Positive skeletal effects of cladrin, a naturally occurring dimethoxydaidzein, in osteopenic rats that were maintained after treatment discontinuation.

UNLABELLED: Effects of cladrin treatment and withdrawal in osteopenic rats were studied. Cladrin improved trabecular microarchitecture, increased lumbar vertebral compressive strength, augmented coupled remodeling, and increased bone osteogenic genes. A significant skeletal gain was maintained 4 weeks after cladrin withdrawal. Findings suggest that cladrin has significant positive skeletal effects. INTRODUCTION: We showed that a standardized extract of Butea monosperma preserved trabecular bone mass in ovariectomized (OVx) rats. Cladrin, the most abundant bioactive compound of the extract, promoted peak bone mass achievement in growing rats by stimulating osteoblast function. Here, we studied the effects of cladrin treatment and withdrawal on the osteopenic bones. METHODS: Adult female Sprague-Dawley rats were OVx and left untreated for 12 weeks to allow for significant estrogen deficiency-induced bone loss, at which point cladrin (1 and 10 mg/kg/day) was administered orally for another 12 weeks. Half of the rats were killed at the end of the treatments and the other half at 4 weeks after treatment withdrawal. Sham-operated rats and OVx rats treated with PTH or 17ß-estradiol (E2) served as various controls. Efficacy was evaluated by bone microarchitecture using microcomputed tomographic analysis and fluorescent labeling of bone. qPCR and western blotting measured mRNA and protein levels in bone and uterus. Specific ELISA was used for measuring levels of serum PINP and urinary CTx. RESULTS: In osteopenic rats, cladrin treatment dose dependently improved trabecular microarchitecture, increased lumbar vertebral compression strength, bone formation rate (BFR), cortical thickness (Cs.Th), serum PINP levels, and expression of osteogenic genes in bones; and reduced expression of bone osteoclastogenic genes and urinary CTx levels. Cladrin had no uterine estrogenicity. Cladrin at 10 mg/kg maintained acquired skeletal gains 4 weeks after withdrawal. CONCLUSION: Cladrin had positive skeletal effects in osteopenic rats that were maintained after treatment withdrawal.

Molecular cloning and characterization of a glutathione S-transferase in the tropical liver fluke, Fasciola gigantica.

Glutathione S-transferase from an Indian isolate of Fasciola gigantica of buffalo origin was isolated and characterized. Total RNA was transcribed to cDNA by reverse transcription and an amplicon of 657 bp glutathione S-transferase gene was obtained by polymerase chain reaction (PCR). The present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin, with six nucleotide changes causing an overall change of four amino acids. Glutathione S-transferase protein was expressed in Escherichia coli using a prokaryotic expression vector pPROEXHTb. The recombinant protein was purified under non-denaturing and denaturing conditions by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. Recombinant GST protein detected F. gigantica infection in naturally infected buffaloes by dot-ELISA.

Is serum cystatin C an accurate endogenous marker of glomerular filteration rate for detection of early renal impairment in patients with type 2 diabetes mellitus?

BACKGROUND: Researches have recently reported that serum cystatin C is a more sensitive marker of changes in glomerular filtration rate (GFR) than serum creatinine. We conducted this study to evaluate the significance of serum cystatin C as a more sensitive marker of GFR for early detection of renal impairment in special groups of patients with type 2 diabetes mellitus (DM). METHODS: The present study included 40 patients with type 2 DM divided into four equal groups based on their urinary albumin excretion and renal function: group 1 was normoalbuminuric, group 2 was microalbuminuric, group 3 was macroalbuminuric, and group 4 was macroalbuminuric with renal dysfunction. All patients underwent a thorough history, full clinical examination, fasting, and renal function tests. Post-prandial blood glucose levels, glycosylated hemoglobin A1c (HbA1c), proteins, albumin in 24 hr urine, and serum cystatin C were collected. RESULTS: Serum cystatin C and creatinine were significantly higher in macrolbuminuric type 2 diabetic patients with renal dysfunction (group 4: 2.26 +/- 1.28, 4.21 +/- 2.38 mg/dl, respectively; p < 0.001) than macrolbuminuric type 2 diabetic patients with normal renal function (group 3: 1.04 +/- 0.24, 0.96 +/- 0.20 mg/dl, respectively), the microalbuminuric group (0.87 +/- 0.28, 0.71 +/- 0.12 mg/dl, respectively), as well as the normoalbuminuric group (0.55 +/- 0.41, 0.60 +/- 0.18 mg/dl, respectively). ROC plots demonstrated that area under the curve (AUC) of cystatin C (0.74) was greater than that for creatinine clearance (cr.cl) (0.67) and serum creatinine (s-cr) (0.74); therefore, the sensitivity and diagnostic accuracy of cystatin c was better than cr. cl., and both were better than s-cr. Serum cystatin C showed significant correlation in groups 2-4 with s-cr, cr.cl, and 24 hr urine albumin, but no correlation was found in group 1. CONCLUSION: Serum cystatin C is a reliable and easily performed marker for GFR to detect renal impairment in patients with type 2 DM.

Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic ß subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.

Isoformononetin, a methoxydaidzein present in medicinal plants, reverses bone loss in osteopenic rats and exerts bone anabolic action by preventing osteoblast apoptosis.

PURPOSE: Daidzein (Daid) has been implicated in bone health for its estrogen-'like' effects but low bioavailability, unfavorable metabolism and uterine estrogenicity impede its clinical potential. This study was aimed at assessing isoformononetin (Isoformo), a naturally occurring methoxydaidzein, for bone anabolic effect by overcoming the pitfalls associated with Daid. METHODS: Sprague-Dawley ovariectomized (OVx) rats with established osteopenia were administered Isoformo, 17ß-oestradiol (E2) or human parathyroid hormone. Efficacy was evaluated by bone microarchitecture using microcomputed tomography and determination of new bone formation by fluorescent labeling of bone. Osteoblast apoptosis was measured by co-labeling of bone sections with Runx-2 and TUNEL. Biochemical markers of bone metabolism were measured by ELISA. Plasma and bone marrow levels of Isoformo and Daid were determined by LC-MS-MS. Rat bone marrow stromal cells were harvested to study osteoblastic differentiation by Isoformo and Daid. New born rat pups were injected with Isoformo and Daid to study the effect of the compounds on the expression of osteogenic genes in the calvaria by real time PCR. RESULTS: In osteopenic rats, Isoformo treatment restored trabecular microarchitecture, increased new bone formation, increased the serum osteogenic marker (procollagen N-terminal propeptide), decreased resorptive marker (urinary C-terminal teleopeptide of type I collagen) and diminished osteoblast apoptosis in bone. At the most effective osteogenic dose of Isoformo, plasma and bone marrow levels were comprised of ~90% Isoformo and the rest, Daid. Isoformo at the concentration reaching the bone marrow achieved out of its most effective oral dosing induced stromal cell mineralization and osteogenic gene expression in the calvaria of neonatal rats. Isoformo exhibited uterine safety. CONCLUSIONS: Our study demonstrates that Isoformo reverses established osteopenia in adult OVx rats likely via its pro-survival effect on osteoblasts. Given its bone anabolic and anti-catabolic effects accompanied with safety at uterine level we propose its potential in the management of postmenopausal osteoporosis.

Is there a role for endothelin-1 in the hemodynamic changes during hemodialysis?

BACKGROUND: The etiology of hemodialysis (HD)-induced hypotension and hypertension remains speculative. There is mounting evidence that endothelin-1 (ET-1) may play a vital role in these hemodynamic changes. We examined the possible role of intradialytic changes of ET-1 in the pathogenesis of hypotension and rebound hypertension during HD. METHODS: The present study included 45 patients with end-stage renal disease (ESRD) on regular HD. They were divided according to their hemodynamic status during HD into three groups (group I had stable intradialytic hemodynamics, group II had dialysis-induced hypotension, and group III had rebound hypertension during HD). In addition, 15 healthy volunteers were included as a control group. Pulse and blood pressure were monitored before, during (every half hour), and after HD session. ET-1 level was measured at the beginning, middle, and end of HD. ET-1 was measured in the control group for comparison. RESULTS: Pre-dialysis levels of ET-1 were significantly higher in dialysis patients compared to the controls (P < 0.001); however, they were comparable in the three HD groups. The post-dialysis ET-1 level was not changed significantly in group I compared with predialysis values (14.49 +/- 2.04 vs. 14.33 +/- 2.23 pg/ml; P = NS), while the ET-1 concentration decreased significantly in group II and increased in group III in comparison to predialysis values (8.56 +/- 1.44 vs. 11.75 +/- 2.51; 16.39 +/- 3.12 vs. 11.93 +/- 2.11 pg/ml, respectively; P < 0.001). CONCLUSION: Altered ET-1 levels may be involved in the pathogenesis of rebound hypertension and hypotension during HD.

Outpatient diagnostic hysteroscopy.

STUDY OBJECTIVE: To evaluate the feasibility, validity, indications, and results of a large series of diagnostic hysteroscopies performed without anesthesia. DESIGN: Retrospective analysis of hysteroscopy charts performed between 1989 and 1996 (Canadian Task Force classification II-2). SETTING: University-affiliated endoscopy unit. PATIENTS: Four thousand consecutive women referred for different indications. INTERVENTIONS: Diagnostic hysteroscopy was performed in 91% of patients without premedication or anesthetics. In some women premedication or general or local anesthesia was required to access the uterine cavity. MEASUREMENTS AND MAIN RESULTS: The success rate, validity indication, complication rate, and number of biopsies were critically evaluated and assessed in relation to increased experience of operators. In 91% of women we accessed the uterine cavity at the first attempt without premedication, whereas 207 (5. 1%) patients required local anesthesia and 99 (2.4%) premedication. Only 1.6% required general anesthesia. In 52% intrauterine pathology was diagnosed and in 21% further surgical treatment was suggested. CONCLUSION: Hysteroscopy was feasible when performed in an outpatient setting without general or local anesthesia in more than 90% of women. The operator's experience seems a key factor both for accurate endometrial evaluation and to reduce failure and endometrial biopsy rates. The low frequency of further surgical treatment justifies performing the procedure in the office.

Direct demonstration of iron in a term placenta in two cases of beta-thalassemia.

The aim of this study was to quantify iron in the placentas at the end of pregnancy in two patients suffering from beta-thalassemia and compare the data to that recorded from the placentas of healthy women. Iron was quantified in placental specimens taken after the delivery. The specimens were subjected to chemical treatments in order to remove extracellular iron and leave the intracellular iron intact. After coloration, each specimen was subjected to quantitative analysis of images in order to identify and quantify iron. Our results demonstrated that in beta-thalassemia there is an accumulation of iron in the outer basal cytoplasm of the epithelial cells of placental cotyledons.