Decreased AdipoR1 signaling and its implications for obesity-induced male infertility.

Obesity is among the risk factors for male infertility. Although several mechanisms underlying obesity-induced male subfertility have been reported, the entire mechanism of obesity-induced male infertility still remains unclear. Here, we show that sperm count, sperm motility and sperm fertilizing ability were decreased in male mice fed a high-fat diet and that the expression of the AdipoR1 gene and protein was decreased, and the expression of pro-apoptotic genes and protein increased, in the testis from mice fed a high-fat diet. Moreover, we demonstrate that testes weight, sperm count, sperm motility and sperm fertilizing ability were significantly decreased in AdipoR1 knockout mice compared to those in wild-type mice; furthermore, the phosphorylation of AMPK was decreased, and the expression of pro-apoptotic genes and proteins, caspase-6 activity and pathologically apoptotic seminiferous tubules were increased, in the testis from AdipoR1 knockout mice. Furthermore, study findings show that orally administrated AdipoRon decreased caspase-6 activity and apoptotic seminiferous tubules in the testis, thus ameliorating sperm motility in male mice fed a high-fat diet. This was the first study to demonstrate that decreased AdipoR1/AMPK signaling led to increased caspase-6 activity/increased apoptosis in the testis thus likely accounting for male infertility.

AdipoR agonist increases insulin sensitivity and exercise endurance in AdipoR-humanized mice.

Adiponectin receptors, AdipoR1 and AdipoR2 exert anti-diabetic effects. Although muscle-specific disruption of AdipoR1 has been shown to result in decreased insulin sensitivity and decreased exercise endurance, it remains to be determined whether upregulation of AdipoR1 could reverse them in obese diabetic mice. Here, we show that muscle-specific expression of human AdipoR1 increased expression levels of genes involved in mitochondrial biogenesis and oxidative stress-detoxification to almost the same extents as treadmill exercise, and concomitantly increased insulin sensitivity and exercise endurance in obese diabetic mice. Moreover, we created AdipoR-humanized mice which express human AdipoR1 in muscle of AdipoR1·R2 double-knockout mice. Most importantly, the small-molecule AdipoR agonist AdipoRon could exert its beneficial effects in muscle via human AdipoR, and increased insulin sensitivity and exercise endurance in AdipoR-humanized mice. This study suggests that expression of human AdipoR1 in skeletal muscle could be exercise-mimetics, and that AdipoRon could exert its beneficial effects via human AdipoR1.

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients.

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-ß-lactam ß-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major ß-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of (2 ± 1) × 106 µM-1 s-1 and a slow koff of (2 ± 1) × 10-3 s-1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA-avibactam revealed several interactions that stabilized the acyl-enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime-avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime-avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime-avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.

Insights into ß-lactamases from Burkholderia species, two phylogenetically related yet distinct resistance determinants.

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to ß-lactams among Burkholderia spp. is attributable to expression of ß-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum ß-lactamase. PenA hydrolyzes ß-lactams with k(cat) values ranging from 0.38 ± 0.04 to 460 ± 46 s(-1) and possesses high k(cat)/k(inact) values of 2000, 1500, and 75 for ß-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s(-1). Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k(cat) values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of ß-lactamases, which can lead to the rational design of novel agents against these pathogens.

Novel hybrid laparoscopic sacrocolpopexy for pelvic organ prolapse with a severe paravaginal defect.

Abdominal sacrocolpopexy is the gold standard for treating pelvic organ prolapse (POP) because of safety and durable good results. More recently laparoscopic sacrocolpopexy (LSC), a less invasive approach, has become popular. Although these surgeries are versatile and can treat almost all patients with POP, these techniques have shortcomings. Specifically, reinforcement of lateral vaginal defects are not very strong, thus patients with POP and a severe paravaginal defect are not good candidates for abdominal or laparoscopic sacrocolpopexy. To overcome this problem, we developed a novel type of LSC, which can reinforce severe paravaginal defects by using a reversed T-shaped anterior mesh combining the advantage of transvaginal mesh surgery. We refer to this novel surgery as 'hybrid LSC'. Thus far, eight patients have successfully undergone this surgery. Hybrid LSC is a simple and secure method, and is an alternative treatment for POP with a severe paravaginal defect.

Novel hybrid mesh surgery combines sacrocolpopexy with transvaginal mesh placement for pelvic organ prolapse.

Mesh surgeries, such as sacrocolpopexy and transvaginal mesh surgery, are commonly used to treat pelvic organ prolapse. Although mesh surgeries have a high success rate, they are unsuitable for some patients. For a patient with pelvic organ prolapse and highly calcified multiple fibroids, we performed hybrid sacrocolpopexy combined with transvaginal mesh surgery with a method modified for the patient's condition. Three months after surgery, the results were highly satisfactory. This approach is simple, secure, and versatile for patients who are not good candidates for conventional mesh surgeries. This novel hybrid mesh surgery is an option for treating various types of pelvic organ prolapse.

Evaluation of laparoendoscopic single-site gynecologic surgery with a multitrocar access system.

OBJECTS: Laparoendoscopic single-site surgery (LESS) is an ideal approach for gynecologic surgery and yields better cosmetic results. However, a standard umbilical approach with LESS is not appropriate for gynecologic surgery requiring intra-abdominal suturing and dissection requiring traction. Therefore, we have developed a new multitrocar access system for gynecologic LESS. The purpose of this study was to evaluate the efficacy of gynecologic LESS using this access system. METHODS: This access system consists of one 12-mm trocar, two 5-mm trocars, and a 5-mm flexible laparoscope. Two 5-mm trocars with small port heads were inserted cross-wise on opposite sides of the sleeve of the centrally positioned 12-mm trocar to maintain triangulation. Thirty-eight patients with various gynecologic conditions underwent LESS with this access system. The results of these surgeries were retrospectively compared to those of conventional laparoscopic procedures. RESULTS: Of the 38 LESS procedures performed with this access system, none was up-converted, converted to an open laparotomy, or required blood transfusion. The Salpingo-oophorectomy with LESS had several benefits, such as no extension of the skin incision of the trocar site and no leakage of the contents of the ovarian cyst into the peritoneal cavity, over that with conventional laparoscopy. A comparison of LESS (11 patients) and conventional laparoscopy (16 patients) for total hysterectomy showed no significant difference in total blood loss (234.0 mL vs. 221.6 mL) or the weight of the resected uterus (276.0 g vs. 285.0 g), although the mean total operative time was greater with LESS (199.0 min vs. 168.5 min). CONCLUSION: Our multitrocar access system is safe and secure, and can be adapted for various gynecologic surgeries involving complicated procedures. LESS with this access system achieves results comparable to those of conventional laparoscopy with 4 ports, although the operative time is longer.

Thrombin-stimulated proliferation is mediated by endothelin-1 in cultured rat gingival fibroblasts.

Abstract Endothelin-1 (ET-1) appears to be involved in drug-induced proliferation of gingival fibroblasts. Thrombin induces proliferation of human gingival fibroblasts via protease-activated receptor 1 (PAR1). In this study, using cultured rat gingival fibroblasts, we investigated whether thrombin-induced proliferation of gingival fibroblasts is mediated by ET-1. Thrombin-induced proliferation (0.05-2.5 U/mL). Proliferation was also induced by a PAR1-specific agonist (TFLLR-NH(2,) 0.1-30 microm), but not by a PAR2-specific agonist (SLIGRL-NH(2)). Thrombin (2.5 U/mL) induced an increase in immunoreactive ET-1 expression, which was inhibited by cycloheximide (10 microg/mL), and an increase in preproET-1 mRNA expression, as assessed by reverse transcription polymerase chain reaction. TFLLR-NH(2) increased ET-1 release into the culture medium in both a concentration (0.01-10 microm)- and time (6-24 h)-dependent manner, as assessed by solid phase sandwich enzyme-linked immunosorbent assay. The thrombin (2.5 U/mL)-induced proliferation was inhibited by a PAR1-selective inhibitor, SCH79797 (0.1 microm) and an ET(A) antagonist, BQ-123 (1 microm), but not by an ET(B) antagonist, BQ-788 (1 microm). These findings suggest that thrombin, acting via PAR1, induced proliferation of cultured rat gingival fibroblasts that was mediated by ET-1 acting via ET(A).

Identification of AUF1 as a rapamycin-responsive binding protein to the 5'-terminal oligopyrimidine element of mRNAs.

In vertebrates, mRNAs containing a 5'-terminal oligopyrimidine (TOP) motif are coordinately post-transcriptionally regulated. Binding of specific proteins to this element has been proposed to downregulate expression of TOP mRNAs at the level of translational initiation. We previously reported that rapamycin induces binding activity to the TOP element of ribosomal protein (r-protein) L32 mRNA. In this study, we adapt DEAE-cellulose/oligo dT-cellulose tandem column chromatography to purify TOP element-binding proteins from bovine submaxillary lymph nodes (SLN). We also show by northwestern blot analysis that two proteins of molecular weight 47kDa (47BP) and 43kDa (43BP) specifically bind to a (32)P-labeled riboprobe containing TOP regulatory element of the r-protein L32. Microsequencing of the purified 47BP revealed an internal sequence of 15 amino acids identical to the consensus sequence of the 2x RBD-Gly family. Western blot analysis of the cytoplasm fractions using an AUF1 antibody revealed that these two proteins are p45 AUF1 and p42 AUF1. Increases of the four isoforms of AUF1 protein were observed in 100,000g supernatant fractions of rapamycin-administered rat SLN. Furthermore, decreases of p45 AUF1 and p42 and/or p40 AUF1 were observed in the polysomal fractions of BJAB cells in which translation of TOP mRNAs was selectively suppressed by rapamycin treatment. Taken together, these results suggest that AUF1 is a TOP mRNA-binding protein that may participate in the translational suppression of TOP mRNAs resulting from rapamycin treatment.

Inhibition of angiotensin II- and endothelin-1-stimulated proliferation by selective MEK inhibitor in cultured rabbit gingival fibroblastsdagger.

We investigated the implication of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the proliferation stimulated by angiotensin II (Ang II) and endothelin-1 (ET-1) in cultured rabbit gingival fibroblasts (CRGF). Ang II stimulated activation of ERK1/2 and the activation was inhibited by CV-11974, an AT1 antagonist, and saralasin, an AT1/AT2 antagonist, but not by PD123,319, an AT2 antagonist in the CRGF. Ang II-stimulated proliferation was inhibited by PD98059 or U0126, selective MEK inhibitors. Furthermore, ET-1 stimulated proliferation via G-protein-coupled ETA receptors, which were identified by Western blot analysis of membrane protein from the CRGF. ET-1 also stimulated activation of ERK1/2 and the activation was inhibited by BQ-123, an ETA inhibitor, and TAK044, an ETA/ETB inhibitor, but not by BQ-788, an ETB inhibitor. ET-1-stimulated proliferation was inhibited by PD98059 or U0126. These findings suggest that ERK1/2 play a role in the signaling process leading to proliferation stimulated by Ang II and ET-1 via G-protein-coupled receptors, AT1 and ETA in CRGF.

Proliferative response to phenytoin and nifedipine in gingival fibroblasts cultured from humans with gingival fibromatosis.

The response of gingival fibroblasts cultured from humans with gingival fibromatosis to phenytoin (PHT) and nifedipine (NIF) was investigated. PHT and NIF induced proliferation, and increased the expression of immunoreactive endothelin-1 (ET-1). ET-1 (0.1 nm-1 microm) itself also induced proliferation in a concentration-dependent manner. The proliferation was inhibited by BQ-123 (ETA receptor antagonist; 1 microm) and TAK044 (ETA/ETB receptor antagonist; 1 microm), but not by BQ-788 (ETB receptor antagonist; 1 microm). The proliferation induced by PHT (0.25 microm) and NIF (0.25 microm) was inhibited by BQ-123 (1 microm). In addition, the results of Western blot analysis indicated the presence of ETA and ETB receptors in/on the fibroblasts. These findings suggest that PHT- and NIF-induced gingival proliferation may be mediated by endogenously generated ET-1, possibly via ETA receptors.

Pharmacological properties of angiotensin II receptors in cultured rabbit gingival fibroblasts.

We demonstrated that angiotensin II (Ang II, 10-1000 nM) induced proliferation of cultured rabbit gingival fibroblasts in a concentration-dependent manner. The Ang II-induced proliferation was inhibited by CV-11974 (AT1 antagonist; 1 microM) and saralasin (AT1/AT2 antagonist; 1 microM), but not by PD123,319 (AT2 antagonist; 1 microM), suggesting that Ang II-induced proliferation was mediated via AT1 receptors present in and/or on gingival fibroblasts. The results of Western blot analysis indicated the presence of AT1 and AT2 receptors in/on the fibroblasts. In a subsequent radioligand binding assay, the binding of [3H]Ang II to the fibroblasts was specific and saturable with both high- and low-affinity sites. Competition binding experiments indicated that Ang II completely displaced [3H]Ang II binding, and CV-11974 and PD123,319 maximally displaced up to approximately 63% and 37% of the total binding, respectively. Ang II and CV-11974 completely displaced the [3H]DuP753 binding but PD123,319 did not, indicating a single population of binding site. These findings demonstrate that gingival fibroblasts contain both AT1 and AT2 receptor subtypes for Ang II, and support that Ang II stimulation of AT1 receptors results in proliferation of the fibroblasts.

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture.

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.