Alterations in nitric oxide synthase isoforms in acute lower limb ischemia and reperfusion

Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

Nitric oxide synthase in critically ischaemic muscle and alterations in isoform expression during revascularization surgery

BACKGROUND: Dysfunction of the nitric oxide pathway is implicated in peripheral arterial disease. Nitric oxide synthase (NOS) isoforms and NOS activity were studied in muscle from patients with critical leg ischaemia (CLI). Alterations in NOS during revascularization surgery were also assessed. METHODS: Muscle biopsies were taken from patients with CLI undergoing amputation and also from patients undergoing femorodistal bypass at the start of surgery, after arterial clamping and following reperfusion. The presence of NOS within muscle sections was confirmed using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. NOS isoform distribution was studied by immunohistochemistry. NOS mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blotting. NOS activity was assessed with the citrulline assay. RESULTS: All three NOS isoforms were found in muscle, associated with muscle fibres and microvessels. NOS I and III protein expression was increased in CLI (P = 0.041). During revascularization, further ischaemia and reperfusion led to a rise in NOS III protein levels (P = 0.008). NOS activity was unchanged. CONCLUSION: Alterations in NOS I and III occurred in muscle from patients with CLI and further changes occurred during bypass surgery.

Molecular characterization of aromatic peroxygenase from Agrocybe aegerita

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions--for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine--which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes' affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO's widespread occurrence in the fungal kingdom.

The kinetics of feline leukaemia virus shedding in experimentally infected cats are associated with infection outcome

Feline leukaemia virus (FeLV) infection in felids results mainly from oronasal exposure to infectious saliva and nasal secretions, but the potential for viral transmission through faeces and urine has not been completely characterized. In order to assess and compare potential FeLV transmission routes, we determined the viral kinetics in plasma, saliva, faeces and urine during early experimental FeLV infection (up to week 15 post-exposure) in specific pathogen-free cats. In addition to monitoring p27 antigen levels measured by ELISA, we evaluated the presence of infectious particles by cell culture assays and quantified viral RNA loads by a quantitative real-time TaqMan polymerase chain reaction. RNA load was associated with infection outcome (high load-progressive infection; low load-regressive infection) not only in plasma, but also in saliva, faeces and urine. Infectious virus was isolated from the saliva, faeces and urine of infected cats with progressive infection as early as 3-6 weeks post-infection, but usually not in cats with regressive infection. In cats with progressive infection, therefore, not only saliva but also faeces and to some extent urine might represent potential FeLV transmission routes. These results should be taken into account when modelling FeLV-host interactions and assessing FeLV transmission risk. Moreover, during early FeLV infection, detection of viral RNA in saliva may be used as an indicator of recent virus exposure, even in cats without detectable antigenaemia/viraemia. To determine the clinically relevant outcome of FeLV infection in exposed cats, however, p27 antigen levels in the peripheral blood should be measured.

Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors

BACKGROUND Bone-to-tendon healing after rotator cuff repairs is mainly impaired by poor tissue quality. The tenocytes of chronic rotator cuff tendon tears are not able to synthesize normal fibrocartilaginous extracellular matrix (ECM). We hypothesized that in the presence of platelet-released growth factors (PRGF), tenocytes from chronically retracted rotator cuff tendons proliferate and synthesize the appropriate ECM proteins. MATERIALS AND METHODS Tenocytes from 8 patients with chronic rotator cuff tears were cultured for 4 weeks in 2 different media: standard medium (Iscove's Modified Dulbecco's Media + 10% fetal calf serum + 1% nonessential amino acids + 0.5 μg/mL ascorbic acid) and media with an additional 10% PRGF. Cell proliferation was assessed at 7, 14, 21, and 28 days. Messenger (m)RNA levels of collagens I, II, and X, decorin, biglycan, and aggrecan were analyzed using real time reverse-transcription polymerase chain reaction. Immunocytochemistry was also performed. RESULTS The proliferation rate of tenocytes was significantly higher at all time points when cultured with PRGF. At 21 days, the mRNA levels for collagens I, II, and X, decorin, aggrecan, and biglycan were significantly higher in the PRGF group. The mRNA data were confirmed at protein level by immunocytochemistry. CONCLUSIONS PRGFs enhance tenocyte proliferation in vitro and promote synthesis of ECM to levels similar to those found with insertion of the normal human rotator cuffs. CLINICAL RELEVANCE Biologic augmentation of repaired rotator cuffs with PRGF may enhance the properties of the repair tissue. However, further studies are needed to determine if application of PRGF remains safe and effective in long-term clinical studies. LEVEL OF EVIDENCE Basic Science Study, Cell Biology.

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES: We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND: CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS: In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS: ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

The Pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.

Metabolomics reveals that aldose reductase activity due to AKR1B10 is upregulated in hepatitis C virus infection

To understand the changes in the metabolome of hepatitis C virus (HCV)-infected persons, we conducted a metabolomic investigation in both plasma and urine of 30 HCV-positive individuals using plasmas from 30 HCV-negative blood donors and urines from 30 healthy volunteers. Samples were analysed by gas chromatography-mass spectrometry and data subjected to multivariate analysis. The plasma metabolomic phenotype of HCV-positive persons was found to have elevated glucose, mannose and oleamide, together with depressed plasma lactate. The urinary metabolomic phenotype of HCV-positive persons comprised reduced excretion of fructose and galactose combined with elevated urinary excretion of 6-deoxygalactose (fucose) and the polyols sorbitol, galactitol and xylitol. HCV-infected persons had elevated galactitol/galactose and sorbitol/glucose urinary ratios, which were highly correlated. These observations pointed to enhanced aldose reductase activity, and this was confirmed by real-time quantitative polymerase chain reaction with AKR1B10 gene expression elevated sixfold in the liver. In contrast, AKR1B1 gene expression was reduced 40% in HCV-positive livers. Interestingly, persons who were formerly HCV infected retained the metabolomic phenotype of HCV infection without reverting to the HCV-negative metabolomic phenotype. This suggests that the effects of HCV on hepatic metabolism may be long lived. Hepatic AKR1B10 has been reported to be elevated in hepatocellular carcinoma and in several premalignant liver diseases. It would appear that HCV infection alone increases AKR1B10 expression, which manifests itself as enhanced urinary excretion of polyols with reduced urinary excretion of their corresponding hexoses. What role the polyols play in hepatic pathophysiology of HCV infection and its sequelae is currently unknown.

Real-time ultra-high optical coherence tomography monitoring of optical tissue effects caused by selective retina therapy

Purpose: Selective retina therapy (SRT) has shown great promise compared to conventional retinal laser photocoagulation as it avoids collateral damage and selectively targets the retinal pigment epithelium (RPE). Its use, however, is challenging in terms of therapy monitoring and dosage because an immediate tissue reaction is not biomicroscopically discernibel. To overcome these limitations, real-time optical coherence tomography (OCT) might be useful to monitor retinal tissue during laser application. We have thus evaluated a proprietary OCT system for its capability of mapping optical changes introduced by SRT in retinal tissue. Methods: Freshly enucleated porcine eyes, covered in DMEM upon collection were utilized and a total of 175 scans from ex-vivo porcine eyes were analyzed. The porcine eyes were used as an ex-vivo model and results compared to two time-resolved OCT scans, recorded from a patient undergoing SRT treatment (SRT Vario, Medical Laser Center Lübeck). In addition to OCT, fluorescin angiography and fundus photography were performed on the patient and OCT scans were subsequently investigated for optical tissue changes linked to laser application. Results: Biomicroscopically invisible SRT lesions were detectable in OCT by changes in the RPE / Bruch's complex both in vivo and the porcine ex-vivo model. Laser application produced clearly visible optical effects such as hyperreflectivity and tissue distortion in the treated retina. Tissue effects were even discernible in time-resolved OCT imaging when no hyper-reflectivity persisted after treatment. Data from ex-vivo porcine eyes showed similar to identical optical changes while effects visible in OCT appeared to correlate with applied pulse energy, leading to an additional reflective layer when lesions became visible in indirect ophthalmoscopy. Conclusions: Our results support the hypothesis that real-time high-resolution OCT may be a promising modality to obtain additional information about the extent of tissue damage caused by SRT treatment. Data shows that our exvivo porcine model adequately reproduces the effects occurring in-vivo, and thus can be used to further investigate this promising imaging technique.

Time-Resolved Ultra–High Resolution Optical Coherence Tomography for Real-Time Monitoring of Selective Retina Therapy

Purpose: Selective retina therapy (SRT) is a novel treatment for retinal pathologies, solely targeting the retinal pigment epithelium (RPE). During SRT, the detection of an immediate tissue reaction is challenging as tissue effects remain limited to intracellular RPE photodisruption. Time-resolved ultra-high axial resolution optical coherence tomography (OCT) is thus evaluated for the monitoring of dynamic optical changes at and around the RPE during SRT. Methods: An experimental OCT system with an ultra-high axial resolution of 1.78 µm was combined with an SRT system and time-resolved OCT M-scans of the target area were recorded from four patients undergoing SRT. OCT scans were analyzed and OCT morphology was correlated with findings in fluorescein angiography, fundus photography and cross-sectional OCT. Results: In cases where the irradiation caused RPE damage proven by fluorescein angiography, the lesions were well discernible in time-resolved OCT images but remained invisible in fundus photography and cross-sectional OCT acquired after treatment. If RPE damage was introduced, all applied SRT pulses led to detectable signal changes in the time-resolved OCT images. The extent of optical signal variation seen in the OCT data appeared to scale with the applied SRT pulse energy. Conclusion: The first clinical results proved that successful SRT irradiation induces detectable changes in the OCT M-scan signal while it remains invisible in conventional ophthalmoscopic imaging. Thus, real-time high-resolution OCT is a promising modality to monitor and analyze tissue effects introduced by selective retina therapy and may be used to guide SRT in an automatic feedback mode.

Canakinumab in adults with steroid-refractory pyoderma gangrenosum

BACKGROUND Pyoderma gangrenosum (PG) is a rare, neutrophilic, ulcerative skin disease that is difficult to treat, especially when unresponsive to steroids. OBJECTIVES To determine whether canakinumab is an effective and safe treatment in PG. METHODS Five adult patients with clinically and histologically confirmed steroid-refractory PG were enrolled in this prospective open-label study. They received canakinumab 150 mg subcutaneously at week 0 with an optional 150 mg at week 2 in case of an inadequate response [Physician's Global Assessment (PGA) ≥ 2], and an optional 150-300 mg at week 8 depending on PGA. The primary clinical end point was clinical improvement (PGA at least -1 from baseline) and/or complete remission (PGA 0 or 1) at week 16. Real-time quantitative polymerase chain reaction was performed on skin samples to quantify cytokine mRNA levels. RESULTS Interleukin (IL)-1β and its known target genes IL6, CXCL8 and IL36A were significantly increased in lesional skin of PG. Under canakinumab therapy, four of five patients showed a decrease in target-lesion size, PGA and Dermatology Life Quality Index (DLQI), and three of five achieved complete remission. The mean diameter of target lesions decreased from 4·32 ± 2·6 cm at visit 1 to 0·78 ± 1·3 cm at visit 7 (P = 0·03). Mean DLQI decreased from 15 ± 5 at visit 1 to 8 ± 4 by visit 7 (P = 0·01). Adverse effects were reported in two patients: fatigue in one and worsening of disease at a nontarget lesion in the other. CONCLUSIONS Our data indicate that IL-1β plays a key pathogenic role in PG and canakinumab may represent a therapeutic option for steroid-refractory PG.

Regulation of bovine IL-12R beta 2 subunit mRNA expression in bovine lymph node cells

The beta 2 subunit of the interleukin (IL)-12 receptor (IL-12R beta 2) has been shown to play an essential role in differentiation of T helper 1 (Th1) cells in the murine and human system, and antibodies raised against IL-12R beta 2 recognized this molecule on human Th1 but not Th2 cells. However, while the cytokines secreted by clones of murine cells allowed the definition of distinct T helper cell subsets, bovine clones with polarized Th1 and Th2 cytokine profiles were rarely found. This raised important questions about the regulation of immune responses in cattle. We therefore cloned bovine IL-12R beta2 (boIL-12R beta 2) DNA complementary to RNA (cDNA) from the start codon to the 3' end of the mRNA. Comparison of boIL-12R beta 2 cDNA with human and murine IL-12R beta 2 cDNA sequences revealed homologies of 85 and 78%, respectively. The deduced protein sequence showed the hallmark motifs of the cytokine receptor superfamily including the four conserved cysteine residues, the WSXWS motif and fibronectin domains in the extracellular part as well as a STAT4 binding site in the intracellular part of the molecule. Using real-time reverse transcription-polymerase chain reaction, upregulation of mRNA expression of this molecule could be demonstrated in cultured bovine lymph node cells stimulated with phytohemagglutinin. Furthermore, cells with upregulated boIL-12R beta 2 mRNA responded with enhanced expression of interferon gamma to treatment with interleukin 12.