-sided occurrence. RESULTS: The most common anatomic variation was absence/hypoplasia of the posterior communicating artery. Preterm infants at term had a higher prevalence of a complete CoW and a lower prevalence of anatomic variations compared with term-born infants; this finding was significant for the anterior cerebral artery (P = .02). There was increased prevalence of variations of the major cerebral arteries in those infants with vascular-related abnormalities, statistically significant for the posterior cerebral artery (P = .004). There was no statistically significant difference between boys and girls and left/right variations. CONCLUSIONS: Prematurity is associated with more complete CoWs and fewer anatomic variations. In vascular-related abnormalities, more variations involved major arterial segments, but fewer variations occurred in the communicating arteries. Overall reference values of the variations match those of the general adult population.

A spectrum of unusual neuroimaging findings in patients with suspected Sturge-Weber syndrome.

BACKGROUND AND PURPOSE: Sturge-Weber syndrome (SWS) is frequently associated with neurologic complications such as seizures, so diagnosing this condition has important implications for patient management. The purpose of this study was to report unusual neuroimaging findings in patients with facial port-wine stain (PWS) and clinically suspected SWS. MATERIALS AND METHODS: Cranial MR imaging was reviewed for all children with facial port-wine stain (PWS) involving the upper face and eyelids who were referred to Great Ormond Street Hospital between 2003 and 2007 for investigation of suspected SWS. Patients were excluded from further analysis if the imaging findings were normal on initial and subsequent scans and the subject remained free of neurologic disease, or if the imaging showed the well-recognized pattern of exclusively supratentorial pial enhancement representing the pial angioma of SWS. For the remaining patients, the neurologic, dermatologic, and ophthalmologic records were examined and all available imaging was reviewed by a neuroradiologist. We documented the presence and distribution of pial enhancement; corroborative features of SWS, such as atrophy, calcification, choroid plexus changes, and ocular abnormalities; and all other intracranial abnormalities. RESULTS: Of the 62 patients referred for assessment, imaging findings were considered typical of SWS in 32 (52%) and were normal or showed abnormalities attributable to an unrelated pathology in 20 (32%). Of the remaining 10 patients, in 7 (11%), there was evidence of a pial angioma in an unusual distribution involving infratentorial structures, with the angioma in 1 patient being diagnosed at postmortem only; in 2 (3%), there were imaging abnormalities with some features in common with typical SWS, such as subcortical calcification, but with no evidence of pial enhancement; in 1 (1.6%), the initial MR imaging finding was normal, but repeat imaging subsequently revealed pial enhancement. CONCLUSIONS: Involvement of infratentorial structures is common but may be relatively subtle and should be actively sought. Cases in which there are certain patterns of imaging abnormalities but an apparent absence of supratentorial pial enhancement on MR imaging may represent formes frustes of SWS; visualization of pial angiomatosis may also be delayed until later in childhood than expected.

Complex steroid-peptide-receptor cascade controls insect ecdysis.

Insect ecdysis sequence is composed of pre-ecdysis, ecdysis and post-ecdysis behaviors controlled by a complex cascade of peptide hormones from endocrine Inka cells and neuropeptides in the central nervous system (CNS). Inka cells produce pre-ecdysis and ecdysis triggering hormones (ETH) which activate the ecdysis sequence through receptor-mediated actions on specific neurons in the CNS. Multiple experimental approaches have been used to determine mechanisms of ETH expression and release from Inka cells and its action on the CNS of moths and flies. During the preparatory phase 1-2 days prior to ecdysis, high ecdysteroid levels induce expression of ETH receptors in the CNS and increased ETH production in Inka cells, which coincides with expression of nuclear ecdysone receptor (EcR) and transcription factor cryptocephal (CRC). However, high ecdysteroid levels prevent ETH release from Inka cells. Acquisition of Inka cell competence to release ETH requires decline of ecdysteroid levels and beta-FTZ-F1 expression few hours prior to ecdysis. The behavioral phase is initiated by ETH secretion into the hemolymph, which is controlled by two brain neuropeptides-corazonin and eclosion hormone (EH). Corazonin acts on its receptor in Inka cells to elicit low level ETH secretion and initiation of pre-ecdysis, while EH induces cGMP-mediated ETH depletion and consequent activation of ecdysis. The activation of both behaviors is accomplished by ETH action on central neurons expressing ETH receptors A and B (ETHR-A and B). These neurons produce numerous excitatory or inhibitory neuropeptides which initiate or terminate different phases of the ecdysis sequence. Our data indicate that insect ecdysis is a very complex process characterized by two principal steps: (1) ecdysteroid-induced expression of receptors and transcription factors in the CNS and Inka cells. (2) Release and interaction of Inka cell peptide hormones and multiple central neuropeptides to control consecutive phases of the ecdysis sequence.

The UK case-control study of cerebral oedema complicating diabetic ketoacidosis in children.

AIMS/HYPOTHESIS: Cerebral oedema complicating diabetic ketoacidosis (DKA) remains the major cause of morbidity and mortality in children with type 1 diabetes, but its aetiology remains unknown. Our objective was to determine the impact of baseline biochemical factors and of treatment-related variables on risk of the development of cerebral oedema in children with DKA. MATERIALS AND METHODS: This was a national UK case-control study. Through the British Paediatric Surveillance Unit we identified 43 cases of cerebral oedema. Through a parallel reporting system, we also identified 2,940 episodes of DKA and selected 169 control subjects on the basis of comparable age, sex, numbers of new or known cases of diabetes and date of admission. Baseline biochemical data and treatment-related variables were extracted from the clinical notes of cases and control subjects. RESULTS: Allowing for differences in age, sex and new or known diabetes, cases were more acidotic at diagnosis of DKA (odds ratio [OR] for events in the least acidotic compared with the most acidotic tertile=0.02 [95% CI: 0.002-0.15], p<0.001). In addition, cases had higher potassium and urea levels at baseline. Calculated osmolality and baseline glucose were not significantly different. After allowing for severity of acidosis, insulin administration in the first hour (OR 12.7 [1.41-114.5], p=0.02) and volume of fluid administered over the first 4 h (OR 6.55 [1.38-30.97], p=0.01) were associated with risk. Low baseline plasma sodium and an elevated p(a)CO(2) also contributed to risk in the final regression model. Bicarbonate administration was not associated with increased risk of an event when corrected for acidosis. CONCLUSIONS/INTERPRETATION: In this case-control study of DKA, baseline acidosis and abnormalities of sodium, potassium and urea concentrations were important predictors of risk of cerebral oedema. Additional risk factors identified were early administration of insulin and high volumes of fluid. These observations should be taken into account when designing treatment protocols.

Differential effect of alpha-syntrophin knockout on aquaporin-4 and Kir4.1 expression in retinal macroglial cells in mice.

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.

Functional electrical therapy: retraining grasping in spinal cord injury.

OBJECTIVE: To determine the clinical efficacy of functional electrical therapy in the rehabilitation of grasping function for quadriplegics. STUDY DESIGN: Randomized intervention-versus-control trial. SETTING: Rehabilitation hospital for spinal cord injury in Toronto, Canada. METHODS: A total of 21 people with new spinal cord injuries ranging from C3 to C7 were randomly assigned to two groups: Control (N=9) and Intervention (N=12). The intervention was functional electrical therapy, which consisted of repetitive grasping exercises using a neuroprosthesis that applied surface electrical stimulation to the arm to generate and/or assist grasping movements. It was applied by registered Occupational Therapists in a clinical setting. Main outcome measures were: Functional Independence Measure (FIM), Spinal Cord Independence Measure (SCIM), and the Rehabilitation Engineering Laboratory Hand Function Test. Consumer perceptions of functional electrical therapy were assessed via qualitative interviews. RESULTS: Differences between the Control and Intervention groups could be observed although they are not significant due to an insufficient number of participants. Consumer perceptions were positive, including improved Activities of Daily Living and self-satisfaction. CONCLUSION: Functional electrical therapy has the potential to be an effective treatment modality to restore grasping function in quadriplegia. It can be implemented by occupational therapists in a clinical setting. Further research is required to establish suitable indications for participant selection. In addition, a larger number of participants is needed to demonstrate statistical significance of the Functional Electrical Therapy.

Prolonged cardiac repolarisation during spontaneous nocturnal hypoglycaemia in children and adolescents with type 1 diabetes.

AIMS/HYPOTHESIS: It has been postulated that hypoglycaemia-related cardiac dysrhythmia and, in particular, prolonged cardiac repolarisation, may contribute to increased mortality rates in children and adolescents with type 1 diabetes. METHODS: We examined the prevalence of prolonged QT interval on ECG during spontaneous hypoglycaemia in 44 type 1 diabetic subjects (aged 7-18 years), and explored the relationships between serial overnight measurements of QT interval corrected for heart rate (QTc) and serum glucose, potassium and epinephrine levels. Each subject underwent two overnight profiles; blood was sampled every 15 min for glucose measurements and hourly for potassium and epinephrine. Serial ECGs recorded half-hourly between 23.00 and 07.00 hours were available on 74 nights: 29 with spontaneous hypoglycaemia (defined as blood glucose <3.5 mmol/l) and 45 without hypoglycaemia. RESULTS: Mean overnight QTc was longer in females than in males (412 vs 400 ms, p=0.02), but was not related to age, diabetes duration or HbA(1)c. Prolonged QTc (>440 ms) occurred on 20 out of 74 (27%) nights, with no significant differences between male and female subjects, and was more prevalent on nights with hypoglycaemia (13/29, 44%) than on nights without (7/45, 15%, p=0.0008). Potassium levels were lower on nights when hypoglycaemia occurred (minimum potassium 3.4 vs 3.7 mmol/l, p=0.0003) and were inversely correlated with maximum QTc (r=-0.40, p=0.03). In contrast, epinephrine levels were not higher on nights with hypoglycaemia and were not related to QTc. CONCLUSIONS/INTERPRETATION: In young type 1 diabetic subjects, prolonged QTc occurred frequently with spontaneous overnight hypoglycaemia and may be related to insulin-induced hypokalaemia.

Insulin lispro: a potential role in preventing nocturnal hypoglycaemia in young children with diabetes mellitus.

AIMS: The long duration of action of soluble insulin given in the evening could contribute to the high prevalence of nocturnal hypoglycaemia seen in young children with Type 1 diabetes mellitus (T1DM). We examined whether replacing soluble insulin with insulin lispro reduced this risk in children on a three times daily insulin regimen. METHODS: Open crossover study comparing insulin lispro vs. soluble insulin in 23 (16 boys) prepubertal children (age 7-11 years) with T1DM on three injections/day; long-acting isophane insulin remained identical. At the end of each 4-month treatment arm, an overnight 15-min venous sampled blood glucose profile was performed. RESULTS: Despite similar blood glucose levels pre-evening meal (lispro vs. soluble: mean +/- se 6.5 +/- 1.0 vs. 7.1 +/- 1.1 mmol/l, P = 0.5), post-meal (18.00-22.00 h) blood glucose levels were lower on insulin lispro (area under curve 138 +/- 12 vs. 170 +/- 13 mmol min-1 l-1, P = 0.03). In contrast, in the early night (22.00-04.00 h) the prevalence of low blood glucose levels (< 3.5 mmol/l) was lower on lispro (8% of blood glucose levels) than on soluble insulin (13%, P = 0.01). In the early morning (04.00-07.00 h) mean blood glucose and prevalence of low levels were no different between the two treatment groups, and fasting (07.00 h) blood glucose levels were similar (6.1 +/- 0.8 vs. 6.3 +/- 0.9 mmol/l, P = 0.8). At the end of each treatment arm there were no differences in HbA1c (lispro vs. soluble 8.6% vs. 8.4%, P = 0.3), or in insulin doses (mean, range 0.97, 0.68-1.26 vs. 0.96, 0.53-1.22 U/kg per day, P = 0.2). CONCLUSIONS: The shorter duration of action of insulin lispro given before the evening meal may reduce the prevalence of early nocturnal hypoglycaemia without compromising HbA1c in young children with T1DM.

Conservation of ecdysis-triggering hormone signalling in insects.

Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, we found Inka cells in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviours in pharate larvae of Bombyx, indicating functional similarity of these peptides. We isolated several PETH-immunoreactive peptides from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and identified the gene encoding two putative ETHs in the mosquito Anopheles gambiae. Inka cells also are stained with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, our enzyme immunoassays show that these antisera cross-react with PETH and ETH. Our results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioural sequence.

Impact of feeding problems on nutritional intake and growth: Oxford Feeding Study II.

Poor nutritional status and growth failure are common in children with cerebral palsy (CP). The aim of this study was to assess, within a subgroup of a large and clearly defined population of children with disabilities, the impact of feeding difficulties on (1) the quality (micronutrient intake) and quantity (macronutrient intake) of their diet and (2) their growth. One hundred children with disabilities (40 females, 60 males; mean age 9 years, SD 2 years 5 months; range 4 years 6 months to 13 years 7 months) underwent a detailed dietetic analysis and a comprehensive anthropometric assessment. Diagnostic categories of disability were: CP (n=90); global developmental delay (n=3); Marfan syndrome (n=1); intractable epilepsy (n=2); agenesis of the corpus callosum (n=2); methyl malonic aciduria (n=1); and congenital rubella (n=1). Neurological impairment was classified according to difficulty with mobility which was graded as mild (little or no difficulty walking), moderate (difficulty walking but does not need aids or a helper), and severe (needs aids and/or a helper or cannot walk). Results confirmed the significant impact of neurological impairment in children on body growth and nutritional status becoming worse in those with a greater degree of motor impairment. The major nutritional deficit was in energy intake, with only one fifth reportedly regularly achieving over 100% estimated average requirement (EAR), whilst micronutrient intake was less markedly impaired and protein intake was normal in this group (96% above EAR). Many children with neurological impairment would benefit from individual nutritional assessment and management as part of their overall care.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein.

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, beta-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking alpha-syntrophin (alpha-Syn(-/-)). The total level of AQP4 expression appears normal in brains of alpha-Syn(-/-) mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of alpha-Syn(-/-) mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in alpha-Syn(-/-) mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse-chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires alpha-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Dual ecdysteroid action on the epitracheal glands and central nervous system preceding ecdysis of Manduca sexta.

Initiation of the ecdysis behavioural sequence in insects requires activation of the central nervous system (CNS) by pre-ecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which are released from the Inka cells of the epitracheal glands. Here, we show that the developmental events preceding larval and pupal ecdysis of Manduca sexta involve a dual action of ecdysteroids on the epitracheal glands and CNS. The low steroid levels in freshly ecdysed and feeding larvae are associated with small-sized epitracheal glands, reduced peptide production in Inka cells and insensitivity of the CNS to ETH. The elevated ecdysteroid levels before each ecdysis lead to a dramatic enlargement of Inka cells and increased production of peptide hormones and their precursors. As blood ecdysteroids reach peak levels, the CNS becomes responsive to Inka cell peptides. These effects of natural ecdysteroid pulses can be experimentally induced by injection of 20-hydroxyecdysone or the ecdysteroid agonist tebufenozide (RH-5992) into ecdysed larvae, thus stimulating peptide production in Inka cells and inducing CNS sensitivity to ETH. A direct steroid action on the CNS is demonstrated by subsequent treatment of isolated nerve cords from ecdysed larvae with 20-hydroxyecdysone and ETH, which results in pre-ecdysis or ecdysis bursts. Our data show that ecdysteroid-induced transcriptional activity in both the epitracheal glands and the CNS are necessary events for the initiation of the ecdysis behavioural sequence.

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4.

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.

Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL-deficient knee joints obtained by RNase protection assay.

Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post-traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post-surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 microg RNA/g wet wt in unoperated, control joints to 200-350 microg RNA/g wet wt in ACL-deficient joints. No significant changes were detected in the concentration of DNA (900-1200 microg DNA/g wet wt). Low concentrations of COL1A1 (2-3 pmol mRNA/g DNA) and COL6A3 (0.3-0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL-deficiency induced a 20-38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11-19 fold increase at 12 weeks post-surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL.

Subtotal parathyroidectomy in renal failure: still needed after all these years.

There are scant data on the frequency of parathyroidectomy (PTX) for end-stage renal disease (ESRD). Medical therapy for ESRD and secondary hyperparathyroidism has evolved to include better dialytic urea removal and the use of calcitriol. The aim of this study was to determine whether medical therapy has changed the frequency or indications for PTX in the management of renal failure. Hospital and clinic records were analyzed to gather information on all patients undergoing PTX for secondary hyperparathyroidism (2HPT) (n = 48) and tertiary hyperparathyroidism (3HPT) (n = 26) from 1986 through 1998 at our institution. Prospective computer databases were queried for information concerning both chronic dialysis and renal transplant patients at our center. The patients were divided based on date of operation before or after 1991, a divider that separated the patients into groups before or after the widespread adoption of intravenous calcitriol treatment during hemodialysis at our institution. Over the 12 year period, the proportion of our chronic dialysis patients undergoing PTX did not change significantly, ranging from 0% to 2.5% per year. Comparing all patients undergoing PTX for 2HPT during 1986-1991 versus 1992-1998, there was no significant difference in time on dialysis [7.0 +/- 4.2 (n = 11) vs. 7.5 +/- 4.6 (n = 36) years, mean +/- SD]. The later group had higher intact parathyroid hormone (iPTH) levels [765 +/- 415 (n = 6) vs. 1377 +/- 636 (n = 28) pg/ml; p = 0.03], lower serum calcium [11.2 +/- 1.0 (n = 12) vs. 9.9 +/- 1.5 (n = 34) mg/dl; p = 0.006], and higher serum phosphate [5.7 +/- 1.6 (n = 12) vs. 7.2 +/- 2.3 (n = 31) mg/dl; p = 0.042]. Among the population of patients with transplants undergoing PTX for 3HPT, the average percent per year undergoing PTX ranged from 0% to 4.2% and did not change during the study period. Comparing the 1986-1991 group to the 1992-1998 group, the time from transplantation to PTX did not change during the study period (3.3 +/- 2.3 vs. 2.9 +/- 3.0 years; p = 0.391), and there were no significant differences between preoperative calcium levels or iPTH levels. Despite advances in dialysis technique and pharmacologic therapy, there has been no change in the proportion of dialysis patients requiring PTX for 2HPT or 3HPT. There was also no change in the time on dialysis for patients with 2HPT or the time from transplant to PTX for patients with 3HPT. Analysis of preoperative biochemical markers as evidence of disease severity suggests there was no change in indications for PTX during our study. From this information we conclude that parathyroid pathophysiology is incompletely understood and medical therapy is not optimal, resulting in a continuing need for PTX in some patients.

Signal transduction in eclosion hormone-induced secretion of ecdysis-triggering hormone.

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.