Expression and mutation analysis of Tpit in the canine pituitary gland and corticotroph adenomas.

Pituitary-dependent hyperadrenocorticism (PDH) in dogs is caused by a pituitary corticotroph adenoma. Although PDH is a common disorder in dogs, little is known about the underlying pathogenesis. In the pituitary glands of humans and mice, the pro-opiomelanocortin (POMC)-expressing cell lineages, the corticotrophs and melanotrophs, have a specific marker in common, the T-box transcription factor Tpit (Tbx19), which is obligate for POMC expression. Tpit also regulates the late differentiation of the corticotrophs and melanotrophs, and therefore may contribute to the pathogenesis of the corticotroph adenomas. The aim of this study was to perform an expression and mutation analysis of Tpit in the normal canine pituitary and in corticotroph adenomas. The distribution of the Tpit protein in the pituitary gland was studied with immunohistochemistry and the expression of the gene with RT-PCR. The coding region of Tpit cDNA from 14 dogs with PDH was screened for mutations. Tpit was expressed in corticotroph and melanotroph cells of the normal and adenomatous canine pituitary, and remained present in non-adenomatous corticotrophs of pituitaries from PDH dogs. No tumor-specific mutation in the Tpit cDNA from the corticotroph adenomas was found. However, a missense polymorphism in the highly conserved DNA-binding domain, the T-box, was discovered in one dog. It is concluded that Tpit can be used as a reliable marker for the corticotroph and melanotroph cells in the canine pituitary tissue and that mutations in the Tpit gene are unlikely to play a major role in the pathogenesis of canine corticotroph adenomas.

Chronic hypocalcemia of vitamin D deficiency leads to lower intracellular calcium concentrations in rat hepatocytes.

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Hypocalcemia modifies the intracellular calcium response to the alpha 1-adrenergic agent phenylephrine in rat hepatocytes.

In vivo, extracellular calcium ([Ca2+]e) homeostasis is maintained within a very narrow range by the calcium regulating hormones. At the cellular level, the response to many agents is transduced by changes in cytosolic Ca2+ ([Ca2+]i) which involves both mobilization of cellular pools and entry of [Ca2+]e through plasma membrane channels. To investigate the cellular effects of chronic hypocalcemia (Ca-) on [Ca2+]i homeostasis, hepatocytes, a cell type well characterized for its [Ca2+]i response, were used. Data indicate that Ca- leads to a significant shift to the left in the basal resting cytosolic Ca2+ concentration distribution curve with half-maximum cumulative frequency of 119 versus 149 nM in Ca- and normal rats (N) respectively (P < 0.0001). The response to the alpha 1-adrenergic agonist phenylephrine (Phe) was also influenced by Ca- with a dampening of the dose-response curve, a significant decrease in the frequency of sustained responses (P < 0.001), and significant changes in the oscillation pattern. Indeed, hepatocytes obtained from Ca- exhibited a higher frequency of large amplitude, low frequency oscillations than N most particularly at the 2 and 5 microM Phe dose while N predominantly exhibited low amplitude, high frequency oscillations on sustained plateaus (P < 0.001). IP3 receptor (IP3R) binding studies and Ca2+ mobilization from IP3-sensitive pools showed that IP3R was highly sensitive to the prevailing Ca2+ with, in the range of resting [Ca2+]i, R affinity significantly lower in Ca- than in N. Upon exposure of permeabilized cells to 25 microM IP3, Ca2+ mobilization from the IP3-sensitive intracellular pool was significantly reduced by Ca- (P < 0.05) suggesting a decrease in the IP3-mobilizable Ca2+ pool in Ca-. Our results indicate that hypocalcemia significantly alters [Ca2+]i signalling by perturbing the initial response to agonist and the [Ca2+]i response pattern. In addition, the decrease in Ca2+ mobilization from IP3-sensitive pools suggests that hypocalcemia may also lead to a decrease in the Ca2+ content of intracellular pools.

A motor reeducation program aimed to improve strength and coordination of the upper limb of a hemiparetic subject.

The objective of the study is to describe a new reeducation program based on a multi-directional and multi-articular dynamometer and to evaluate its applicability in one chronic right hemiparetic subject. The treatment sessions lasted 1 h and were conducted three times per week for a period of 8 weeks. During these sessions, the subject was asked to exert 10 repetitions of 16 torque combinations exerted at the shoulder, elbow and forearm or combined with handgrip exertion. The sequence of torques and force progressed from proximal to distal joints, and were realized in and out of the typical 'synergy patterns' described in this population. In addition, the levels of torque and force requested were increased progressively throughout the treatment period. The coordination of both upper extremities, tested using the finger to nose test, and the dexterity of the affected side, evaluated using the Box and Blocks assessment, tended to improve as treatments progressed. These results indicate the feasibility of this approach and suggest that it may be worthwhile examining the effectiveness of this approach on improving the functional performance of the upper extremity in a larger population of hemiparetic subjects.

Lower limb prosthesis utilisation by elderly amputees.

The goal of prosthetic rehabilitation is to compensate for the loss of a limb by amputation by, in the case of a lower limb, encouraging walking, and to achieve the same level of autonomy as prior to the amputation. However, because of difficulties walking, elderly amputees may use their prosthesis to a greater or lesser degree or simply stop using it during the rehabilitation period. The objective of this research was to study factors such as physical and mental health, rehabilitation, physical independence and satisfaction with the prosthesis to understand why amputees use their prosthesis or not. The sample was composed of 65 unilateral vascular amputees 60 years old or over living at home. The information was collected from medical records, by telephone interview and by mail questionnaire. Prosthesis use was measured by a questionnaire on amputee activities developed by Day (1981). Eighty-one per cent (81%) of the subjects wore their prosthesis every day and 89% of this group wore it 6 hours or more per day. Less use of the prosthesis was significantly related to age, female gender, possession of a wheelchair, level of physical disability, cognitive impairment, poorer self-perceived health and the amputee's dissatisfaction. A multiple regression analysis showed that satisfaction, not possessing a wheelchair and cognitive integrity explained 46% of the variance in prosthesis use.

[Questionnaire on the satisfaction of persons with lower-limb amputations towards their prosthesis: development and validation]. / Questionnaire sur la satisfaction des personnes amputées du membre inférieur face à leur prothèse: développement et validation.

The satisfaction of persons with lower-limb amputations towards their prosthesis constitutes a critical factor in the use of the prosthesis. In order to evaluate a person's satisfaction, the SAT-PRO, a self-administrated questionnaire was developed. The questionnaire includes 15 items developed on the basis of the most significant criteria used by the person when selecting a technical aid. These criteria were measured using an ordinal categorical four-level scale. The validation of the SAT-PRO was established from a sample consisting of 61 people with below-knee or above-knee amputations, aging from 60 years and older. The internal consistency of the instrument is high (Cronback's alpha coefficient is 0.90) and the test-retest reliability coefficient (0.97) indicates a very good consistency of the questionnaire over time. Simple and multiple correlations were used to evaluate construct validity. The degree of use of the prosthesis and the feelings of depression, amongst the measured variables, are the best indicators of the satisfaction of the amputees toward their prosthesis.

Biocompatibility and physicochemical characteristics of alginate-polycation microcapsules.

There is a need for better understanding of the biocompatibility of alginate-polycation microcapsules based on their physicochemical characteristics. Microcapsules composed of alginate with 44% (IntG) or 71% (HiG) guluronate, gelled with calcium (Ca) or barium (Ba) and coated with poly-L-lysine (PLL) or poly-l-ornithine (PLO), followed by IntG alginate were compared. For microcapsules with an IntG(Ca) gel core, using PLO instead of PLL resulted in less immune cell adhesion after 2 days in C57BL/6J mice. The PLO microcapsules were also characterized by greater hydrophilicity and superior resistance to swelling and damage under osmotic stress. For microcapsules with a PLL membrane, replacing the IntG(Ca) gel core with IntG(Ba) or HiG(Ca) gel resulted in stronger immune responses (p<0.05). This was explained by poor penetration of PLL into the gel, as demonstrated by Fourier transform infrared spectroscopy analyses and membrane rupturing during osmotic swelling. X-ray photoelectron spectroscopy analyses show that all microcapsules had the same amount of polycation at their surface. Moreover, alginate coatings had non-significant effects on the biocompatibility and physicochemical properties of the microcapsules. Thus, alginate-polycation interactions for membrane formation are more important for biocompatibility than either the quantity of polycation at the surface or the alginate coating.

Of old and new diseases: genetics of pituitary ACTH excess (Cushing) and deficiency.

The pituitary gland orchestrates our endocrine environment: it produces hormones in response to hypothalamic factors that integrate neural inputs and its activity is balanced by the feedback action of peripheral hormones. Disruption of this equilibrium has severe consequences that affect multiple systems and may be fatal. Genetic analysis of pituitary function led to discovery of critical transcription factors that cause hormone deficiencies when mis-expressed. This review will summarize recent findings that led to the first complete clinical description of inherited, isolated corticotropin (ACTH) deficiency (IAD) and to the first molecular mechanism for excessive ACTH production in Cushing's disease. Indeed, mutations in TPIT, a positive or negative regulator of cell fates for different pituitary lineages, cause neonatal IAD, a condition considered anecdotic before discovery of this transcription factor. Cushing's disease is caused by corticotroph adenomas that produce excess ACTH as a result of resistance to glucocorticoids (Gc). Molecular investigation of the normal mechanism of Gc feedback led to identification of two essential proteins for pro-opiomelanocortin repression that are often mis-expressed in corticotroph adenomas thus providing a molecular explanation for Gc resistance. These two proteins, Brg1 and histone deacetylase 2 (HDAC2), are involved in chromatin remodeling and may also participate in the tumorigenic process, as Brg1 is a tumor suppressor. These recent advances have provided improved diagnosis and opened new perspectives for patient management and therapies.

Pulmonary edema of the right upper lobe associated with acute mitral regurgitation.

To determine the association between mitral regurgitation and pulmonary edema localized in the right upper lobe, the authors reviewed 21 cases of mitral regurgitation secondary to dysfunction or rupture of the papillary muscle or rupture of the chordae tendineae cordis. The patients, 12 men and 9 women ranging in age from 36 to 92 (mean 64) years, had been admitted to a tertiary care hospital between July 1985 and July 1990. Three independent observers, who were unaware of the patients' identity or the diagnoses, reviewed the chest radiographs. In eight of the patients pulmonary edema was localized preferentially in the right upper lobe, an unusual pattern that can simulate neoplasia, hemorrhage or infection. All eight patients had myocardial infarction, five had papillary muscle dysfunction, and three had rupture of the posterior papillary muscle. Mitral regurgitation toward the orifices of the veins of the right upper lobe seems to play a role in the preferential distribution of edema to that lobe. Awareness of edema in the right upper lobe in association with mitral regurgitation might lead to earlier diagnosis of papillary rupture or dysfunction and perhaps affect the outcome.

Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro.

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of granulosa cell co-culture on in-vitro meiotic resumption of bovine oocytes.

This study was undertaken to create an in-vitro model using granulosa cell monolayers to replace the role of the follicle in the maturation of bovine oocytes. Cumulus-oocyte complexes were co-incubated with fresh or 7-day granulosa cell cultures (with new or conditioned medium) or with conditioned medium alone, in the presence or absence of IBMX (isobutylmethylxanthine), adenosine or heparin. Progression to the metaphase-II stage was significantly affected by the co-culture of oocytes with bovine granulosa cell monolayers and to a lesser degree when cultured with supernatant alone (conditioned medium). The oocytes attached rapidly to the monolayer, suggesting that the intimate contact between the granulosa cells and the cumulus-oocyte complexes is an important signal for the maintenance of meiotic arrest. Heparin did not prevent maturation itself, but prevented attachment of cumulus-oocyte complexes to monolayers, thereby reducing their inhibitory effect. Adenosine prevented cumulus expansion and reduced maturation and IBMX was an effective inhibitor only in the presence of additional granulosa cells.

Effect of adenylate cyclase stimulation on meiotic resumption and cyclic AMP content of zona-free and cumulus-enclosed bovine oocytes in vitro.

The effect of adenylate cyclase stimulation via the components of the enzyme on nuclear maturation in bovine cumulus-enclosed and zona-free oocytes was examined. The stimulating agents were cholera toxin, pertussis toxin, forskolin, sodium fluoride and prostaglandin E2. Cyclic AMP contents were measured in cumulus-oocyte complexes, cumulus-enclosed oocytes and in zona-free oocytes after stimulation, to establish the relationship between cumulus cell and oocyte cAMP concentrations and the meiotic status of the oocyte. In cumulus-enclosed oocytes, forskolin alone and 3-isobutyl-1-methylxanthine (IBMX), at 0.5 mmol l-1, inhibited the resumption of meiosis after 8 h of culture; the other agents were without effect. After 24 h of culture, IBMX at 0.5 mmol l-1 was without effect, but at 2 mmol l-1 reduced the percentage of oocytes at the mature stage (51 versus 82% in control medium). Forskolin alone reduced the proportion of oocytes at the mature stage from 82 to 58%. Forskolin plus IBMX at 2 mmol l-1 and sodium fluoride plus IBMX at 2 mmol l-1 significantly diminished the maturation rate (6 and 17% mature oocytes, respectively). Cholera toxin (with IBMX) and forskolin (alone or with IBMX) stimulated the synthesis of high amounts of cAMP in complexes, but only forskolin had a significant effect on the cAMP contents of oocytes derived from complexes. Forskolin was more effective in zona-free oocytes than in cumulus-enclosed oocytes in inhibiting nuclear maturation (24% mature oocytes versus 73% in control medium) even after 24 h of culture; its effect was potentiated by IBMX; forskolin also stimulated cAMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Description of a new motor re-education programme for the paretic lower limb aimed at improving the mobility of stroke patients.

OBJECTIVE: To describe and examine the feasibility of a new treatment approach for the paretic lower limb and to explore its effectiveness in one chronic hemiparetic stroke subject. DESIGN: Case report. The treatment was conducted three times per week over a period of six weeks. The mobility of the patient was assessed prior to the treatment, at the end of the treatment and at a six-week follow-up. SETTINGS: The study was carried out at the research centre of the Institut de réadaptation de Montréal. The treating therapist was an experienced rehabilitation professional as was the assessor, who worked at a different rehabilitation centre. INTERVENTIONS: The motor re-education programme was based on the use of a static dynamometer that measures the linear external forces produced at the ankle level. A computer program provided the subject with constant feedback on the direction and intensity of the applied force. In each treatment session, the subject was asked to produce several submaximal efforts in 16 specific directions. Both the intensity and the number of repetitions were gradually increased. OUTCOME MEASURES: In addition to force production measurements, three clinical assessments of mobility were used: the Timed 'Up and Go', the comfortable gait speed and a 2-minute walk test. RESULTS: The maximal static linear forces produced by the subject increased through the treatment for all directions of effort, but differences were observed amongst directions. During the treatment programme, the subject improved his performance at the three clinical assessments. Even if some of the functional gain was lost at the follow-up, the mobility was still considerably improved as compared to baseline values. CONCLUSION: This study demonstrated the applicability of the treatment programme to a stroke subject. The results seem very promising and encourage further investigation in order to assess more rigorously the effectiveness of this new approach.