Plasma fibrin clots of pulmonary embolism patients present increased amounts of factor XIII and alpha2-antiplasmin at 3 months' anticoagulation since the acute phase.

Fibrin cross-linking by coagulation factor (F)XIII leads to clot stabilization. Reduced plasma FXIII levels have been reported in acute pulmonary embolism (PE) patients. We investigated the impact of anticoagulant therapy on clot-bound amounts of FXIII and α2-antiplasmin and their associations with fibrin clot properties in patients with PE. Clots generated from plasma of 18 acute symptomatic patients on admission and after a 3-month treatment with rivaroxaban were assessed off anticoagulation using mass spectrometry. Plasma FXIII and α2-antiplasmin activity were determined at the 2 time points along with thrombin generation markers, plasma fibrin clot permeability (Ks), and clot lysis time (CLT). Following anticoagulant therapy, clot-bound FXIII increased from 2.97 (interquartile range, 1.98 - 4.08) to 4.66 (3.5 - 6.9) mg/g protein and α2-antiplasmin from 9.4 (7.2 - 10.6) to 11 (9.5 - 14) mg/g protein (both p < 0.0001). The two parameters showed positive correlation at baseline only (r = 0.63, p = 0.0056). Similarly to clot-bound amounts, plasma FXIII (+25.8%) and α2-antiplasmin activity (+12%) increased at 3 months. Plasma FXIII activity on admission, but not after 3 months since the index PE, was associated with amounts of clot-bound FXIII (r = 0.35, p = 0.043) and α2-antiplasmin (r = 0.47, p = 0.048). At baseline, clot-bound FXIII correlated with plasma F1+2 prothrombin fragments levels (r = 0.51, p = 0.03), while clot-bound α2-antiplasmin correlated with CLT (r = 0.43, p = 0.036). At 3 months associations of clot-bound FXIII and α2-antiplasmin were abolished. This study assessed for the first time changes in the fibrin clot composition following acute PE, suggesting an increase of clot-bound and plasma FXIII and α2-antiplasmin levels after 3 months.

Systematic analysis of GSK-3 signaling pathways in aging of cerebral tissue.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase, involved in regulation of multiple physiological processes. In brain, changes in GSK-3 signaling are related to neurodegenerative issues, including Alzheimer's disease. Due to the wide range of GSK-3 cellular targets, a therapeutic use of the enzyme inhibitors entails significant risk of side effects. Thus, altering the ratio of specific pool of GSK-3 or specific substrates instead of changing the global activity of GSK-3 in brains might be a more appropriate strategy. This paper provides a comprehensive data on abundances of proteins involved in GSK-3 signaling in three regions of young and old mouse brains. It might help to identify novel protein targets with the highest therapeutic potential for treatment of age-related neurodegenerative diseases.

Management of Renin-Angiotensin-Aldosterone System Blockade in Kidney Transplant Recipients.

Angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are the cornerstone treatment in chronic kidney disease patients. Despite facilitating a reduction in blood pressure and albuminuria, there are insufficient data in kidney transplant recipients (KTRs). They are often administered for hypertension and polycythemia treatment. The aim of this study was to investigate the frequency and route of administration of ACEIs and ARBs and their early clinical effects in the KTR population. In a cross-sectional, retrospective study we analyzed 874 medical records of all KTRs treated in our unit in 2014. A total of 391 KTRs (44.7%) using ARBs or ACEIs were qualified for the study. The primary reasons for renin-angiotensin-aldosterone system antagonist administration were hypertension (59.1%), polycythemia (19.2%), and proteinuria (18.2%). Among the studied KTRs, 86.7% of patients were treated with ACEIs and 12.2% were treated with ARBs. The majority of patients treated with ACEIs and ARBs received these agents in a dose range below 25% and between 25% and 49% of their maximal dose, respectively. Both the mean serum creatinine level and estimated glomerular filtration rate (chronic kidney disease epidemiology collaboration) remained fairly stable and urine protein excretion (g/24 hours) was significantly reduced after 3 months of ACEI and ARB therapy. The serum potassium level increased significantly, while hemoglobin concentration dropped significantly. In KTRs, renin-angiotensin-aldosterone system antagonists were applied mainly due to hypertension, proteinuria, and polycythemia. ACEIs and ARBs were effective in the reduction of proteinuria and hemoglobin, but graft function was stable and the increase of serum potassium was not of clinical significance.

Improvement of Blood Pressure Control in Renal Transplant Recipients-Retrospective Longitudinal Study.

BACKGROUND: Hypertension is a very common complication in renal transplant recipients (RTRs). It has been identified as a potent cardiovascular risk factor associated with impaired patient and graft survival. METHODS: A longitudinal retrospective analysis was performed to evaluate adherence to recommended blood pressure (BP) targets and to estimate the tendency in the management of hypertension from 2001 to 2015. A total of 96 RTRs (55 male, 41 female; overall mean age (2001), 41.66 ± 11.08 years; mean serum creatinine level, 1.45 ± 0.3 mg/dL; 41.2 ± 34.9 months after kidney transplantation) with diagnoses of hypertension and monitored continuously in the unit from 2001 to 2015 were included in the study. RESULTS: The average diastolic BP decreased (P < .01) and the average systolic BP did not change in this period. The target values of BP (ie, <140/90 mm Hg) were accomplished by 45.8% (2001) and 53.1% (2015) of patients. When the target BP was corrected by age (<150/90 mm Hg for people >65 years old) the adherence improved to 57.29% in 2015. The average number of antihypertensive agents used per patient increased significantly (P < .001): 2.03 ± 1.0 (2001) versus 2.69 ± 1.26 (2015). The most commonly used antihypertensive agents were beta-blockers: 69% and 74% in 2001 and 2015, respectively. There was a significant increase in the percentage of RTRs treated with the use of alpha-blockers (P < .01), angiotensin-converting enzyme inhibitors (P < .001), and angiotensin II receptor blockers (P < .05). CONCLUSIONS: The study showed modest improvement of the hypertension control rate from 2001 to 2015 in RTRs. Greater efforts are needed to implement the guidelines, which would further improve patient and graft outcomes.

Filter-Aided Sample Preparation: The Versatile and Efficient Method for Proteomic Analysis.

Filter-aided sample preparation (FASP) is a versatile and efficient way of processing protein extracts for bottom-up proteomic analysis. The method repurposes centrifugal ultrafiltration concentrators for removal of detergents, protein cleavage, and isolation of pure peptide fractions. FASP can be used for protein cleavage with different proteinases either with single enzymes or in a mode of successive multienzyme digestion (MED)-FASP. The FASP methods are useful for processing of samples ranging in their sizes from submicrogram to several milligram amounts of total protein. They also allow peptide fractionation, and isolation and quantitation of total RNA and DNA acid contents. This chapter describes principles, limitations, and applications of FASP. Additionally detailed FASP and MED-FASP protocols are provided.

A Protocol for Large-Scale Proteomic Analysis of Microdissected Formalin Fixed and Paraffin Embedded Tissue.

Extensive use of biomarkers in diagnostic and therapeutic procedures is probably the hallmark of future medicine. Biomarkers can be of different chemical nature-most frequently DNA, RNA, and proteins are considered as molecules of interest. As the most rational approach appears analysis of proteins, because these are the "effector" molecules directly involved for cell homeostasis. There is no commonly accepted standard procedure in proteomic-based biomarker search. In this chapter, we described a protocol for proteomic analysis of formalin-fixed and paraffin-embedded tissue, which has been proven to be highly effective for proteomics-based biomarker discovery.

Label-Free and Standard-Free Absolute Quantitative Proteomics Using the "Total Protein" and "Proteomic Ruler" Approaches.

Understanding biological systems and their variation upon stimuli requires knowledge on their composition, primarily including information on organization and dynamics of proteomes. The total protein approach (TPA) is a label- and standard-free method for absolute protein quantitation of proteins using large-scale proteomic data. The method relies on the assumption that the total MS signal from all identified proteins in the dataset reflects-in a biochemical sense-the total protein and the MS signal from a single protein corresponds its abundance in the studied sample. The method offers an easy way to quantify thousands of protein per sample. A related method, the "Proteomic Ruler," enables conversion of the protein abundance data calculated by TPA to compute numbers of protein copies per cell. TPA and the Proteomic Ruler are powerful tools for studying dynamics of cell architecture.

Analysis of cytokine production by peanut-reactive T cells identifies residual Th2 effectors in highly allergic children who received peanut oral immunotherapy.

BACKGROUND: Only limited evidence is available regarding the cytokine repertoire of effector T cells associated with peanut allergy, and how these responses relate to IgE antibodies to peanut components. OBJECTIVE: To interrogate T cell effector cytokine populations induced by Ara h 1 and Ara h 2 among peanut allergic (PA) children in the context of IgE and to evaluate their modulation during oral immunotherapy (OIT). METHODS: Peanut-reactive effector T cells were analysed in conjunction with specific IgE profiles in PA children using intracellular staining and multiplex assay. Cytokine-expressing T cell subpopulations were visualized using SPICE. RESULTS: Ara h 2 dominated the antibody response to peanut as judged by prevalence and quantity among a cohort of children with IgE to peanut. High IgE (> 15 kU(A)/L) was almost exclusively associated with dual sensitization to Ara h 1 and Ara h 2 and was age independent. Among PA children, IL-4-biased responses to both major allergens were induced, regardless of whether IgE antibodies to Ara h 1 were present. Among subjects receiving OIT in whom high IgE was maintained, Th2 reactivity to peanut components persisted despite clinical desensitization and modulation of allergen-specific immune parameters including augmented specific IgG4 antibodies, Th1 skewing and enhanced IL-10. The complexity of cytokine-positive subpopulations within peanut-reactive IL-4(+) and IFN-γ(+) T cells was similar to that observed in those who received no OIT, but was modified with extended therapy. Nonetheless, high Foxp3 expression was a distinguishing feature of peanut-reactive IL-4(+) T cells irrespective of OIT, and a correlate of their ability to secrete type 2 cytokines. CONCLUSION: Although total numbers of peanut-reactive IL-4(+) and IFN-γ(+) T cells are modulated by OIT in highly allergic children, complex T cell populations with pathogenic potential persist in the presence of recognized immune markers of successful immunotherapy.

Infection with human rhinovirus 16 promotes enhanced IgE responsiveness in basophils of atopic asthmatics.

BACKGROUND: Rhinovirus and IgE act in concert to promote asthma exacerbations. While basophils are the principal cell type in the blood that is activated by IgE, their role in virus-induced asthma episodes remains elusive. OBJECTIVE: To monitor IgE responsiveness in circulating basophils of rhinovirus-infected atopic asthmatics during acute infection and convalescence. METHODS: The capacity for basophils to respond to IgE was assessed by testing the effects of allergen, or cross-linking anti-FcεRI and anti-IgE antibodies, on surface TSLP receptor in 24-hour PBMC cultures. Activation profiles of basophils from atopic asthmatics challenged intranasally with human rhinovirus 16 were monitored directly ex vivo or else in 24-hour cultures, at baseline (day 0), and then at days 4 and 21 post-challenge. RESULTS: Basophils in atopic asthmatics, but not in non-atopic controls, upregulated TSLP receptor upon IgE receptor ligation. The magnitude of this response was correlated with the proportion of serum total IgE that was allergen-specific (r = 0.615, P < 0.05). Following rhinovirus infection, all subjects developed nasal symptoms that peaked 3-5 days after viral challenge. Basophils displayed maximal IgE responsiveness 3 weeks post-challenge as judged by TSLP receptor levels in 24-hour cultures. No significant change in total IgE or specific IgE antibodies was detected during rhinovirus infection. By contrast, levels of IgE receptor-associated spleen tyrosine kinase, Syk, were increased on day 4 (P < 0.05), and elevated levels were also detected three weeks post-challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating basophils display increased IgE responsiveness 3 weeks after rhinovirus infection in atopic asthmatics. This observation, coupled with increased expression of Syk, implicates basophils in promoting, or else prolonging, rhinovirus-induced inflammation in atopic asthmatics.

Sensitization to food and inhalant allergens in relation to age and wheeze among children with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is common in children; however, persistence of AD with or without asthma is less common. Longitudinal studies remain limited in their ability to characterize how IgE antibody responses evolve in AD, and their relationship with asthma. OBJECTIVE: To use a cross-sectional study design of children with active AD to analyse age-related differences in IgE antibodies and relation to wheeze. METHODS: IgE antibodies to food and inhalant allergens were measured in children with active AD (5 months to 15 years of age, n = 66), with and without history of wheeze. RESULTS: Whereas IgE antibodies to foods persisted at a similar prevalence and titre throughout childhood, IgE antibodies to all aeroallergens rose sharply into adolescence. From birth, the chance of sensitization for any aeroallergen increased for each 12-month increment in age (OR ≥ 1.21, P < 0.01), with the largest effect observed for dust mite (OR = 1.56, P < 0.001). A steeper age-related rise in IgE antibody titre to dust mite, but no other allergen was associated with more severe disease. Despite this, sensitization to cat was more strongly associated with wheeze (OR = 4.5, P < 0.01), and linked to Fel d 1 and Fel d 4, but not Fel d 2. Comparison of cat allergic children with AD to those without, revealed higher IgE levels to Fel d 2 and Fel d 4 (P < 0.05), but not Fel d 1. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in sensitization to cat and dust mite among young children with AD may aid in identifying those at increased risk for disease progression and development of asthma. Early sensitization to cat and risk for wheeze among children with AD may be linked to an increased risk for sensitization to a broader spectrum of allergen components from early life. Collectively, our findings argue for early intervention strategies designed to mitigate skin inflammation in children with AD.

Mechanisms of tolerance induction in allergic disease: integrating current and emerging concepts.

The prevalence of atopy and allergic disease continues to escalate worldwide. Defining immune mechanisms that suppress the underlying Th2-driven inflammatory process is critical for the rational design of new treatments to prevent or attenuate disease. Allergen immunotherapy has provided a useful framework for evaluating changes in the immune response that occur during the development of tolerance. Despite this, elucidating the phenotypic and functional properties of regulatory cells, has proven challenging in humans with allergic disease. This article provides an overview of our current understanding of the immune pathways that orchestrate allergen tolerance, with an emphasis on emerging concepts related to human disease. A variety of regulatory cell types, including IL-10-secreting T and B cells, play a pivotal role in suppressing allergic responses to inhaled, ingested and injected allergens. These cells may inhibit Th2 effectors directly, or else indirectly, through other cell types and mediators. Protective antibodies, including IgG4, Fc sialylated IgG, and IgA, have the capacity to modulate the response by preventing allergen binding to surface-bound IgE, or inhibiting dendritic cell maturation. Immune cell plasticity may augment suppression of Th2 cells by T regulatory cells, through mechanisms that involve T cell conversion, or else unconventional roles of classical effector cells. These actions depend upon external cues provided by the in vivo milieu. As such, specific anatomical sites may preferentially favour tolerance induction. Recent scientific advances now allow a global analysis of immune parameters that capture novel markers of tolerance induction in allergic patients. Such markers could provide new molecular targets for assessing tolerance, and for designing treatments that confer long-lasting protection in a safe and efficacious fashion.

Cytotoxicity of listeriolysin O produced by membrane-encapsulated Bacillus subtilis on leukemia cells.

Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.

Acupuncture for treatment of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS), a disorder of altered bowel habits associated with abdominal pain or discomfort. The pain, discomfort, and impairment from IBS often lead to healthcare medical consultation (Talley 1997) and workplace absenteeism, and associated economic costs (Leong 2003). A recent randomized controlled trial shows variable results but no clear evidence in support of acupuncture as an effective treatment for IBS (Fireman 2001). OBJECTIVES: The objective of this systematic review is to determine whether acupuncture is more effective than no treatment, more effective than 'sham' (placebo) acupuncture, and as effective as other interventions used to treat irritable bowel syndrome. Adverse events associated with acupuncture were also assessed. SEARCH STRATEGY: The following electronic bibliographic databases were searched irrespective of language, date of publication, and publication status: MEDLINE, the Cochrane Central Register of Controlled Trials (CENTRAL) on The Cochrane Library, EMBASE, the Chinese Biomedical Database, the Cumulative Index to Nursing and Allied Health (CINAHL), and the Allied and Complementary Medicine Database (AMED). References in relevant reviews and RCTs were screened by hand. The last date for searching for studies was 7 February 2006. SELECTION CRITERIA: Published reports of randomized controlled trials (RCTs) and quasi-randomised trials of acupuncture therapy for IBS. DATA COLLECTION AND ANALYSIS: All eligible records identified were dually evaluated for eligibility and dually abstracted. Methodological quality was assessed using the Jadad scale and the Linde Internal Validity Scale. Data from individual trials were combined for meta-analysis when the interventions were sufficiently similar. Heterogeneity was assessed using the I squared statistic. MAIN RESULTS: Six trials were included. The proportion of responders, as assessed by either the global symptom score or the patient-determined treatment success rate, did not show a significant difference between the acupuncture and the sham acupuncture group with a pooled relative risk of 1.28 (95% CI 0.83 to 1.98; n=109). Acupuncture treatment was also not significantly more effective than sham acupuncture for overall general well-being, individual symptoms (e.g., abdominal pain, defecation difficulties, diarrhea, and bloating), the number of improved patients assessed by blinded clinician, or the EuroQol score. For two of the studies without a sham control, acupuncture was more effective than control treatment for the improvement of symptoms: acupuncture versus herbal medication with a RR of 1.14(95% CI 1.00 to 1.31; n=132); acupuncture plus psychotherapy versus psychotherapy alone with a RR of 1.20 (95% CI 1.03 to 1.39; n=100). When the effect of ear acupuncture treatment was compared to an unclearly specified combination of one or more of the drugs diazepam, perphenazine or domperidone, the difference was not statistically significant with a RR of 1.49(95% CI 0.94 to 2.34; n=48). AUTHORS' CONCLUSIONS: Most of the trials included in this review were of poor quality and were heterogeneous in terms of interventions, controls, and outcomes measured. With the exception of one outcome in common between two trials, data were not combined. Therefore, it is still inconclusive whether acupuncture is more effective than sham acupuncture or other interventions for treating IBS.

Prism coupling method to excite and analyze Floquet-Bloch modes in linear and nonlinear waveguide arrays.

We demonstrate an experimental method for the direct measurement of band structures of one-dimensional periodic optical media. With the help of a prism coupler that is used in a retroreflective scheme, the allowed bands of a waveguide array in lithium niobate are determined and the results are compared with numerical calculations. Furthermore, we demonstrate the suitability of this method to measure propagation constants of extended nonlinear modes inside the forbidden gap.

Consecutive steps of phosphorylation affect conformation and DNA binding of the chironomus high mobility group A protein.

The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dA-dT).poly(dA-dT) and a fragment of the interferon-beta promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin.

Distinct organization of DNA complexes of various HMGI/Y family proteins and their modulation upon mitotic phosphorylation.

High mobility group (HMG) proteins HMGI, HMGY, HMGI-C, and Chironomus HMGI are DNA-binding proteins thought to modulate the assembly and the function of transcriptional complexes. Each of these proteins contains three DNA-binding domains (DBD), properties of which appear to be regulated by phosphorylation. High levels of these proteins are characteristic for rapidly dividing cells in embryonic tissues and tumors. On the basis of their occurrence, specific functions for each of these proteins have been postulated. In this study we demonstrate differences in the nature of contacts of these proteins with promoter region of the interferon-beta gene. We show that HMGI and HMGY interact with this DNA via three DBDs, whereas HMGI-C and Chironomus HMGI bind to this DNA using only two domains. Phosphorylation of HMGY protein by Cdc2 kinase leads to impairing of contacts between the N-terminally located DBD and a single promoter element. The perturbations in the architecture of the protein.DNA complexes involve changes in the degree of unbending of the intrinsically bent IFNbeta promoter. Our results provide first insights into the molecular basis of functional specificity of proteins of the HMGI/Y family and their regulation by phosphorylation.

[Single use insulin injector evaluated by patients (questionnaire study)]. / Jednorazowy wstrzykiwacz insuliny w ocenie chorych (badanie ankietowe).

The aim of the study was the analysis of perception of the disposable pen injector HumaJect by diabetic patients. Selected features of the insulin delivery systems were evaluated, and on that basis, the comparison between HumaJect and other insulin injectors was made. Research material was collected in questionnaires filled out by doctors after interviewing patients who were using HumaJect for at least one month. 1802 diabetic individuals aged 9 to 88 participated in the study. HumaJect was ranked "very good" (which is the highest possible rank) by 71% of patients. Among specific features, the highest ranks were assigned to "Ease of dose setting", "Dosing range" and "Disposable form". Most of patients assigned higher ranks to HumaJect than to other insulin delivery systems. 89% of patients indicated desire to continue treatment with this injector.

Point mutations within AT-hook domains of the HMGI homologue HMGIYL1 affect binding to gene promoter but not to four-way junction DNA.

High-mobility group I/Y (HMGI/Y) proteins are chromosomal proteins involved in gene and chromatin regulation. Elevated levels of HMGI/Y proteins were reported in diverse malignant tumors, and rearrangements of their genes are casually involved in the development of benign tumors. In humans, the chromosomal locus Xp22 has been often found to be affected in diverse benign mesenchymal tumors. Recent studies revealed that this region contains a retropseudogene HMGIYL1 which potentially can be activated in a way of "exonization" upon aberrations involving this region. The coding sequence of the HMGIY-L1 is highly homologous to the HMGI(Y) gene. On the protein level, both HMGIYL1 and HMGI differ at few amino acid residues, including their putative DNA-binding domains (DBDs). Here we have approached the question of whether the HMGIYL1 product would be able to adopt a role of HMGI in the context of binding to gene promoters and chromatin. Comparative binding studies, employing protein footprinting technique, revealed that HMGIYL1 has lost the ability to bind to the promoter of the interferon beta gene, but retained its high affinity for the four-way junction DNA. Our results stress the importance of particular residues within the DBDs for DNA binding and demonstrate that tight binding of HMGI/Y proteins to the four-way junction DNA can be achieved in alternative ways. The binding of HMGIYL1 to four-way junction DNA suggests that activation of the HMGIYL1 gene would yield a protein sharing some binding properties with HMG1-box proteins and histone H1. Thus, the HMGIYL1 could interplay together with these components in chromatin regulation.

Generic isolated horizons and their applications

The notion of isolated horizons is extended to allow for distortion and rotation. Space-times containing a black hole, itself in equilibrium but possibly surrounded by radiation, satisfy these conditions. The framework has three types of applications: (i) it provides new tools to extract physics from strong field geometry; (ii) it leads to a generalization of the zeroth and first laws of black hole mechanics and sheds new light on the "origin" of the first law; and (iii) it serves as a point of departure for black hole entropy calculations in nonperturbative quantum gravity.

Insertional knock-out of protein translocation systems common for Yersinia enterocolitica and Listeria monocytogenes.

To carry out efficient insertional mutagenesis in Listeria monocytogenes and to facilitate the characterisation of disrupted genes, a novel derivative of plasmid pACYC 184 was constructed, pLIV virA3, carrying a fragment from the virA region of the of Y. enterocolitica plasmid pYVe 0:9. After transformation of this plasmid into L. monocytogenes it was possible to select for its integration into the host DNA at 42 degrees C. Insertional mutants of L. monocytogenes obtained by using pLIV vector containing plasmid DNA fragments from Y. enterocolitica were constructed and are described.