Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.     

Cytogenetic comparison of the sensitivity to mutagens in mice selected for high (HA) and low (LA) swim stress-induced analgesia

Sensitivity to mutagens was studied in mouse lines selectively bred for high analgesia (HA) and for low analgesia (LA) induced by 3-min swimming in 20 degrees C water. Apart from pain-related traits HA mice also manifest, as compared to the LA line, higher emotionality in various behavioural tests, and cope worse with the hypothermic challenge of swimming in cold water. In the present study HA mice appeared more susceptible to the mutagenic effect of whole-body gamma-radiation and mitomycin-C injection. Both treatments caused higher frequencies of chromosomal aberrations and micronucleus in bone marrow cells in the HA than in the LA line. The results are discussed in terms of a genetic correlation between animals' susceptibility to environmental stressors and the mechanism of mutagenesis. As shown by our recent molecular study, the selection for magnitude of swim analgesia has differentiated the parental outbred population into two distinct genotypes characterised by specific minisatellite and microsatellite sequences for each line, which may be genetic markers of particular traits. We conceive that the breeding strategy, along with the differentiation of stress-related phenomena, has altered the frequencies of genes controlling DNA repair in each line.     

The 33 kDa protein of photosystem II is a low-affinity calcium- and lanthanide-binding protein

We have shown that the isolated 33 kDa protein of photosystem II contains one calcium and one lanthanide low-affinity binding site with binding constants (K(D)) on the order of 10(-5) M. Binding of calcium or lanthanides to this site induces conformational changes in the protein that manifest in fluorescence emission spectra of the protein, circular dichroism spectra, and calorimetric thermograms where the phase transitions are shifted to lower temperatures. The role of calcium binding to the 33 kDa protein in the attainment of its native structure and the significance of this interaction for the oxygen evolution process are discussed.     

Structural and functional implications of the instability of the ADP/ATP transporter purified from mitochondria as revealed by FTIR spectroscopy

The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies. When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates. By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process. To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein. From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands. We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed.     

Strip test for bedside detection of interleukin-6 in cervical secretions is predictive for impending preterm delivery

Inflammatory cytokines in amniotic fluid are markers of prematurity which could characterize preterm labour of infectious origin. To avoid amniocentesis, we analyzed IL-6, IL-8, IL-10, and IL-13 by RT-PCR in cervical secretions (CS) of 307 women with preterm labour. IL-6 was detected in 26.3% patients who delivered at less than 34 weeks (specificity: 95.8%). In addition, IL-6 was associated with delivery within 7 days (specificity: 91.6%). To render the detection more rapid and cheaper, a strip test was designed and evaluated comparatively with RT-PCR in 76 women. This bedside strip test was twice more sensitive than RT-PCR, with little decrease in specificity.     

Assessment of the genetic diversity among strains of Xanthomonas cynarae by randomly amplified polymorphic DNA analysis and development of specific characterized amplified regions for the rapid identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

Cost-effective PET investigations in oncology

The authors have reviewed the financial considerations of oncological FDG PET examinations by the guidelines of the Health Care Financing Administration (USA). By critical assessment of large number of clinical investigations,the cost-effectiveness of FDG PET scans has been confirmed in the following cases: differential diagnosis of solitary pulmonary nodule, diagnosis,staging and restaging of non-small cell lung cancer, colorectal cancer, malignant lymphomas, melanoma malignum, esophageal neoplasms and cancers of the head and neck. The role of this method in breast cancer is currently under intensive investigation. Due to the correct staging, PET examinations in these indications enable the clinicians to choose the optimal treatment ensuring the maximum probability of recovery and being cost-effective as unnecessary medical interventions become avoidable.     

Chlamydial infection of cats and human health

Chlamydia psittaci var.felis is considered as a primary and important agent in the etiology of infectious diseases of the upper respiratory tract and eyes in cats, having zoonotic potential. We investigated 13 cats aged between 2 months and 7 years, in which conjunctivitis, rhinitis, laryngotracheitis, bronchopneumonia and lymph adenopathy was clinically diagnosed. To detect the antigen ofC. psittaci the Clearview Chlamydia Direct Test was used for the first time. The presence ofC. psittaci in 10 of 13 investigated cats and in 8 nasal mucosa smears from 10 investigated cats were confirmed in conjunctiva. Using bacteriological examination different species of the genusStaphylococcus in conjunctiva from all 13 investigated animals were also confirmed. Serological investigation using complement fixation test was negative in all animals.     

Stimulation of oxygen evolution in photosystem II by copper(II) ions

We have found that copper(II) ions at about equimolar Cu2+/photosystem II (PS II) reaction center proportions stimulate oxygen evolution nearly twofold. This high affinity Cu-binding site is different from the binding sites of Mn and Ca ions. The analysis of the Cu2+ content in PS II preparations isolated from wild-type tobacco and a tobacco mutant deficient in light-harvesting complex suggests that Cu2+ may be a native component of PS II and may take part in the oxygen evolution process. At higher concentrations, Cu2+ ions inhibit oxygen evolution and quench fluorescence.