Notch signalling pathway mediates hair cell development in mammalian cochlea.

The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair cell is separated from the next by an interceding supporting cell, forming an invariant and alternating mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence group in which developing hair cells suppress differentiation in their immediate neighbours through lateral inhibition. These interactions may be mediated through the Notch signalling pathway, a molecular mechanism that is involved in the determination of a variety of cell fates. Here we show that genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role for Notch in the development of the cochlear mosaic.

Derivation of a risk assessment tool for emergency department patients with sickle cell disease.

INTRODUCTION: Sickle cell patients commonly present to the emergency department (ED). Identifying those requiring admission and those who can safely be discharged is difficult. It was hypothesised that ED variables predictive of 96-h adverse sickle cell patient outcomes are identifiable. METHODS: This observational cohort study included all adult sickle cell patient visits (1 June 2004-31 May 2005) to two ED. Patients were identified by ICD-9 codes of vaso-occlusive crisis and lists from treating haematologists. ED charts were abstracted for history, physical examination, laboratory/imaging data and outcomes. Outcomes were hospitalisation within 96 h of ED presentation for transfusion/antibiotic treatment, acute chest syndrome, or aplastic or sequestration crisis. Logistic regression was used to derive a risk score, which was tested in a validation cohort. The area under the receiver operating curve (AUC) was used to measure score performance. RESULTS: There were 884 ED visits by 125 patients (mean age 36 years/55% female/58% homozygous sickle cell disease). 199 ED visits had one or more outcome (197 transfusion/antibiotic treatment, 71 acute chest syndrome, and one aplastic crisis). The risk score included sickle variant, chest pain, chills, pain dissimilar to past, temperature (<36 degrees C/>38 degrees C), oxygen saturation (<95%), haemoglobin (<10 g/dl), urine nitrites and chest x ray abnormality. The score had an AUC of 0.816 (95% CI 0.778 to 0.854) in the derivation cohort, 0.824 (95% CI 0.760 to 0.889) in the validation cohort. CONCLUSION: Those ED variables predictive of 96-h adverse sickle cell patient outcomes can be identified and combined into a risk score. Prospective validation is necessary before any clinical decision-making based on this score.

Comparison of characteristics of admitted emergency department patients requiring cardiopulmonary resuscitation in the ICU and non-ICU setting.

BACKGROUND: Hospitalised patients requiring cardiopulmonary resuscitation (CPR) have better outcomes in intensive care units (ICUs) than wards. Survival could potentially be improved for patients at high risk for CPR if they can be identified while in the emergency department (ED) and admitted to an ICU setting. It is currently unknown whether patients requiring CPR who are admitted to the ward show a similar pattern of physiological deterioration to those admitted to the ICU, and thus whether future research should consider these two patients groups as distinct. It is hypothesised that, since both groups of patients decompensate to the point of requiring acute resuscitation shortly after hospital admission, they should also share similar premonitory signs of deterioration in their basic physiological parameters. METHODS: A retrospective chart review was performed of adult patients at an urban ED requiring CPR within 72 h of admission from March 2002 to March 2005. Data were compared between subjects admitted to ICU and non-ICU beds. RESULTS: 45 patients (58% women) of mean age 59 years met the inclusion criteria; 40% required CPR in a non-ICU ward. There were no differences in demographic characteristics, ED chief complaint or admission diagnosis between the two groups. Blood pressure was significantly higher in the non-ICU subjects at ED arrival (129/75 vs 100/50), time of admission (122/74 vs 103/58) and before CPR (117/70 vs 92/50) (p

Initial risk stratification and presenting characteristics of patients with evolving myocardial infarctions.

OBJECTIVES: To describe the presenting characteristics and risk stratification of patients presenting to the emergency department with chest pain who have a normal initial troponin level followed by a raised troponin level within 12 h (evolving myocardial infarction (EMI)). METHODS: Data from the Internet Tracking Registry for Acute Coronary Syndromes (i*trACS), a registry of patients presenting with undifferentiated chest pain, were used. This analysis included patients without ST segment elevation with at least two troponin assay results < or = 12 h apart. Patients were stratified into three groups: EMI (initial troponin assay negative, second troponin assay positive), non-ST elevation myocardial infarction (NSTEMI) (initial troponin assay positive) and no MI (all troponin assays negative). RESULTS: Of 4136 eligible patients, 5% had EMI, 8% had NSTEMI and 87% had no MI. Patients with EMI were more similar to those with NSTEMI than those with no MI with respect to demographic characteristics, presentation, admission patterns and revascularisation. The initial ECG in patients with EMI was most commonly non-diagnostic (51%), but physicians' initial impressions commonly reflected MI, unstable angina or high-risk chest pain (76%). This risk assessment was followed by a high rate of critical care admissions (32%) and revascularisation (percutaneous coronary intervention 17%) among patients with EMI. CONCLUSION: Patients with EMI appear similar at presentation to those with NSTEMI. Patients with EMI are perceived as being at high risk, evidenced by similar diagnostic impressions, admission practices and revascularisation rates to patients with NSTEMI.

Independent development of pancreatic alpha- and beta-cells from neurogenin3-expressing precursors: a role for the notch pathway in repression of premature differentiation.

The nature and identity of the pancreatic beta-cell precursor has remained elusive for many years. One model envisions an early multihormonal precursor that gives rise to both alpha- and beta-cells and the other endocrine cell types. Alternatively, beta-cells have been suggested to arise late, directly from the GLUT2- and pancreatic duodenal homeobox factor-1 (PDX1)-expressing epithelium, which gives rise also to the acinar cells during this stage. In this study, we have identified a subset of the PDX1+ epithelial cells that are marked by expression of Neurogenin3 (Ngn3). Ngn3, a member of the basic helix-loop-helix (bHLH) family of transcription factors, is suggested to act upstream of NeuroD in a bHLH cascade. Detailed analysis of Ngn3/paired box factor 6 (PAX6) and NeuroD/PAX6 co-expression shows that the two bHLH factors are expressed in a largely nonoverlapping set of cells, but such analysis also suggests that the NeuroD+ cells arise from cells expressing Ngn3 transiently. NeuroD+ cells do not express Ki-67, a marker of proliferating cells, which shows that these cells are postmitotic. In contrast, Ki-67 is readily detected in Ngn3+ cells. Thus, Ngn3+ cells fulfill the criteria for an endocrine precursor cell. These expression patterns support the notion that both alpha- and beta-cells develop independently from PDX1+/Ngn3+ epithelial cells, rather than from GLU+/INS+ intermediate stages. The earliest sign of alpha-cell development appears to be Brain4 expression, which apparently precedes Islet-1 (ISL1) expression. Based on our expression analysis, we propose a temporal sequence of gene activation and inactivation for developing alpha- and beta-cells beginning with activation of NeuroD expression. Endocrine cells leave the cell cycle before NeuroD activation, but re-enter the cell cycle at perinatal stages. Dynamic expression of Notch1 in PDX+ epithelial cells suggests that Notch signaling could inhibit a Ngn-NeuroD cascade as seen in the nervous system and thus prevent premature differentiation of endocrine cells.

Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels.

Mice with targeted mutations in genes required for Notch signal transduction die during embryogenesis, displaying overt signs of hemorrhage due to defects in their vascular development. Surprisingly, directed expression of a constitutively active form of Notch4 within mouse endothelial cells produces a similar vascular embryonic lethality. Moreover, patients with mutations in Notch3 exhibit the cerebral vascular disorder, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These findings underscore the importance of Notch signaling in vascular development; however, they do not identify the specific functional defect. Here, we report that Notch1, Notch3, Notch4, Delta4, Jagged1 and Jagged2 are all expressed in arteries, but are not expressed by veins. These findings identify an aspect of Notch signaling that could contribute to the mechanism by which this pathway modulates vascular morphogenesis.

Regulation of follistatin messenger ribonucleic acid in porcine granulosa cells by epidermal growth factor and the protein kinase-C pathway.

Follistatin is a 35-kilodalton monomer isolated from follicular fluid by virtue of its ability to suppress FSH secretion from cultured pituitary cells. Experiments were designed to test the hypothesis that the accumulation of follistatin RNA in the ovary is regulated by epidermal growth factor (EGF) and activation of the protein kinase-C (PKC) pathway. Follistatin mRNA was quantitated by slot blot hybridization of total RNA from primary cultures of porcine granulosa cells treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PMA (0.1, 1.0, 10, and 100 nM) induced a dose-dependent increase in follistatin mRNA accumulation after 2 h, with a maximal increase of 40-fold over that in untreated control cultures at a dose of 10 nM. PMA (10 nM) induced a time-dependent increase in follistatin mRNA levels, with a maximal response at 2 h. Follistatin gene expression was induced by a 2-h incubation with EGF (3 nM), but not by LH (100 ng/ml), GnRH (10 nM) or prostaglandin F2 alpha (80 micrograms/ml). EGF (0.01, 0.1, 1, and 10 nM) induced a dose-dependent induction of follistatin gene expression in granulosa cells after 2-h incubation, with maximal stimulation of 33-fold at a dose of 1 nM. The time course of induction of follistatin mRNA by EGF was very similar to that induced by PMA, with maximal stimulation occurring at 2 h and declining thereafter. Pretreatment of granulosa cells for 24 h with PMA abrogated the EGF-induced stimulation of follistatin mRNA accumulation. However, cotreatment of granulosa cells with EGF and PMA for 2 h resulted in additive stimulation of follistatin mRNA. These results demonstrate that 1) follistatin gene expression in cultured porcine granulosa cells is acutely stimulated by PMA and EGF in a time- and dose-dependent manner; 2) follistatin gene expression may be regulated by the PKC pathway; and 3) the stimulatory effect of EGF on follistatin gene expression may require PKC.

Duration of acute exposures to vibration and finger circulation.

OBJECTIVES: This study investigated changes in finger circulation after different durations of exposure to hand-transmitted vibration. METHODS: Finger skin temperature (FST), finger blood flow (FBF), and finger systolic blood pressure (FSBP) were measured in the middle fingers of both hands of 10 healthy men. Finger vascular resistance was also estimated. The right hand was exposed for 7.5, 15, and 30 minutes (static load 10 N) to 125-Hz vibration (root-mean-square acceleration 87 m/s2). Static load only was used as a control. Finger circulation was measured before the vibration and static load exposure and at fixed intervals during exposure and a 45-minute recovery period. RESULTS: No significant changes were found with the static load. The FST and FSBP did not change significantly during vibration exposure, whereas vibration produced significant reductions in FBF and increases in vascular resistance at each duration when compared with preexposure and contralateral (non-vibrated) finger values. Temporary vasodilation occurred in the vibrated finger immediately after each vibration exposure. Recovery was complete for FBF and vascular resistance after the 7.5-minute vibration, whereas a progressive FBF reduction occurred in both the vibrated and the nonvibrated fingers after 15- and 30-minute exposure. The longer the duration of vibration exposure, the stronger the vasoconstriction in the vibrated finger during recovery. CONCLUSIONS: Vasoregulatory mechanisms mediated by both intrinsic (local) and extrinsic (neural or endocrine) control systems seem to be related to digital circulatory changes during 125-Hz vibration. It is concluded that, not only the frequency and magnitude of vibration, but also its duration contributes to the reaction of the digital vessels to acute vibration.

Magnitude of acute exposures to vibration and finger circulation.

OBJECTIVES: Changes in finger circulation were studied during and after acute exposure to increasing magnitudes of hand-transmitted vibration. METHODS: Finger skin temperature (FST) and finger blood flow (FBF) were measured in the middle fingers of both hands of 10 healthy men. The right hand was exposed for 15 minutes to 125-Hz vibration with acceleration magnitudes of either 5.5, 22, 44, or 62 m/s2 root-mean-square. The measures of finger circulation were taken before the vibration, at fixed intervals during exposure, and during a 45-minute recovery period. RESULTS: The FST did not change during vibration exposure, whereas vibration of any magnitude provoked significant reductions in the FBF of the vibrated finger when compared with the preexposure FBF and the contralateral (nonvibrated finger) FBF. Vasoconstrictor aftereffects (i.e., during recovery) were observed in both fingers after the end of exposure to vibration magnitudes greater than 22 m/s2 root-mean-square. The higher the vibration magnitude, the stronger the reduction of FBF in either finger during both vibration exposure and the recovery period. This effect was stronger in the vibrated finger than in the nonvibrated finger during both periods. CONCLUSIONS: Acute exposure to 125-Hz vibration can reduce FBF in both the vibrated and the nonvibrated finger, and the degree of digital vasoconstriction is related to the magnitude of the vibration. The pattern of the hemodynamic changes during and after vibration exposure suggests that complex vasomotor mechanisms are involved in the response of digital vessels to acute vibration.

Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle.

Forty-two Holstein heifers were superovulated with FSH-P (total dose, 30 mg) and cloprostenol. Treatment was initiated on Day 3 (Group D3, n = 11), Day 6 (Group D6, n = 11), Day 9 (Group D9, n = 10) or Day 12 (Group D12, n = 10) of the estrous cycle. Heifers were bled daily for serum progesterone and estradiol-17beta determinations and every 6 h for a 48-h duration at the expected time of estrus for luteinizing hormone (LH) assay. Ova and embryos were flushed from the reproductive tracts and the number of corpora lutea (CL) were recorded after slaughter on Day 7 post-estrus. Mean (+/- SEM) numbers of observed CL were higher (P < 0.05) in Group D9 (33.3 +/- 4.8) than in Group D3 (15.3 +/- 3.8), with Group D6 (17.0 +/- 2.9) and Group D12 (23.9 +/- 7.3) being intermediate. Similarly, mean (+/- SEM) numbers of fertilized embryos were highest (P < 0.05) in Group D9 (13.3 +/- 2.2). There was also a nonsignificant trend for the number of transferable embryos to be greatest in Group D9. Neither serum progesterone concentrations 3 d after the LH peak nor peak serum estradiol 17beta concentrations differed among groups, but both were significantly correlated with numbers of observed CL and total ova and embryos.

A retrospective clinical study of osteochondrosis dissecans in 21 horses.

Osteochondrosis dissecans was diagnosed clinically and radiographically in 31 joints of 21 horses. The horses ranged in age from 8 months to 5 years at the time of presentation. The usual age of onset of clinical signs was 18 to 24 months. Presenting complaints included joint effusion and lameness of either gradual or sudden onset. In Thoroughbred horses, the stifle joint was the most common site of lesions and in Standardbred horses lesions occurred more commonly in the hock. In 16 of the 21 horses, the contralateral joint was radiographed and 9 of these horses had bilateral lesions. Thoroughbred horses were affected most commonly, followed by Standardbred horses. The prevalence was higher in males than females, the male: female ratio being 2.5:1.

Finger systolic blood pressures: effects of cold provocation on the reference finger.

Finger systolic blood pressure measured after cold provocation and ischemia of a digit is used to assist in the diagnosis of vibration-induced white finger, VWF. A reduction in finger systolic blood pressure after cooling is assumed to indicate vascular dysfunction. The percentage pressure change observed in the tested finger is often corrected for whole body effects (systemic systolic pressure changes) according to the pressure change measured in a reference finger. The commonly used method of correction is based on assumptions as to the causes of any changes occurring in the reference finger. It is assumed that the reference finger is not differentially susceptible to the cold provocation of the test finger, arising from either close proximity to the cold provocation or from a vascular disorder in the reference finger. An experiment has been undertaken to investigate the repeatability, over three days, of measurements of the arm systolic pressures of both arms and the finger systolic pressures in air of four fingers of both hands. The systolic pressures of both arms and of four fingers of one hand were also measured whilst the fifth finger of the same hand was subjected to cold provocation at 10 degrees C. Twelve healthy male subjects were rested in a supine position for 15 minutes in a room at 21-24 degrees C before measurements were taken. Finger systolic blood pressures were recorded using strain gauge plethysmography. The results show that the systolic blood pressure measurements were generally repeatable, but differed with measurement location. Cold provocation of the test finger had little consistent effect on the systolic pressures measured at other locations. The results are interpreted with regard to the correction of finger systolic pressure using a reference measurement.

The adverse effect of spasticity on 3-month poststroke outcome using a population-based model.

Several devices and medications have been used to address poststroke spasticity. Yet, spasticity's impact on outcomes remains controversial. Using data from a cohort of 460 ischemic stroke patients, we previously published a validated multivariable regression model for predicting 3-month modified Rankin Score (mRS) as an indicator of functional outcome. Here, we tested whether including spasticity improved model fit and estimated the effect spasticity had on the outcome. Spasticity was defined by a positive response to the question "Did you have spasticity following your stroke?" on direct interview at 3 months from stroke onset. Patients who had expired by 90 days (n = 30) or did not have spasticity data available (n = 102) were excluded. Spasticity affected the 3-month functional status (ß = 0.420, 95 CI = 0.194 to 0.645) after accounting for age, diabetes, leukoaraiosis, and retrospective NIHSS. Using spasticity as a covariable, the model's R (2) changed from 0.599 to 0.622. In our model, the presence of spasticity in the cohort was associated with a worsened 3-month mRS by an average of 0.4 after adjusting for known covariables. This significant adverse effect on functional outcomes adds predictive value beyond previously established factors.

Thermal thresholds, vibrotactile thresholds and finger systolic blood pressures in dockyard workers exposed to hand-transmitted vibration.

OBJECTIVES: To quantify neurological dysfunction in workers exposed to hand-transmitted vibration using alternative neurological tests. To relate the neurological findings to the results of vascular tests and the symptoms reported by subjects with vibration-induced white finger. METHODS: Thermal thresholds (for perception of heat and cold), vibrotactile thresholds (for perception of vibration at 31.5 and 125 Hz) and finger systolic blood pressures were measured in 107 dockyard workers, including 31 controls and 76 workers exposed to hand-transmitted vibration (50 reporting finger blanching consistent with vibration-induced white finger). A history of vibration exposure and symptoms associated with hand-transmitted vibration were obtained for each subject. RESULTS: Increased duration of exposure to vibration resulted in a deterioration of both thermal thresholds and vibrotactile thresholds. Finger systolic blood pressures were lower in subjects reporting finger blanching and were related to the extent of blanching on the measured finger. Reported sensations of tingling were not correlated with any of the threshold measures; thermal thresholds and vibrotactile thresholds showed evidence of deterioration with reports of increasing numbness. Both numbness and tingling were correlated with reports of finger blanching. Finger systolic blood pressures were not correlated with either thermal or vibrotactile thresholds. CONCLUSIONS: Vascular and neurological signs produced by hand-transmitted vibration can occur independently, but the principal vascular symptom (i.e. attacks of blanching) and some commonly reported neurological symptoms (i.e. sensations of numbness and tingling) may be related.

Interpretation of the finger skin temperature response to cold provocation.

OBJECTIVES: To compare alternative methods of interpreting the response of finger skin temperature (FST) to cold provocation for the detection of the abnormal cold response observed in vibration-induced white finger (VWF). METHOD: The FST response to cold provocation was measured in 36 male subjects: 12 office workers, 12 manual workers and 12 manual workers with symptoms of VWF. The FSTs were monitored continuously on the distal phalanges of all five fingers of a test hand for 2 min before, for 5 min during, and for 10 min following, immersion of the test hand in water at 15 degrees C. Of the fingers investigated, 147 were reported not to exhibit blanching and 33 were reported to exhibit blanching. Twenty-one alternative methods of interpreting the response of FSTs to cold provocation were assessed. These were grouped as: (1) areas above the response profile (i.e. the area above the curve showing the FSTs as a function of time during cooling and recovery), (2) areas below the response profile, (3) absolute temperatures during and following cold provocation, (4) percentage differences in FSTs, (5) the times taken for FSTs to rise by specified amounts and (6) rates of change of FSTs. Differences in the response to cooling between those fingers reported to blanch and the fingers not reported to blanch were tested, and receiver operating characteristics (ROCs) were used to compare the sensitivity and specificity of the various measures to symptoms of VWF. RESULTS: The areas above the response profile, areas below the response profile, percentage FSTs, absolute FSTs and rates of change of FSTs tended to discriminate between healthy and unhealthy subjects on a group basis. However, some of these methods of interpreting the FST response to cold provocation did not show a high sensitivity or specificity to vascular dysfunction on individual fingers. The area above the response profile, the percentage of initial temperature at the fifth minute of recovery and the maximum temperature during the 10-min recovery period, were found to show the highest sensitivity and specificity to symptoms of vascular dysfunction. CONCLUSIONS: The method chosen to interpret the FST response to cold provocation affects the ability of the test to detect an abnormal cold response. The area above the response profile, the percentage of initial temperature at the fifth minute of recovery and the maximum temperature achieved during a 10-min recovery period appear to be the most suitable measures for monitoring vascular function in workers exposed to hand-transmitted vibration. It is suggested that the FST response to cold provocation should be interpreted with respect to the state of initial blood flow.

Acute vascular responses to the frequency of vibration transmitted to the hand.

OBJECTIVES: To investigate the acute effects of the frequency of hand transmitted vibration on finger circulation. A further aim was to investigate whether the frequency weighting assumed in current standards for hand transmitted vibration reflects the haemodynamic changes which occur in the fingers exposed to vibration with different frequencies but with the same frequency weighted acceleration magnitude. METHODS: Finger skin temperature (FST) and finger blood flow (FBF) were measured in the middle fingers of both hands of 10 healthy men. With a static load of 10 N, the right hand was exposed for 15 minutes to the following root mean square (rms) acceleration magnitudes and frequencies of vertical vibration: 5.5 m/s(2) at 16 Hz; 11 m/s(2) at 31.5 Hz; 22 m/s(2) at 63 Hz; 44 m/s(2) at 125 Hz; and 88 m/s(2) at 250 Hz. These exposures to vibration produce the same frequency weighted acceleration magnitude (5.5 m/s(2) rms) according to the frequency weighting included in the international standard ISO 5349. A control condition consisted of exposure to the static load only. Finger circulation was measured before application of the vibration and static load and at fixed intervals during exposure to vibration and a 45 minute recovery period. RESULTS: No significant changes in finger circulation were found with only the static load. The FST did not change significantly during or after acute exposure to vibration. In the vibrated right finger, exposures to vibration with frequencies of 31. 5-250 Hz provoked a greater reduction in FBF than did vibration of 16 Hz or the static load only. In the non-vibrated left finger, the FBF measured with vibration at each frequency of 63-250 Hz was significantly lower than that measured with static load only. The reduction in FBF during exposure to vibration with any frequency was stronger in the vibrated finger than in the non-vibrated finger. In both fingers, there was a progressive decrease in FBF after the end of exposure to vibration with frequencies of 31.5-250 Hz. The higher the frequency of vibration, the stronger the decrease in FBF in both fingers during recovery. CONCLUSIONS: Acute exposures to vibration with equal frequency weighted magnitude reduce the FBF in both vibrated and non-vibrated fingers for frequencies between 31.5 and 250 Hz. The extent of digital vasoconstriction after exposure to vibration increases with increasing frequency. The frequency weighting given in current standards tends to overestimate the vasoconstriction associated with acute exposures to vibration frequencies around 16 Hz.

Regulation of follistatin gene expression in the ovary and in primary cultures of porcine granulosa cells.

Experiments were designed to test the hypotheses that (1) follistatin gene expression in granulosa cells is regulated during follicular growth, and (2) that alteration of follistatin mRNA concentration can be hormonally induced in primary cultures of porcine granulosa cells. RNA isolated from granulosa cells from small (1-3 mm diameter), medium (3-5 mm) and large (> 5 mm) follicles of prepubertal and postpubertal sows was analysed by hybridization to a porcine follistatin cDNA probe. Amounts of follistatin mRNA increased with follicular diameter, but no differences in follicular follistatin mRNA were detected between prepubertal and postpubertal sows. Treatment of cultured porcine granulosa cells with FSH or LH for 20 h stimulated follistatin mRNA concentration by a factor of two (100 ng FSH ml-1) and a factor of 1.5 (10 ng LH ml-1), respectively, over untreated controls. Treatment of cultured granulosa cells with 200 ng FSH ml-1, 200 ng LH ml-1, 10 mumol dibutyryl cAMP l-1, 30 mumol forskolin l-1 and 100 ng cholera toxin ml-1 stimulated follistatin mRNA accumulation in granulosa cells by factors of 4.9, 3.7, 1.6, 13.7 and 3.5, respectively, compared with control cultures. Stimulation of follistatin mRNA accumulation in cultured granulosa cells by dibutyryl cAMP (30, 100 and 300 mumol l-1) and forskolin (3, 10 and 100 mumol l-1) was dose dependent. FSH and forskolin induced time-dependent increases in follistatin mRNA concentration in cultured granulosa cells, with maximal induction occurring 72 h after treatment (a factor of 4.5 for FSH and 15.5 for forskolin).(ABSTRACT TRUNCATED AT 250 WORDS)

Follistatin has characteristics of a primary response gene in porcine granulosa cells.

To explore the regulation of follistatin gene expression, porcine granulosa cells were incubated with the translational inhibitor, cycloheximide (CHX), for periods from 6-24 h. This resulted in a 3 to 10-fold increase in follistatin mRNA accumulation compared to vehicle treated control cultures. At 20 h, CHX augmented the follicle stimulating hormone (FSH) induced stimulation of follistatin mRNA accumulation by a mean of more than sixfold. Over 6 h, CHX elevated follistatin mRNA abundance twofold, while epidermal growth factor (EGF) increased the message threefold. CHX in the presence of EGF produced an effect additive to the EGF response. Results in the longer term differed, as pretreatment of granulosa cells with CHX for 20 h suppressed the induction of follistatin gene expression by both EGF and phorbol 12-myristate-13-acetate. By blockade of transcription with Actinomyocin D, an estimate of the half-life of follistatin mRNA between 4 and 8 h was made. Half-life did not appear to be affected by the CHX suppression of protein translation. From the observations of the occurrence of follistatin gene expression independent of protein synthesis, superinduction in the presence of CHX and FSH, and the interactions between CHX and EGF, it is concluded that follistatin is a primary response gene in porcine granulosa cells.

Jagged2: a serrate-like gene expressed during rat embryogenesis.

DSL (Delta, Serrate, Lag-2) ligands activate Notch signaling and thereby regulate the differentiation of many different cell types during development. We have isolated a novel Serrate-like gene, Jagged2, whose amino acid sequence and expression pattern during rat embryogenesis suggest that it functions as a ligand for Notch. In contrast to previously described DSL ligands for Notch, Jagged2 is not widely expressed in the developing central nervous system. However, Jagged2 and Notch1 are coexpressed in the apical ectodermal ridge (AER), suggesting a role for this ligand-receptor pair in limb development. Furthermore, unlike Jagged1, Jagged2 is coexpressed with Notch in the developing thymus, where it may induce Notch signaling to direct T-cell fate. Our data are consistent with the idea that the diversity of cell types regulated by Notch signaling is a consequence of activation of unique Notch isoforms by different DSL ligands.

Response of finger circulation to energy equivalent combinations of magnitude and duration of vibration.

OBJECTIVES: To investigate the acute response of finger circulation to vibration with different combinations of magnitude and duration but with the same "energy equivalent" acceleration magnitude according to current standards for hand transmitted vibration. METHODS: Finger skin temperature (FST) and finger blood flow (FBF) were measured in the middle fingers of both hands of 10 healthy men who had not used hand held vibrating tools regularly. With a static load of 10 N, the right hand was exposed to 125 Hz vibration with the following unweighted root mean square (rms) acceleration magnitudes and durations of exposure: 44 m/s(2) for 30 minutes; 62 m/s(2) for 15 minutes; 88 m/s(2) for 7.5 minutes; 125 m/s(2) for 3.75 minutes; and 176 m/s(2) for 1.88 minutes. These vibration exposures produce the same 8 hour energy equivalent frequency weighted acceleration magnitude (approximately 1.4 m/s(2) rms) according to international standard ISO 5349 (1986). Finger circulation was measured in both the right (vibrated) and the left (non-vibrated) middle fingers before application of the vibration, and at fixed intervals during exposure to vibration and during a 45 minute recovery period. RESULTS: The FST did not change during exposure to vibration, whereas vibration with any combination of acceleration magnitude and duration produced significant percentage reductions in the FBF of the vibrated finger compared with the FBF before exposure (from -40.1% (95% confidence interval (95% CI) -24.3% to -57.2%) to -61.4% (95% CI -45.0% to -77.8%). The reduction in FBF during vibration was stronger in the vibrated finger than in the non-vibrated finger. Across the five experimental conditions, the various vibration stimuli caused a similar degree of vasoconstriction in the vibrated finger during exposure to vibration. There was a progressive decrease in the FBF of both fingers after the end of exposure to vibration with acceleration magnitudes of 44 m/s(2) for 30 minutes and 62 m/s(2) for 15 minutes. Significant vasoconstrictor after effects were not found in either finger after exposure to any of the other vibration stimuli with greater acceleration magnitudes for shorter durations. CONCLUSIONS: For the range of vibration magnitudes investigated (44 to 176 m/s(2) rms unweighted; 5.5 to 22 m/s(2) rms when frequency weighted according to ISO 5349), the vasoconstriction during exposure to 125 Hz vibration was independent of vibration magnitude. The after effect of vibration was different for stimuli with the same energy equivalent acceleration, with greater effects after longer durations of exposure. The energy equivalent acceleration therefore failed to predict the acute effects of vibration both during and after exposure to vibration. Both central and local vasoregulatory mechanisms are likely to be involved in the response of finger circulation to acute exposures to 125 Hz vibration.