[DNA vaccine encoding alpha-fetoprotein with fused ornithine decarboxylase degradation signal significantly suppresses hepatocellular carcinoma growth in mice].

Hepatocellular carcinoma (HCC) is among the most frequent malignancies in humans. HCC therapy is not efficient and the usual outcome is poor. In this regard, novel approaches to treat and prevent HCC are urgently needed. The Alpha-fetoprotein (AFP) is a serological marker of HCC. Recently it has been shown, that the DNA vaccines expressing AFP are capable in generating immune response against AFP. However, both, the immunization procedures and DNA vaccines used before were complex and not always very effective. We have shown that DNA vaccine encoding HIV-1 reverse transcriptase (RT) with fused ornithine decarboxylase (ODC) degradation signal induced a strong Th-1 immune response against RT in mice. Using this approach we designed a set of novel DNA vaccines bearing AFP and ODC degradation signal. Results obtained on transfected cells demonstrated efficient expression and fast proteasomal degradation of the recombinant AFP. The anti-tumor immune response stimulation was shown in immunized animals and most importantly a notable retardation of tumor growth was observed as a result of protective vaccination.

[Critical amino acids of ornitin decarboxylase degron: the presence and C-terminal arrangement is insufficient for alfa-fetoprotein degradation].

Mouse ornithine decarboxylase (ODC) degrades in proteasome in an ubiquitin-independent manner with an averagehalf-life of 2 h. The 37 amino acid long C-terminal fragment known as a degradation signal (degron) is responsible for the effective degradation of ODC. Recently, amino acids being critical for degradation in the ODC-degron have been mapped. Mutations of Cys441 and Ala442 led to protein stabilization, while a substitution of other amino acids composing ODC-degron had almost no effect on the protein turnover; whereas insertions or deletions in region between Ala442 and ODC C-terminus diminished greatly rate of protein degradation, e.g. positioning of the key amino acids from the C-terminus was shown to be crucial. Using these data we introduced both key amino acids into the alfa-fetoprotein with truncated exportation signal (deltaAFP), at the same distance from the C-terminus as they being in the ODC (deltaAFPCAG and deltaAFPLCAG). Removal of N-terminal exportation signal prevented secretion of modified proteins. Using in silico approach we demonstrated no significant difference in hydrophobicity or secondary structure between C-terminus of deltaAFP and mutated proteins. The degradation kinetics of deltaAFP, deltaAFPCAG, deltaAFPLCAG in cyloheximide-chase and proteasome inhibition assay (using MG132) was identical. Obtained results suggest that introduced substitutions are insufficient for effective recognition of mutated deltaAFP by26S proteasome. We assume thatadditional amino aci ds composing ODC-degron or their combine action could also affect degradation. Besides that, one cannot exclude that conformation of the mutated deltaAFP limits its C-terminus accessibility to proteasome.

Short communication: Simultaneous substitutions of V38M and N43T-N44K in the gp41 heptad repeat 1 (HR1) disrupt HIV type 1 gPr160 endoproteolytic cleavage (*).

We cloned and sequenced gp41 HIV-1 from plasma of AIDS patients under HAART and T-20 (enfuvirtide, Fuzeon) therapy and revealed several T-20 resistance-associated mutations. Two mutations, a single V38A and a double N43T-N44K were the most frequent; however, they were not found together in one clone. We anticipated that simultaneous mutations of these three residues might play a vital role in the viral life cycle. To address this problem, we introduced N43T-N44K and V38M + N43T-N44K substitutions to a cloned gp41 and introduced modified gp41 into the pNL4-3 molecular clone. HEK293T cells were transfected with the obtained vectors and released viruses were examined for reverse transcriptase (RT) activity, infectivity on reporter TZM-bl cells, and in Western blotting. Nearly equal RT activity was demonstrated in viruses with and without mutations. However, viruses with the V38M + N43T-N44K mutations were not infectious and, as shown by Western blotting, gPr160 cleavage was impaired. These data suggest that V38M + N43T-N44K mutations perturbed the natural conformation of gPr160 in a way that access of furin to the cleavage site (REKR) was blocked. Therefore, the residues V38 + N43-N44 retain the gPr160 conformation in proximity to the furin cleavage site and, as a consequence, are critical for virus infectivity. These data may explain why viruses with V38M + N43T-N44K mutations were not previously detected in the plasma of T-20-experienced patients.

[Alpha oscillations of human brain elicited by illusory contour perception].

We examined the changes in the phase-locked and total alpha oscillations of EEG during perception of illusory (Kanizsa square) and non-illusory images in 16 healthy adults. We applied a wavelet-based time-frequency analysis to compute alpha band power, statistical evaluation was performed using repeated-measures ANOVA and the non-parametric multi-way analysis. Results showed that both stimuli provoked an initial increase of total alpha power emerging not later than in 100 ms after the stimulus onset. The increase was caused by the phase-locked alpha response, and the illusory contour was systematically followed by a higher level of alpha power than the non-illusory one. Further suppression of total alpha power occurred within the time window of 200-500 ms after the stimulus onset and did not depend on the stimulus type. Both stages of the total alpha response were more prominent over the parieto-occipito-temporal scalp areas. We hypothesize that the first stage of alpha response is related to the modulation of the activity of neural circuits participating in processing of a coherent pattern whereas a later suppression of total alpha power links to the orienting processes.

Endogenous JSRV-like proviruses in domestic cattle: analysis of sequences and transcripts.

Jaagsiekte retrovirus is an exogenous (exJSRV) beta-retrovirus with a simple genome. It causes lower airway epithelial cell tumors in small ruminants. Endogenous (enJSRV) counterparts of exJSRV are present in different copy numbers in numerous Bovidae family members. This work has focused on enJSRV in Simmental (Germany) and Limousine (France) beef breeds of domestic cattle and domestic goat. Of the enJSRV sequences in cattle, the orf-x sequences were about 99% identical, the LTR sequences were about 97% identical and the env sequences were nearly 95% identical to the corresponding endogenous sequences in sheep. A significant polymorphism of the proviral sequences between the cattle breeds was noted. Clonal analyses of the amplicons suggest two enJSRV proviruses in cattle genome. The endogenous sequences revealed in goat were closer to enzootic nasal tumor virus (ENTV) from goat rather than to enJSRV from sheep. The expression of enJSRV in cattle was partial (env only) and detected exclusively in bone marrow.

Detection and characterization of betaretroviral sequences, related to sheep Jaagsiekte virus, in Africans from Nigeria and Cameroon.

Jaagsiekte retrovirus (JSRV) causes ovine pulmonary adenomatosis (OPA) that resembles bronchioloalveolar carcinoma (BAC) in humans. To test the possible role of JSRV in human diseases, DNA specimens from 103 individuals either healthy or suffering from lung carcinomas were analyzed for JSRV sequences. orf-x sequences were detected in 19 of 64 samples and gag-prt sequences in 4 of 38 samples, predominantly in individuals from Africa. Sequences obtained from orf-x amplimers varied in-between each other and differed from control endogenous ovine JSRV sequence. No association with lung cancer was found. This is the first report of JSRV-like sequences detected in humans.

Defective human T-cell leukaemia virus type 1 (HTLV-1) genomes: no evidence in serologically indeterminate german blood donors but new type detected in established cell lines.

Individuals reactive in antibody screening tests (ELISA) and with one or more reactions to HTLV-1 proteins on Western blotting, but lacking the criteria of a confirmed HTLV infection, are not exceptional in regions with a low prevalence of HTLV-1/-2 infections. PCR analysis of these indeterminate samples, using "diagnostic" pol and tax sets of primers, give negative results. However, expression of HTLV-1 defective proviruses with internal deletions undetectable by PCR with diagnostic primers could have taken place. Seven German HTLV-1 ELISA-reactive blood donors, who showed reactivity also in Western blots against several viral proteins, and twenty haemophiliacs, were examined by nested PCR and/or PCR/Southern hybridisation with primers designed for detection of HTLV-1 defective proviruses. No HTLV-1-specific amplification products were obtained. However, HTLV-1 defective proviruses with large internal deletions were detected in four out of five cell lines established from symptomatic HTLV-1 cases and two in HUT-102 cells. In two amplicons, short inverted rRNA sequences between gag and env fragments of HTLV-1 defective proviruses were revealed. These results do not exclude the presence of defective HTLV-1 proviruses in individuals with indeterminate serology although this is unlikely.

[Defective proviruses of the human T-cell leukemia virus: structure and classification]. / Defektnye provirusy virusa T-kletochnogo leikoza cheloveka: struktura i klassifikatsiia.

Peripheral blood mononuclear cells of 24-70% individuals infected with HTLV-1 contain defective proviruses (dp) in addition to the full size ones. Most of them remain silent lacking regions sufficient for viral genes transcription except those activated under cell promoter or retaining viral open reading frames (orfs). It is still unclear whether these proviruses are associated with the development of T-cell leukemia in adults, tropic spastic paresis, or myelopathy. Classification of previously reported dp is presented, their origin and possible function in human HTLV-1 associated diseases are discussed.

[C-reactive protein and defect in lipid transport blood system in patients with rheumatoid arthritis]. / S-reaktivnyi belok i narusheniia lipid-transportnoi sistemy krovi u bol'nykh revmatoidnym artritom.

In 88 patients with rheumatoid arthritis lipid-protein spectrum of the blood serum were determined biochemically and by means of nefelometric technique, C-reactive protein (CRP) was measured with enzyme immunoassay. An increase in CRP concentration was associated with lowering of apoA1 and HDL FL, triglycerides. Thus, elevation of CRP in RA reflects not only activity of inflammation but also defects in serum lipid-transport system by the atherogenic type.

Path-dependent neutralization of multiply charged Ar ions incident on Au(110).

Using a time-of-flight technique we have investigated the backscattering of 5 keV Ar(2+) and Ar(11+) projectiles from Au(110). A strong dependence on target azimuth is found for the neutral flux resulting from (quasi)binary projectile collisions with target atoms, while for the charged components only a weak dependence is seen. A deconvolution of the observed dependences based on trajectory simulations clearly shows site-specific neutralization differences between the various possible binary collisions occurring with surface atoms. Such differences must be properly accounted for in order to permit meaningful comparison with theory.