Kruppel-like factor 5 (KLF5) is critical for conferring uterine receptivity to implantation
A blastocyst will implant only when the uterus becomes receptive. Following attachment, luminal epithelial cells undergo degeneration at the site of the blastocyst. Although many genes critical for uterine receptivity are primarily regulated by ovarian hormones, Kruppel-like factor 5 (KLF5), a zinc finger-containing transcription factor, is persistently expressed in epithelial cells independently of ovarian hormones. Loss of uterine Klf5 causes female infertility due to defective implantation. Cox2 is normally expressed in the luminal epithelium and stroma at the site of blastocyst attachment, but luminal epithelial COX2 expression is absent with loss of Klf5. This is associated with the retention of the epithelium around the implantation chamber with arrested embryonic growth. These results suggest that Klf5 is indispensable for normal implantation.

title: In pursuit of leucine-rich repeat-containing G protein-coupled receptor-5 regulation and function in the uterus.
abstract: Leucine-rich repeat-containing G protein-coupled receptor (LGR)-5 is a recently identified marker of stem cells in adult intestinal epithelium and hair follicles. Because of this characteristic, we studied the status of Lgr5 expression in the mouse uterus under various conditions. Lgr5 is highly expressed in the uterine epithelium of immature mice and is dramatically down-regulated after the mice resume estrous cycles. Surprisingly, whereas its expression is up-regulated in uteri of ovariectomized mice, the expression is down-regulated by estrogen and progesterone via their cognate nuclear receptors, estrogen receptor-alpha and progesterone receptor, respectively. Using a mouse endometrial cancer model, we also found that Lgr5 is highly expressed in the epithelium during the initial stages of tumorigenesis but is remarkably down-regulated in fully developed tumors. Lgr5 is a downstream target of Wnt signaling in the intestine. Genetic evidence shows that either excessive or absence of Wnt signaling dampens Lgr5 expression in the uterus. Collectively, our results show that Lgr5 expression in the mouse uterine epithelium is unique and dynamically regulated under various physiological and pathological states of the uterus, suggesting that this orphan receptor has important functions in uterine biology. However, identifying definitive uterine function of LGR5 will require further investigation using conditional deletion of uterine Lgr5 because systemic deletion of this gene is neonatally lethal.

title: [Isolation of a metsulfuron-methyl-resistant bacteria and cloning and expression of the acetohydroxyacid synthase genes ilvlH].
abstract: Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine in plant, fungi and bacteria, and also is target of the sulfonylurea, imidazolinone, triazolopyrimidine and other acetohydroxyacid synthase inhibitor herbicides.



The purpose of this study is to get the resistant gene, prepare a functional bacteria with acetohydroxyacid synthase, and investigate the relationship between the mutation sites of the acetohydroxyacid synthase and the herbicides-resistant.



A metsulfuron-methyl-resistant bacterium Lm10 was isolated from metsulfuron-methyl contaminated soil. Acetohydroxyacid synthase genes ilvIH was amplified from the genome DNA of strains Lm10 by PCR. The ilvI and ilvH were cloned into the bacterial expression vector pET29a(+) respectively.



Strain Lm10 was identified preliminarily as Pseudomonas sp.. It can endure 14000 micromol/L metsulfuron-methyl and showed cross resistance to diffenent acetohydroxyacid synthase inhibitor herbicids, such as chlorsulfuron, imazethapyr, flumetsulam and penoxsulam. The alignment result of the ilvIH amino acid sequence showed that ilvI of strains Lm10 differed from that of strain KT2440 by 6 sites, While the ilvH of the two strains were the same. The gene ilvI and ilvH were functional expressed in the Escherichia coli strain BL21(DE3) respectively. The expressed production pET-I displayed acetohydroxyacid synthase activity and showed resistance to high concentration of metsulfuron-methyl.



The ilvI displayed acetohydroxyacid synthase activity. The 6 different sites in ilvI of strains Lml0 probably led to herbicids resistance.

title: Genetic Loss of Faah Compromises Male Fertility in Mice1
abstract: Marijuana is the most commonly used illicit drug. Although there is some indication that reproductive functions in males are impaired in chronic marijuana users, the genetic evidence and underlying causes remain largely unknown. Herein we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah−/− sperm on the egg zona pellucida provides evidence that the capacity of sperm to penetrate the zona barrier is hampered by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.

title: Ovarian LGR5 is critical for successful pregnancy.
abstract: Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in many organs, including female reproductive organs, and is a stem cell marker in the stomach and intestinal epithelium, hair follicles, and ovarian surface epithelium. Despite ongoing studies, the definitive physiological functions of Lgr5 remain unclear. We utilized mice with conditional deletion of Lgr5 (Lgr5(d/d)) in the female reproductive organs by progesterone receptor-Cre (Pgr(Cre)) to determine Lgr5's functions during pregnancy. Only 30% of plugged Lgr5(d/d) females delivered live pups, and their litter sizes were lower. We found that pregnancy failure in Lgr5(d/d) females was due to insufficient ovarian progesterone (P4) secretion that compromised decidualization, terminating pregnancy. The drop in P4 levels was reflected in elevated levels of P4-metabolizing enzyme 20α-hydroxysteroid dehydrogenase in corpora lutea (CL) inactivated of Lgr5. Of interest, P4 supplementation rescued decidualization failure and supported pregnancy to full term in Lgr5(d/d) females. These results provide strong evidence that Lgr5 is critical to normal CL function, unveiling a new role of LGR5 in the ovary.

title: Determination of tetracyclines in food samples by molecularly imprinted monolithic column coupling with high performance liquid chromatography.
abstract: A novel solid phase extraction (SPE) method for determination of tetracyclines (TCs) in milk and honey samples by molecularly imprinted monolithic column was developed. Using tetracycline (TC) as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, methanol as the solvent, cyclohexanol and dodecanol as the mixed porogenic solvents, a TC imprinted monolithic column was prepared by in situ molecular imprinting technique for the first time, and the optimal synthesis conditions and the selectivity of TC imprinted monolithic column were investigated. The interfering substances in food samples and TCs can be separated successfully on imprinted column. Molecularly imprinted solid phase extraction (MISPE) coupling with C18 column was used to determinate the TCs in milk and honey. The recoveries of this method for six tetracyclines antibiotics such as tetracycline (TC), oxytetracycline (OTC), minocycline (MINO), chlortetracycline (CTC), metacycline (MTC) and doxycycline (DTC) were investigated, and high recoveries of 73.3-90.6% from milk samples and 62.6-82.3% from honey samples were obtained. A method for determination of TCs at low concentration level in milk and honey samples was successfully developed by using the monolithic column as the precolumn for solid phase extraction of six TCs compounds.

title: Regression of solid tumors by engineered overexpression of von Hippel–Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1α
abstract: The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1α therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1α and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1α and VHL plasmids. Combination therapy resulted in increased losses of HIF-1α, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1α, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

title: Sonographic evaluation of para- and perirenal fat thickness is an independent predictor of early kidney damage in obese patients
abstract: PurposeTo determine whether para- and perirenal fat ultrasonographic thickness (PFUT) is related to increased urinary albumin excretion and whether PFUT is an independent indicator of early kidney damage in obese subjects.MethodSixty-seven nonhypertensive, nondiabetic obese patients and 34 age- and sex-matched normal healthy volunteers were involved in this study. Clinical characteristics, blood biochemistry, PFUT, and urinary albumin/creatinine ratio (ACR) of the subjects were measured. The intraoperator and interoperator coefficient of variation was 5.6 and 3.2 %, respectively.ResultsACR and PFUT were significantly higher in obese patients than those of normal healthy volunteers. PFUT was higher in obese patients with microalbuminuria than those with normoalbuminuria. Correlation analysis showed PFUT had a positive correlation with body mass index (BMI, r = 0.677, P < 0.01), waist circumference (WC, r = 0.686, P < 0.01), plasma free fatty acids (FFAs, r = 0.589, P < 0.01), and ACR (r = 0.610, P < 0.01). ACR had a positive correlation with BMI (r = 0.444, P < 0.01), WC (r = 0.440, P < 0.01), and plasma FFAs (r = 0.496, P < 0.01). Multivariate regression analyses showed that ACR could be predicted by PFUT.ConclusionsPFUT may be an independent predictor of early kidney damage in nonhypertensive, nondiabetic obese patients, and PFUT could be a useful tool for the assessment of visceral fat and early kidney damage in obese patients.

title: Antisense HIF-1α prevents acquired tumor resistance to angiostatin gene therapy
abstract: Angiostatin is a naturally occurring inhibitor of angiogenesis that is being developed as a drug to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1α (HIF-1α) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1α inhibited the expression of HIF-1α and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1α plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1α can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies.

title: Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1α eradicates gliomas
abstract: The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.