Isolation,structural characterization,and antiviral activity of positional isomers of monopegylated interferon alpha-2a (PEGASYS)

Interferon alpha-2a plays an essential role in the treatment of chronic hepatitis C, but it is limited in its efficacy by the short in vivo half-life. To improve the half-life and efficacy, interferon alpha-2a is conjugated with a 40-kDa branched polyethylene glycol moiety (PEG-IFN, PEGASYS). From this preparation the positional PEG-IFN isomers were isolated and characterized by different analytical methods and antiviral assay. Two chromatographic steps were used to separate and purify nine isomers. The analytical methods IE-HPLC, RP-HPLC, SE-HPLC, SDS-PAGE, and MALDI-TOF MS indicated that each of these nine isomers is conjugated to the branched polyethylene glycol chain at a specific lysine. No isomer with a modification at the amino terminus was observed. All positional isomers induced viral protection of MDBK cells in the antiviral assay. When comparing the quantitative potency of the individual isomers with the whole mixture of PEG-IFN, significant differences in the specific activities were observed: PEG-Lys(31) and PEG-Lys(134) showed higher activities than the mixture, PEG-Lys(164) was equal to the mixture, whereas the activities of PEG-Lys(49), PEG-Lys(70), PEG-Lys(83), PEG-Lys(112), PEG-Lys(121), and PEG-Lys(131) were lower.     

Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.     

Effect of different doses of prostaglandin E2 on intrauterine pressure and uterine motility during diestrus in experimental cows

Studies in human medicine proved the important role of prostaglandin E2, which stimulates uterine contractions in vivo and in vitro and has been extensively used to ripen the cervix around labor. We wanted to demonstrate that increasing the dosage of prostaglandin E2 (1.25 mg, 2.5 mg, 5 mg and 10 mg) provokes an increase in intrauterine pressure and uterine motility in cattle. Five healthy, lactating dairy cows were used as experimental animals for this study. Intrauterine pressure was recorded during the diestrus phase (1 recording per cow and diestrus phase) by means of a transcervically placed intraluminal pressure microtransducer. Physiologic uterine motility was recorded for 30 min, then placebo or one of the prostaglandin E2- dosages was administered through an indwelling catheter in the jugular vein, followed by a 2-h recording period (eight 15-min periods). Area under the curve (AUC), mean amplitude, frequency of pressure waves and intrauterine pressure were analyzed. Furthermore, we recorded protocols for monitoring heart and respiratory rates and side effects at 9 given examination times. Significant differences were found for the AUC, the mean amplitude and the intrauterine pressure (P < or = 0.05), whereas the number of pressure waves per 15 min did not differ significantly among treatments. Peak values for AUC, mean amplitude and intrauterine pressure were found during the first 15 min after administration of 10 mg of prostaglandin E2. Dose-effect curves showed that the 2.5 mg dosage provided the optimal ratio between myometrial stimulation and undesirable side-effects.     

Functional Characterization of the Conserved “GLK” Motif in Mitochondrial Porin from Neurospora crassa

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a glycine-leucine-lysine (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [-³²P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective -barrel that is not directly involved in ATP binding.     

Evidence for the Uptake of Large Anions through Stomatal Pores

Experiments were conducted with leek (Allium porrum L.) leaves to investigate whether aqueous solutions are able to penetrate stomata. Epidermal strips were used for the determination of transport rates. Stomata were opened by fusicoccin or closed by darkness or abscisic acid. A droplet containing the anionic fluorescent dye, uranine, was placed on the physiologically outer side of the epidermis and allowed to dry. With open stomata 30 times more uranine penetrated through the epidermal strips than with closed stomata (comparison of medians). In another experiment droplets of uranine solution were placed on leaf segments and epidermal strips were removed after drying of the droplets. Penetration of uranine through stomata was detectable under the microscope both with epidermal strips from the transport experiments and with strips obtained after application on leaf segments. As maximum uptake rates occurred during the drying process, it is concluded that penetration took place via water films. These results show that the physical restrictions preventing stomatal penetration of static droplets are not decisive for drying droplets and that stomatal uptake of dissolved ionic substances occurs under natural conditions, i.e. without surfactants or applied pressure.     

Evolution of metabolic diversity: Insights from microbial polyketide synthases

Polyketides are a family of complex natural products that are built from simple carboxylic acid building blocks. In microorganisms, the majority of these secondary metabolites are produced by exceptionally large, multifunctional proteins termed polyketide synthases (PKSs). Each unit of a type I PKS assembly line resembles a mammalian type fatty acid synthase (FAS), although certain domains are optionally missing. The evolutionary analysis of microbial PKS has revealed a long joint evolution process of PKSs and FASs. The phylogenomic analysis of modular type I PKSs as the most widespread PKS type in bacteria showed a large impact of gene duplications and gene losses on the evolution of type I PKS in different bacterial groups. The majority of type I PKSs in actinobacteria and cyanobacteria may have evolved from a common ancestor, whereas in proteobacteria most type I PKSs were acquired from other bacterial groups. The modularization of type I PKSs almost unexceptionally started with multiple duplications of a single ancestor module. The repeating modules represent ideal platforms for recombination events that can lead to corresponding changes in the actual chemistry of the products. The analysis of these “natural reprogramming” events of PKSs may assist in the development of concepts for the biocombinatorial design of bioactive compounds.     

γ-H2AX as a Marker for Dose Deposition in the Brain of Wistar Rats after Synchrotron Microbeam Radiation

Objective

Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT).

Methods

Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.

Results

While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.

Conclusion

In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.     

Process evaluation of a tailored intervention programme of cardiovascular risk management in general practices

Background

A tailored implementation programme to improve cardiovascular risk management (CVRM) in general practice had little impact on outcomes. The questions in this process evaluation concerned (1) impact on counselling skills and CVRM knowledge of practice nurses, (2) their use of the various components of the intervention programme and adoption of recommended practices and (3) patients’ perceptions of counselling for CVRM.

Methods

A mixed-methods process evaluation was conducted. We assessed practice nurses’ motivational interviewing skills on audio-taped consultations using Motivational Interviewing Treatment Integrity (MITI). They also completed a clinical knowledge test. Both practice nurses and patients reported on their experiences in a written questionnaire and interviews. A multilevel regression analysis and an independent sample t test were used to examine motivational interviewing skills and CVRM knowledge. Framework analysis was applied to analyse qualitative data.

Results

Data from 34 general practices were available, 19 intervention practices and 14 control practices. No improvements were measured on motivational interviewing skills in both groups. There appeared to be better knowledge of CVRM in the control group. On average half of the practice nurses indicated that they adopted the recommended interventions, but stated that they did not necessarily record this in patients’ medical files. The tailored programme was perceived as too large. Time, follow-up support and reminders were felt to be lacking. About 20% of patients in the intervention group visited the general practice during the intervention period, yet only a small number of these patients were referred to recommended options.

Conclusions

The tailored programme was only partly used by practice nurses and had little impact on either their clinical knowledge and communication skills or on patient reported healthcare. If the assumed logical model of change is valid, a more intensive programme is needed to have an impact on CVRM in general practice at all.

     

Epidemiology of Pelvic Fractures in Germany: Considerably High Incidence Rates among Older People

Epidemiological data about pelvic fractures are limited. Until today, most studies only analyzed inpatient data. The purpose of this study was to estimate incidence rates of pelvic fractures in the German population aged 60 years or older, based on outpatient and inpatient data. We conducted a retrospective population-based observational study based on routine data from a large health insurance company in Germany. Age and sex-specific incidence rates of first fractures between 2008 and 2011 were calculated. We also standardized incidence rates with respect to age and sex in the German population. Multiple Poisson regression models were used to evaluate the association between the risk of first pelvic fracture as outcome and sex, age, calendar year and region as independent variables. The total number of patients with a first pelvic fracture corresponded to 8,041 and during the study period 5,978 insured persons needed inpatient treatment. Overall, the standardized incidence rate of all first pelvic fractures was 22.4 [95% CI 22.0–22.9] per 10,000 person-years, and the standardized incidence rate of inpatient treated fractures 16.5 [16.1–16.9]. Our adjusted regression analysis confirmed a significant sex (RR 2.38 [2.23–2.55], p < 0.001, men as reference) and age effect (higher risk with increasing age, p < 0.001) on first fracture risk. We found a slight association between calendar year (higher risk in later years compared to 2008, p = 0.0162) and first fracture risk and a further significant association with region (RR 0.92 [0.87–0.98], p = 0.006, Westfalen-Lippe as reference). The observed incidences are considerably higher than incidences described in the international literature, even if only inpatient treated pelvic fractures are regarded. Besides which, non-inclusion of outpatient data means that a relevant proportion of pelvic fractures are not taken into account. Prevention of low energy trauma among older people remains an important issue.