YPTB3816 of 'Yersinia pseudotuberculosis' strain IP32953 is a virulence-related metallo-oligopeptidase

Background: although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. Results: in this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. Conclusions: the results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity

Volume CXIV, Number 4, November 7, 1996

Objective: Turner syndrome (TS) is a chromosomal disorder caused by complete or partial X chromosome monosomy that manifests various clinical features depending on the karyotype and on the genetic background of affected girls. This study aimed to systematically investigate the key clinical features of TS in relationship to karyotype in a large pediatric Turkish patient population.Methods: Our retrospective study included 842 karyotype-proven TS patients aged 0-18 years who were evaluated in 35 different centers in Turkey in the years 2013-2014.Results: The most common karyotype was 45,X (50.7%), followed by 45,X/46,XX (10.8%), 46,X,i(Xq) (10.1%) and 45,X/46,X,i(Xq) (9.5%). Mean age at diagnosis was 10.2±4.4 years. The most common presenting complaints were short stature and delayed puberty. Among patients diagnosed before age one year, the ratio of karyotype 45,X was significantly higher than that of other karyotype groups. Cardiac defects (bicuspid aortic valve, coarctation of the aorta and aortic stenosis) were the most common congenital anomalies, occurring in 25% of the TS cases. This was followed by urinary system anomalies (horseshoe kidney, double collector duct system and renal rotation) detected in 16.3%. Hashimoto's thyroiditis was found in 11.1% of patients, gastrointestinal abnormalities in 8.9%, ear nose and throat problems in 22.6%, dermatologic problems in 21.8% and osteoporosis in 15.3%. Learning difficulties and/or psychosocial problems were encountered in 39.1%. Insulin resistance and impaired fasting glucose were detected in 3.4% and 2.2%, respectively. Dyslipidemia prevalence was 11.4%.Conclusion: This comprehensive study systematically evaluated the largest group of karyotype-proven TS girls to date. The karyotype distribution, congenital anomaly and comorbidity profile closely parallel that from other countries and support the need for close medical surveillance of these complex patients throughout their lifespa

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa

Functional analysis of Oligopeptidase A from 'Yersinia pseudotuberculosis'

Proteases have long been studied and analyzed. However, some are more well known than others. Oligopeptidase A (OpdA) is an example of an enzyme that requires particular attention, as preliminary results by Karlyshev ‘et al’ (unpublished results) has demonstrated its absence in ‘Yersinia pseudotuberculosis’ results in a reduction of virulence when introduced into mice. Over-expression, purification and enzymatic activity of OpdA from ‘Y. pseudotuberculosis’ is reported in this study. OpdA-His was successfully over expressed and purified with a molecular weight of 77 kDa. The enzyme displayed no activity against resorufin labeled casein but was able to cleave the short fluorogenic substrates; Abz-NKPRRPQEDDnp and Abz-AAL-EDDnp. The hydrolysis of Abz-NKPRRPQ-EDDnp and Abz- AAL-EDDnp by OpdA-His was enhanced in the presence of Ca2+, C02+ and Mn2+. Interestingly, OpdA-His was inhibited by 0.1 mM and 1 mM Zn2+ and Cu2+, and stimulated by 0.001 mM and 0.01 mM Zn2+. Metal chelating agent EGTA at 1 mM concentration was unable to fully inhibit the hydrolysis of Abz-NKPRRPQ-EDDnp but fully inhibited the hydrolysis of Abz-AAL-EDDnp after 5 minutes. Likewise, the inhibitory effect of 0.1 mM chymostatin, 0.02 M N-[N-(N-Acetyl-L-Ieucyl)-L-Ieucyl]-L-norleucine (ALLN) and 74IJM antipain was more dramatic on the hydrolysis of Abz-AAL-EDDnp than the hydrolysis of Abz-NKPRRPQ-EDDnp. In order to determine the ability of OpdA-His to cleave products of other proteases and give an insight into the role of OpdA-His, several attempts were made to purify the ATP-dependent protease Lon. Unfortunately, these attempts were unsuccessful and Lon was not purified. Glycine extracts of ‘Escherichia coli’ cells over-expressing OpdA-His did not present any bands on SDS-PAGE. Furthermore, to have an understanding whether OpdA-His was involved in antimicrobial peptide breakdown, E. coli cells over-expressing OpdA-His were incubated with different concentrations bradykinin for 24 hr at 37 degrees Celsius. Interestingly, over-expression of OpdA-His reduced cell viability. Overall, these results give strong evidence that OpdA from ‘Y. pseudotuberculosis’ is a Zn-dependent metallo-protease that harbors a concentration dependent inhibitory zinc-binding site

Optociliary Shunt Vessels or Neovascularisation of the Optic Disc: Fluorescein Angiography Versus Optical Coherence Tomography Angiography

A 56-year-old man with a history of gallbladder carcinoma, hypothyroidism and hypertension was examined by us after developing marked visual loss in his left eye. A left ischaemic type of central retinal vein occlusion (CRVO) with macular oedema was diagnosed. Three months later, a non-ischaemic type of CRVO with no macular oedema developed in his right eye. While the left eye received five intravitreal ranibizumab injections and panretinal photocoagulation, the right central retinal vein occlusion improved spontaneously without any treatment. Ten months after his first visit we noticed optociliary shunt vessel formation in the right eye and neovascularisation of the optic disc in the left eye. Fluorescein angiography and optical coherence tomography angiography were performed at the same visit. The place of fluorescein angiography and optical coherence tomography angiography in distinguishing the optociliary shunt vessel from neovascularisation of the optic disc is discussed

Diagnostic and Management Strategies of Bietti Crystalline Dystrophy: Current Perspectives

Bietti crystalline dystrophy (BCD) is a rare, genetically determined chorioretinal dystrophy presenting with intraretinal crystalline deposits and varying degrees of progressive chorioretinal atrophy commencing at the posterior pole. In some cases, there can be concomitant corneal crystals noted first in the superior or inferior limbus. CYP4V2 gene, a member of the cytochrome P450 family is responsible for the disease and more than 100 mutations have been defined thus far. However, a genotype-phenotype correlation has not been established yet. Visual impairment commonly occurs between the second and third decades of life. By the fifth or sixth decade of life, vision loss can become so severe that the patient may potentially become legally blind. Multitudes of multimodal imaging modalities can be utilized to demonstrate the clinical features, course, and complications of the disease. This present review aims to reiterate the clinical features of BCD, update the clinical perspectives with the help of multimodal imaging techniques, and overview its genetic background with future therapeutic approaches

Oxidative status and paraoxonase activity in children with asthma

Objective: To compare paraoxonase activity and changes in oxidative status in asthmatic children and healthy children by determining serum paraoxonase activity and total oxidative status, total antioxidant capacity and lipid hydroperoxidation. Methods: Forty two asthmatic children were compared with 32 healthy children of similar age and sex. To evaluate the paraoxonase and oxidative status, total antioxidant capacity and lipid hydroperoxidation were examined. Serum paraoxonase activity was evaluated by measuring the rate of paraoxon hydrolosis. Oxidative status was evaluated by the method developed by Erel. Lipid hydroperoxide was measured by an iodometric method. Results: In comparison with the healthy control group, the paraoxonase activity of the asthmatic children was found to be low (163.7 ± 73.0 (U/L) and 349.2 ± 153.9 (U/L), P = 0.002) and total oxidant status (9.0 ± 3.5 ?mol H2O2 Eq/L and 13.4 ± 7.0 ?mol H2O2 Eq/L, P =0.002), total antioxidant capacity (5.5 ± 2.5 µmol Trolox Eq/L and 1.0 ± 0.6 µmol Trolox Eq/L, P < 0.001), and lipid hydroperoxidation values (9.9 ± 3.4 ?mol H2O2 Eq/L and 4.4 ± 1.5 ?mol H2O2 Eq/L, P < 0.001) were found to be high. The high density lipoprotein (HDL) concentration of the asthmatic children was lower than that in the control group (40.1 ± 9.2 mg/dl and 54.5 ± 15.9 mg/dl, P < 0.001) Conclusion: In asthmatic children, when total oxidant status, total antioxidant capacity and lipid hydroperoxidation levels increase, paraoxonase activity decreased