Immunohistochemical and functional studies on calcium-sensing receptors in rat uterine smooth muscle

1. Activation of calcium‐sensing receptors (CaS) leads to relaxation of vascular smooth muscle. However, the role of CaS in uterine smooth muscle is unknown. Therefore the aim of the present study was to investigate the expression and function of CaS in the uterus. 2. The expression of CaS in the oestrogen‐dominated rat uterus was investigated using immunohistochemistry. The effects of putative CaS ligands on oxytocin‐induced contractions of longitudinally orientated uterine strips from oestrogen‐dominated rats were determined at reduced extracellular Ca²⁺concentrations using conventional organ bath techniques. 3. Immunohistochemical evidence showed the presence of CaS in the endometrium and smooth muscle layers of the rat uterus. Oxytocin‐induced contractions were inhibited by cations (Gd³⁺ > Ca²⁺ = Mg²⁺), polyamines (spermine > spermidine) and the positive allosteric modulators cinacalcet and calindol. However (R)‐ and (S)‐cinacalcet were equipotent, indicating a lack of stereoselectivity, and the negative allosteric modulator calhex‐231 also caused dose‐dependent relaxation. In addition, although intermediate‐conductance calcium‐activated potassium channels and cytochrome P450‐dependent signal transduction have been implicated in CaS‐induced relaxation of vascular smooth muscle, neither Tram‐34 nor miconazole (1 μmol/L), which block these pathways, respectively, had any effect on the ability of cinacalcet to inhibit oxytocin‐induced contractions. 4. Calcium‐sensing receptors are expressed in smooth muscle layers of the rat uterus and their ligands produce potent relaxation of longitudinally orientated uterine strips. However, the pharmacological profile of inhibition of contractility by CaS ligands is not consistent with a role for CaS in the regulation of uterine contractility in the rat.     

Ccdc66 null mutation causes retinal degeneration and dysfunction

Retinitis pigmentosa (RP) is a group of human retinal disorders, with more than 100 genes involved in retinal degeneration. Canine and murine models are useful for investigating human RP based on known, naturally occurring mutations. In Schapendoes dogs, for example, a mutation in the CCDC66 gene has been shown to cause autosomal recessively inherited, generalized progressive retinal atrophy (gPRA), the canine counterpart to RP. Here, a novel mouse model with a disrupted Ccdc66 gene was investigated to reveal the function of protein CCDC66 and the pathogenesis of this form of gPRA. Homozygous Ccdc66 mutant mice lack retinal Ccdc66 RNA and protein expression. Light and electron microscopy reveal an initial degeneration of photoreceptors already at 13 days of age, followed by a slow, progressive retinal degeneration over months. Retinal dysfunction causes reduced scotopic a-wave amplitudes, declining from 1 to 7 months of age as well as an early reduction of the photopic b-wave at 1 month, improving slightly at 7 months, as evidenced by electroretinography. In the retina of the wild-type (WT) mouse, protein CCDC66 is present at highest levels after birth, followed by a decline until adulthood, suggesting a crucial role in early development. Protein CCDC66 is expressed predominantly in the developing rod outer segments as confirmed by subcellular analyses. These findings illustrate that the lack of protein CCDC66 causes early, slow progressive rod-cone dysplasia in the novel Ccdc66 mutant mouse model, thus providing a sound foundation for the development of therapeutic strategies.     

In vitro antiplasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark of <Emphasis Type="Italic">Kigelia africana</Emphasis> (Lam.) Benth (Bignoniaceae)

In order to assess the potential of the stem bark of Kigelia africana (Lam.) Benth as source of new anti-malarial leads, n-hexane and ethyl acetate (EtOAc) extracts and four compounds isolated from the stem bark were screened in vitro against the chloroquine-resistant W-2 and two field isolates of Plasmodium falciparum using lactate dehydrogenase assay. The products were also tested for their cytotoxicity on LLC/MK2 monkey kidney cells. The EtOAc extract exhibited a significant antiplasmodial activity (IC₅₀ = 11.15 μg/mL on W-2; 3.91 and 4.74 μg/mL on field CAM10 and SHF4 isolates, respectively), whereas the n-hexane fraction showed a weak activity (IC₅₀ = 73.78 μg/mL on W-2 and 21.85 μg/mL on SHF4). Three out of the four compounds showed good activity against all the three different parasite strains (IC₅₀ < 5 μM). Specicoside exhibited the highest activity on W-2 (IC₅₀ = 1.54 μM) followed by 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid (IC₅₀ = 1.60 μM) and atranorin (IC₅₀ = 4.41 μM), while p-hydroxycinnamic acid was the least active (IC₅₀ = 53.84 μM). The EtOAc extract and its isolated compounds (specicoside and p-hydroxycinnamic acid) were non-cytotoxic (CC₅₀ > 30 μg/mL), whereas the n-hexane extract and two of its products, atranorin and 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid showed cytotoxicity at high concentrations, with the last one being the most toxic (CC₅₀ = 9.37 μg/mL). These findings justify the use of K. africana stem bark as antimalaria by traditional healers of Western Cameroon, and could constitute a good basis for further studies towards development of new leads or natural drugs for malaria.     

Hepatic miRNA expression reprogrammed by <Emphasis Type="Italic">Plasmodium chabaudi</Emphasis> malaria

Evidence is accumulating that miRNAs are critically implicated in the outcome of diseases, but little information is available for infectious diseases. This study investigates the hepatic miRNA signature in female C57BL/6 mice infected with self-healing Plasmodium chabaudi malaria. Primary infections result in approximately 50% peak parasitemia on day 8 p.i., approximately 80% survival, and development of protective immunity. The latter is evidenced as 100% survival and 1.5% peak parasitemia upon homolog re-infections of those mice which are still alive on day 56 after primary infection. Such immune mice exhibit increased levels of IgG2a and IgG2b isotypes and still contain P. chabaudi-infected erythrocytes in their livers as revealed by light microscopy and PCR analysis. Primary infections, but not secondary infections, induce an upregulation of hepatic mRNAs encoding IL-1β, TNFα, IFNγ, NF-κB, and iNOS, and a downregulation of mRNAs for CYP7A1 and SULT2A2, respectively. Using miRXplore microarrays containing 634 mouse miRNAs in combination with quantitative RT-PCR, the liver is found to respond to primary infections with an upregulation of the three miRNA species miR-26b, MCMV-miR-M23-1-5p, and miR-1274a, and a downregulation of the 16 miRNA species miR-101b, let-7a, let-7g, miR-193a-3p, miR-192, miR-142-5p, miR-465d, miR-677, miR-98, miR-694, miR-374^*, miR-450b-5p, miR-464, miR-377, miR-20a^*, and miR-466d-3p, respectively. Surprisingly, about the same pattern of miRNA expression is revealed in immune mice, and this pattern is even sustained upon homolog re-infections of immune mice. These data suggest that development of protective immunity against malarial blood stages of P. chabaudi is associated with a reprogramming of the expression of distinct miRNA species in the female mouse liver.     

Immunoglobulin G antibody profiles against Anopheles salivary proteins in domestic animals in Senegal

Although domestic animals may not be permissive for Plasmodium, they could nevertheless play a role in the epidemiology of malaria by attracting Anopheles away from humans. To investigate interactions between domestic animals and mosquitoes, we assayed immunoglobulin G (IgG) antibodies directed against the salivary proteins of Anopheles gambiae in domestic animals living in Senegalese villages where malaria is endemic. By Western blotting, sera from bovines (n=6), ovines (n=36), and caprines (n=36) did not react with Anopheles whole saliva. In contrast, equine sera recognized proteins in both saliva and salivary gland extracts. Two of the major immunogens (32 and 72 kDa) were also reactive in extracts from other major mosquito genera (Aedes and Culex), but reactions toAnopheles-specific antigens were detected in 12 of 17 horses. These data suggest that horses strongly react to Anopheles bites, and further experiments on horses are warranted to investigate the impact of this domestic animal species on the transmission of human malaria.     

Exacerbated potassium-induced paralysis of mouse soleus muscle at 37°C vis-à-vis 25°C: implications for fatigue. K+ -induced paralysis at 37°C

The main aim was to investigate the effects of raised [K⁺]_o on contraction of isolated non-fatigued skeletal muscle at 37°C and 25°C to assess the physiological significance of K⁺ in fatigue. Mouse soleus muscles equilibrated at 25°C had good mechanical stability when temperature was elevated to 37°C. The main findings at 37°C vis-à-vis 25°C were as follows. When [K⁺]_o was raised from 4 to 7?mM, there was greater twitch potentiation, but no significant difference in peak tetanic force. At 10?mM [K⁺]_o there was (1) a faster time course for the decline of peak tetanic force, (2) a greater steady-state depression of twitches and tetani, (3) an increase of peak force over 50?C200?Hz (whereas it decreased at 25°C), (4) significant tetanus restoration when stimulus pulse duration increased from 0.1 to 0.25?ms and (5) greater depolarisation of layer-2 fibres, with no repolarisation of surface fibres. These combined data strengthen the proposal that a large run-down of the K⁺ gradient contributes to severe fatigue at physiological temperatures via depolarisation and impaired sarcolemmal excitability. Moreover, terbutaline, a ??₂-adrenergic agonist, induced a slightly greater and more rapid, but transient, restoration of peak tetanic force at 10?mM [K⁺]_o at 37°C vis-à-vis 25°C. A right shift of the twitch force?Cstimulation strength relationship at 10?mM [K⁺]_o was partially reversed with terbutaline to confer the protective effect. Thus, catecholamines are likely to stimulate the Na⁺?CK⁺ pump more powerfully at 37°C to restore excitability and attenuate, but not prevent, the detrimental effects of K⁺.     

Clinical and radiological outcome of anterior–posterior fusion versus transforaminal lumbar interbody fusion for symptomatic disc degeneration: a retrospective comparative study of 133 patients

Abundant data are available for direct anterior/posterior spine fusion (APF) and some for transforaminal lumbar interbody fusion (TLIF), but only few studies from one institution compares the two techniques. One-hundred and thirty-three patients were retrospectively analyzed, 68 having APF and 65 having TLIF. All patients had symptomatic disc degeneration of the lumbar spine. Only those with one or two-level surgeries were included. Clinical chart and radiologic reviews were done, fusion solidity assessed, and functional outcomes determined by pre- and postoperative SF-36 and postoperative Oswestry Disability Index (ODI), and a satisfaction questionnaire. The minimum follow-up was 24 months. The mean operating room time and hospital length of stay were less in the TLIF group. The blood loss was slightly less in the TLIF group (409 vs. 480 cc.). Intra-operative complications were higher in the APF group, mostly due to vein lacerations in the anterior retroperitoneal approach. Postoperative complications were higher in the TLIF group due to graft material extruding against the nerve root or wound drainage. The pseudarthrosis rate was statistically equal (APF 17.6% and TLIF 23.1%) and was higher than most published reports. Significant improvements were noted in both groups for the SF-36 questionnaires. The mean ODI scores at follow-up were 33.5 for the APF and 39.5 for the TLIF group. The patient satisfaction rate was equal for the two groups. This work is dedicated to the memory of Grace and Julia Hanson.     

Expression and activity of COX‐1 and 2 and 5‐LOX in joint tissues from dogs with naturally occurring coxofemoral joint osteoarthritis

Understanding the neurobiology of pain in naturally occurring models of osteoarthritis (OA) may improve the understanding of human OA pain. Both COX and LOX have been associated with joint pain. This study evaluated COX‐1, COX‐2, and 5‐LOX expression and activity in a naturally occurring canine model of secondary OA. Hip joint capsule with synovial tissue (HJC) and femoral head subchondral bone (FH) was collected from normal dogs and dogs undergoing total hip replacement for coxofemoral joint OA. Tissues were analyzed for COX‐1, COX‐2, and LOX protein, and PGE₂ and LTB₄. Significantly more COX‐2 protein was present in OA HJC than normal joints (p = 0.0009). There was no significant difference in COX‐1 or LOX protein, although LOX protein was increased (p = 0.069). PGE₂ concentration in normal and OA HJC was similar (p = 1.0). LTB₄ concentration in OA HJC was significantly greater than normal HJC (p = 0.028). Significantly more COX‐1 (p = 0.0098), COX‐2 (p = 0.0028), and LOX (p = 0.0095) protein was present in OA FH tissue compared to normal FH tissue. There were no differences in PGE₂ or LTB₄ concentration in normal and OA FH tissue (p = 0.77 and p = 0.11). Together, these data suggest both COX‐2 and 5‐LOX are appropriate targets for the management of pain associated with naturally occurring OA. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Adipose Tissue Plasticity During Catch-Up Fat Driven by Thrifty Metabolism: Relevance for Muscle-Adipose Glucose Redistribution During Catch-Up Growth

OBJECTIVE

Catch-up growth, a risk factor for later type 2 diabetes, is characterized by hyperinsulinemia, accelerated body-fat recovery (catch-up fat), and enhanced glucose utilization in adipose tissue. Our objective was to characterize the determinants of enhanced glucose utilization in adipose tissue during catch-up fat.

RESEARCH DESIGN AND METHODS

White adipose tissue morphometry, lipogenic capacity, fatty acid composition, insulin signaling, in vivo glucose homeostasis, and insulinemic response to glucose were assessed in a rat model of semistarvation-refeeding. This model is characterized by glucose redistribution from skeletal muscle to adipose tissue during catch-up fat that results solely from suppressed thermogenesis (i.e., without hyperphagia).

RESULTS

Adipose tissue recovery during the dynamic phase of catch-up fat is accompanied by increased adipocyte number with smaller diameter, increased expression of genes for adipogenesis and de novo lipogenesis, increased fatty acid synthase activity, increased proportion of saturated fatty acids in triglyceride (storage) fraction but not in phospholipid (membrane) fraction, and no impairment in insulin signaling. Furthermore, it is shown that hyperinsulinemia and enhanced adipose tissue de novo lipogenesis occur concomitantly and are very early events in catch-up fat.

CONCLUSIONS

These findings suggest that increased adipose tissue insulin stimulation and consequential increase in intracellular glucose flux play an important role in initiating catch-up fat. Once activated, the machinery for lipogenesis and adipogenesis contribute to sustain an increased insulin-stimulated glucose flux toward fat storage. Such adipose tissue plasticity could play an active role in the thrifty metabolism that underlies glucose redistribution from skeletal muscle to adipose tissue.The pattern of growth early in life is now recognized to be an important predictor of chronic metabolic diseases. In particular, people who had low birth weight or whose growth faltered during infancy and childhood, but who subsequently showed catch-up growth, had higher propensity for the development of abdominal obesity, type 2 diabetes, and cardiovascular diseases later in life (1–8). The mechanistic basis of the link between catch-up growth and risks for these chronic diseases is poorly understood. There is, however, compelling evidence that mammalian catch-up growth is characterized by a disproportionately higher rate of body fat than lean tissue gain (9) and that an early feature of such “preferential catch-up fat” is concomitant hyperinsulinemia (10).Using a rat model of semistarvation-refeeding (11), in which catch-up fat is studied in the absence of hyperphagia, we previously showed that the hyperinsulinemic state of catch-up fat preceded the development of excess adiposity and could be linked to suppressed thermogenesis, per se, in the absence of hyperphagia (12). Subsequent studies of hyperinsulinemic-euglycemic clamps during catch-up fat showed that in vivo insulin-mediated glucose utilization was diminished in skeletal muscle but enhanced in white adipose tissue (WAT), suggesting that preferential catch-up fat is characterized by glucose redistribution from skeletal muscle to WAT (13). Consistent with this hypothesis are the demonstrations, in this rat model of catch-up fat, of diminished mitochondrial mass and lower insulin receptor substrate (IRS)-1–associated phosphatidylinositol-3-kinase (PI3K) activity in the skeletal muscle (14,15). Furthermore, ex vivo studies in WAT have previously shown that glucose uptake and utilization are enhanced during refeeding after fasting or caloric restriction (16,17).Elucidating the mechanisms that underlie such enhancement in glucose uptake and glucose flux toward lipid synthesis in WAT is therefore of central importance in understanding the mechanisms of glucose redistribution during catch-up fat. In addressing this topic, we have characterized our rat model of catch-up fat for changes in adipose tissue morphometry (adipocyte size and number) and fatty acid composition given their importance as determinants of WAT responsiveness to the action of insulin on glucose utilization. Indeed, it is established that small adipocytes have a greater capacity for insulin-mediated glucose uptake and de novo lipogenesis than larger ones (18–22), while alterations in adipocyte membrane phospholipid composition in favor of a high ratio of polyunsaturated fatty acids (PUFAs) to saturated fatty acids (SFAs) correlates with increased rate of insulin-stimulated glucose transport and glucose flux toward de novo lipogenesis in WAT (23–24). We have therefore investigated here the extent to which differences in adipocyte number and diameter, key gene markers for adipocyte proliferation, as well as the fatty acid composition of phospholipid and triglyceride lipid fractions of WAT, might be involved in the enhanced glucose flux toward lipogenesis. Furthermore, given the importance of insulin signaling in adipocyte growth (25) and in controlling glucose flux toward lipogenesis (26,27), we have also evaluated the in vivo insulinemic response to glucose and investigated proximal insulin signaling in WAT under basal and in vivo insulin-stimulated conditions during catch-up fat.