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Cellular senescence is a stable cell cycle arrest that is the causative process of aging. The PI3K/AKT/mTOR pathway is implicated in the control of cellular senescence and inhibitors of this pathway have been successfully used for life span prolongation experiments in mammals. PTEN is the major regulator of the PI3K/AKT/mTOR pathway and loss of PTEN promotes a senescence response termed PICS. Here we report a novel-screening assay, for the identification of compounds that block different types of senescence response. By testing a library of more than 3000 natural and chemical compounds in PTEN deficient cells we have found that an extract from Salvia haenkei (SH), a native plant of Bolivia is a potent inhibitor of PICS. SH also decreases replicative and UV-mediated senescence in human primary fibroblasts and in a model of in vitro reconstructed human epidermis. Mechanistically, SH treatment affects senescence driven by UV by interfering with IL1-α signalling. Pre-clinical and clinical testing of this extract by performing toxicity and irritability evaluation in vitro also demonstrate the safety of SH extract for clinical use as anti-aging skin treatment.

Infectious diseases during pregnancy can impact the development of fetal immunity, leading to reduced neonatal resistance to infection and decreased responses to pediatric vaccines. Plasmodium falciparum causes placental infection in low parity pregnant women and is among the pathogens that affect fetal immunity. Recognizing the relationship between malaria and gammadelta T lymphocytes in adults, we asked whether neonatal gammadelta T cells would be altered in malaria-endemic regions as a marker for changes in fetal immunity. Our initial studies compared cord blood gammadelta T cells from deliveries to HIV- mothers in Jos (Nigeria) where malaria is endemic, or in Rome (Italy). We noted substantial differences in the Vgamma2 repertoire for cord blood collected in Jos or Rome; differences were consistent with a negative selection mechanism operating on the fetal Vgamma2 chain repertoire in neonates from Jos. A specific disruption affected the fraction of gammadelta T cells that we expect will respond to Bacille Calmette-Guerin (BCG). Fetal gammadelta T cell depletion might be a mechanism for impaired neonatal immunity and lowered responses to pediatric vaccines.

Y‐box proteins are a highly conserved family of gene expression regulatory factors. During gametogenesis they may play a dual role, as both transcriptional activators of germ cell‐specific genes and as translational repressors of stored maternal transcripts. We report the identification of RlYB2, a Y‐box homolog gene specifically expressed in the germ cells of green frogs belonging to Rana esculenta complex, a model system characterized by a hybridogenetic gametogenesis. In developing germ cells of the hybrid R. esculenta, arisen by natural cross of the parental species R. ridibunda and R. lessonae, one set of the parental genomes is excluded and the remaining one, first endoreduplicates and then is transmitted to gametes. In situ hybridizations performed on gonadal tissues showed that the RlYB2 transcript was widely expressed in the ooplasm at early stages of oogenesis in both the parental species and hybrids. Interestingly, a hybridization signal, presumably related to RlYB2‐like nascent transcripts, wa...

One of the most promising strategies for drug design and development is the identification of new molecules able to selectively inhibit those enzymes involved in pathological processes, without affecting other enzymes associated with physiological functions. Nevertheless, some enzymes can show a double-edge aspect of their own inhibition, which can lead to positive as well as negative consequences according to the pathological state. Phospholipase D (PLD), an ubiquitous enzyme nowadays considered as a critical regulator of several aspects of cell biology and signal transduction pathways, is a clear example of those double-dealer enzymes. While a great deal has been learned about PLD structure, biological functions and activation/regulation mechanisms, little yet is known about the derivable effects of its potential negative regulation, also due to the lack of specific inhibitors. Multiple evidences on PLD involvement in many pathological states development and progression, including inflammation, carcinogenesis and metastases, have been supplied, so that a deregulation of its activity could contribute to attenuate or slow down the inflammatory and tumour formation/progression processes. On the other hand, in agreement with other previous observations, we have recently demonstrated the direct contribution of PLD activation in promoting intracellular mycobacterial killing. In this case, PLD inhibition resulted in a significant reduction of antimicrobial innate immune response and, hence, in a possible harmful effect. In the light of the above reported considerations, besides the recent advances in characterising new compounds able to selectively inhibit PLD activity and/or signalling, this review aimed at elucidating the potential dual beneficial/harmful consequences of PLD activity modulation.

We studied the extent of sexual size dimorphism (SSD) in the European pond turtle throughout most of its Italian distribution range using eight morphological parameters of 580 Emys specimens, describing populations from Italy and large Mediterranean islands. Sexual size dimorphism was evident in the whole sample, but not in all the considered areas. SSD in studied Emys likely mirrors different growth trajectories and body size architectures between sexes at adult stage. Italian populations resulted separated into five groups with morphological differences: (i) East Po plain Italy north to Apennines; (ii) Tyrrhenian Italy; (iii) Corse; (iv) Sardinia; and (v) southern Italy and Sicily. These results agree with previously reported data on systematics of Mediterranean populations. Some Italian populations were also tested using restriction endonuclease analysis of 18+28S ribosomal DNA on 43 specimens of eight localities. The occurrence of a restriction fragment length polymorphism of rDNA was also noted. Morp...

A family of interspersed repetitive elements, RlBamHI, with sequence similarity to the transposase of hAT DNA transposons, occurs in genomes of eight western Palaearctic water frog taxa and the brown frog Rana dalmatina, but was not detected in Xenopus laevis or Salamandra salamandra. RlBamHI elements are not tandemly arrayed, are dispersed across all chromosomes although not uniformly distributed, and based on dot-blot hybridizations may constitute as much as approximately 10% of the genomes of Rana lessonae, Rana ridibunda, and Rana perezi, but only approximately 1% of that of Rana saharica. Eleven nucleotide sequences of a 572 bp fragment from the nine taxa are very similar (pairwise differences 0.4–8.1% for nucleotides, 0.6–18.2% for amino acids), and all share a single open reading frame across the whole RlBamHI fragment. The reading frame is maintained despite several indels, most of which are multiples of 3 bp, but a pair of which in one species alters and, after 13 codons, restores the reading frame. It is possible that the reading frame is selectively maintained, suggesting recent or even present transposition capacity. The amino acid sequences encoded by RlBamHI elements, but not the nucleotide sequences themselves, reveal the similarity of RlBamHI to members of the widespread hAT superfamily of DNA transposons. Amino acid sequence comparisons permitted no convincing phylogenetic placement of RlBamHI among 32 representative hAT transposons across organisms, probably reflecting both the genetic divergence of RlBamHI elements and the paucity of aligned residues available for analysis. Among DNA transposons, a genomic fraction of 10% is extraordinarily high. Phylogenetic analyses of Rana RlBamHI sequences match poorly with independent genetic and molecular phylogenies; the elements compared probably are not orthologous, which renders their sequences inadequate for reconstructing organismal phylogenetic histories.

Viral and host factors can influence HIV-1 progression, among them human leucocyte antigen (HLA) has shown the strongest effect. However, studies on the functional contribution of HLA in controlling HIV progression toward AIDS are limited by multiple issues, including the viral strain variability within the study subjects. In this study, in a cohort of children infected with a monophyletic strain (CRF02_AG) during an outbreak, we evaluated the HIV-1 Gag, Vif, Vpr, Tat and hepatitis C virus E1/E2 (as control) proteins circulating in a cohort for the capability to be presented by the HLA molecules in the same population. A total of 70 Non-progressors and 37 Progressors to AIDS were evaluated. In the presence of a constant capability of HIV-1 to mutate in the region containing epitopes of Gag protein, the number of epitopes recognized in silico by the combination of the HLA alleles along the Gag consensus sequence is significantly higher in the Non-progressors compared with Progressors (HLA-A: Non-progressors = 1.532 ± 1.211, Progressors = 0.7714 ± 1.031, P = 0.0016; HLA-B: Non-progressors = 1.556 ± 1.298, Progressors = 1.000 ± 0.817, P = 0.0319; HLA-DR: Non-progressors = 13.30 ± 9.488, Progressors = 7.294 ± 6.952, P = 0.0006). Similar results were obtained for the other HIV-1 proteins Vif and Vpr, whereas no differences were obtained in the number of epitopes for the hepatitis C virus E1/E2 protein sequence or for the scrambled HIV-1 sequence. Finally, the results were confirmed also in a subgroup of subjects where both HLA typing and Gag sequence were available. In conclusion, in the absence of bias due to viral strain diversity, it is the overall fitting of the HLA molecules that are capable of better binding HIV-1 proteins in determining the major role in the control of HIV-1 replication and progression to AIDS.

Alteration in the humoral immune response has been observed during HIV infection. The polymorphisms of enhancer HS1,2, member of the 3′ regulatory region of the Ig heavy chain cluster, may play a role in the variation of the humoral response leading to pathological conditions. To assess the role of the HS1,2 polymorphic variants in the progression of AIDS, the HS1,2-A allelic frequencies were investigated in a cohort of HIV infected pediatric subjects from a nosocomial outbreak with a monophyletic strain of HIV. From a total group of 418 HIV infected children in the outbreak cohort, 42 nonprogressors and 31 progressors without bias due to antiretroviral therapy were evaluated. HS1,2 allele *1 has been associated with nonprogressors (allelic frequency: 51.19% versus 33.87% in progressors, OR 0.5, andP=0.0437), while allele *2 has been associated with progression (allelic frequency: 48.39% versus 30.95% in nonprogressors, OR 2.1, andP=0.0393). Further, only subjects carrying allele *2 in absence of allele *1, either in homozygous condition for allele *2 [nonprogressors 2/42 (4.76%), Progressors 7/31 (22.58%), OR 5.8, andP=0.0315]or in combination with other allelic variants [nonprogressors 7/42 (16.67%), Progressors 13/31 (41.93%), OR 3.61, andP=0.0321], have been associated with HIV progression to AIDS. In conclusion, while the HS1,2 allele *1 has a protective effect on HIV progression when present, allele *2 is associated with progression toward AIDS when allele *1 is absent.

Objective: HLA polymorphisms within the peptide binding pocket have been associated with rapid and slowprogression to AIDS, suggesting that the capability to present efficiently HIV-1 epitopes is crucial for the infection control. To minimize the effects of genetic background due to population coming from different geographic area and viral strain variability in the cohort, an analysis of all the polymorphisms associated with the HLA-A, -B and -DR alleles has been performed in a cohort of children with a monophyletic HIV-1 infection (CRF02_AG) during an outbreak in Libya. Methods: High-resolution HLA-typing has been performed in 58 children infected with a monophyletic strain of HIV-1: 26 Long-Term Non-Progressors (LTNP), 9 Slow-Progressors (SP) and 23 Fast-Progressors (FP). HLA amino acid polymorphism frequency has been compared in the in FP respect to LTNP. Results: HLA-B resulted the most interesting locus of the study; 10 positions located in B- and F-pocket for peptidebinding have been found significant after Bonferroni’s correction: 11S (LTNP=7.69% FP=34.78% OR=0.156 P

To analyze the clinical relevance of AZT resistance mutations in AZT-naive patients, 56 HIV-1 seropositive patients treated for 18 months with stavudine/lamivudine (27 patients) or AZT/lamivudine (29 patients) were studied. AZT-like resistance mutations were found in 13 out of 29 (44%) patients treated with AZT/lamivudine and in 11 out of 27 (40%) patients treated with stavudine/lamivudine. No stavudine or multi-drug resistance mutations were detected. After 26 months of treatment more than 60% of patients showed a virological failure. Among 10 patients failing treatment with stavudine/lamivudine, 9 had AZT-like resistance mutations. The phenotypic test, performed on HIV-1 strains isolated from six of these nine patients, showed a resistance to AZT in five isolates and to stavudine in two isolates. The genotypic pattern of the latter two isolates showed the combined mutations M184V plus R211K and L214F. AZT-like resistance mutations in AZT-naive patients seem to correlate with a virological failure during long-term stavudine therapy. J. Med. Virol. 65:631–636, 2001. © 2001 Wiley-Liss, Inc.

TAFs are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. TAFII60/70 was studied in yeast, Drosophila and humans: in the present work, we analyzed the homologue PwTAFII70 in Pleurodeles. The gene is expressed in ovarian oocytes and throughout development, and the level of expression decreases in late embryos. The transcripts are localized in the animal hemisphere of the fertilized eggs and in the animal blastomeres of embryos at cleavage; later PwTAFII70 mRNA is expressed in the neural plate and folds. TAFII70 protein, which is present in fertilized eggs and throughout development, progressively shows a lower level of expression starting from the neurula stage.

In this study we have examined the phenotypic and functional properties of circulating gamma delta T cells in patients with Guillain Barre syndrome (GBS), in normal healthy controls, and in patients with active multiple sclerosis (MS). Cells expressing the Vdelta2 T cell receptor showed elevated expression of the C-lectin receptor NKRP1A in both GBS and MS, suggestive of an activated state. However, in patients with GBS these cells failed to respond to pyrenil-pyrophosphate derivatives and Vdelta2 + T cell clones derived from these patients released lower levels of IFNgamma than Vdelta2 + clones derived from controls and MS patients. In contrast, in patients with GBS the Vdelta1 + subset was expanded, showed elevated expression of NKRPIA and Vdelta1 + clones derived from these patients secreted high levels of IL-4. Our findings of expanded NKRP-1A +, IL-4-producing Vdelta1 T cells in the GBS patients suggests the possibility that these cells are activated by the recognition of non-protein antigens in an MHC-unrestricted manner and contribute to the humoral response to glycolipids that is a hallmark of this disease.

To evaluate the decay rate of cellular proviral HIV-DNA and viral replication in patients receiving highly active antiretroviral therapy (HAART) in the very early phase of infection.Thirty-four patients treated with HAART and retrospectively selected for progressive decline of plasma viraemia up to undetectable levels (20 copies/ml), were stratified according to CD4+ cell count and plasma viraemia at base line:500 x 10(6) cells/l with5000 copies/ml (group 1) or with5000 copies/ml (group 2),5000 copies/ml with 300-500 x 10(6) cells/l (group 3) or with300 x 10(6) cells/l (group 4). Plasma HIV-RNA and proviral HIV-DNA were analysed at baseline and after 1, 2, 3, 6, 9 and 12 months of treatment.After 1 year of treatment, a significant decrease of proviral DNA titre was observed in all patients and a decrease1 log was achieved in 24 of 29 subjects of the first three groups. The more pronounced decay of HIV-DNA (half-life 28 weeks) up to50 HIV-DNA copies/10(6) CD4+ cells was detected in patients of group 1. At the year's endpoint, five patients (four in group 1 and one in group 2) had20 HIV-DNA copies. However, HIV strains sensitive to antiretroviral drugs were isolated from peripheral lymphocytes of 16 out of 34 patients.In patients with undetectable plasma viraemia after 1 year of HAART, the highest reduction of proviral DNA up to50 copies/10(6) CD4+ cells was obtained only in subjects in the early asymptomatic phase of infection. Nevertheless, a replication-competent virus can be detected in all phases of antiretroviral therapy.

Two repetitive DNA families were detected in the genomes of Palaearctic green water frogs. The first family, named RrS1, is a centromeric satellite DNA, which allows discrimination between the genomes of Rana ridibunda and Rana lessonae, thence representing a useful molecular tool to determine timing and modes of genome exclusion from the germ line cells of the hybrid R. es‐culenta. The second repetitive family, named Rana/Pol III, consists of short, tandemly arrayed sequences, scattered throughout the genomes, and resembling SINE retroposons in their structure. The Rana/Pol III family is present in the genomes of R. lessonae, R. ridibunda, and their hybrid R. esculenta, as well as in R. shqiperica, R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian species R. perezi, although less abundant, but appear to be lacking in the North African species R. saharica. The distribution of Rana/Pol III elements in the genomes of Palaearctic green water frogs is...

Studies of host responses to infection have traditionally focused on the direct antimicrobial activity of effector molecules (antibodies, complement, defensins, reactive oxygen and nitrogen intermediates) and immunocytes (macrophages, lymphocytes, and neutrophils among others). The discovery of the systems for programmed cell death of eukaryotic cells has revealed a unique role for this process in the complex interplay between microorganisms and their cellular targets or responding immunocytes. In particular, cells of the monocyte/macrophage lineage have been demonstrated to undergo apoptosis following intracellular infection with certain pathogens that are otherwise capable of surviving within the hostile environment of the phagosome or which can escape the phagosome. Mycobacterium tuberculosis is a prototypical 'intracellular parasite' of macrophages, and the direct induction of macrophage apoptosis by this organism has recently been reported from several laboratories. This paper reviews the current understanding of the mechanism and regulation of macrophage apoptosis in response to M. tuberculosis and examines the role this process plays in protective immunity and microbial virulence.

Developmental toxicity of chromium(III), aluminium(III) and cadmium(II) were evaluated by examining abnormalities and mortality in embryos belonging to different species of amphibians. Cr(III) and Al(III) are lethal at 1.5 mM concentration, and seriously affect the differentiation of central nervous system, skeleton and eye, and cause cephalic and trunk oedemas at lower concentrations, being aluminium significantly more harmful than chromium. Cd(II), tested only in P. wall, is highly toxic: embryos exposed to concentrations ranging from 0.18 to 50 μM display malformations, delay and arrest of development in a dose dependent manner.

A cDNA encoding a protein (B24) belonging to the Mcm/P1 family was isolated from the newt Triturus carnifex. In eukaryotes, the members of the Mcm/P1 family are essential factors in the DNA replication process. B24 protein (TcMcm3) is present in salamandrid ovarian oocytes and early embryos; its role was tested by injecting specific anti-B24 monoclonal antibodies into the cytoplasm of one blastomere of two-cell stage embryos. The injected blastomere encountered cleavage arrest either soon after the injection or following one or two divisions; later, it degenerated. Instead, the uninjected blastomere went on developing and organizing a hemi-embryo, which does not grow beyond the tailbud stage. These results are consistent with the hypothesis that the B24 protein is involved in DNA replication at cleavage. The B24 protein studied here appears to play a specific role in early development; other variants of the Mcm3 group seem to be employed by different adult tissues.

THE aim of this study was to investigate whether Mycobacterium tuberculosis (MTB) infection affects human immunodeficiency virus (HIV) replication in T67 human astrocytoma cells and whether HIV and MTB infections induce iNOS mRNA expression in T67 cells. T67 cells were susceptible to both HIV Lai and MTB infections, and MTB was able to increase HIV infection in T67 cells, as demonstrated by a marked increase in p24 release. Furthermore, both HIV and MTB infections strongly induced inducible nitric oxide synthase (iNOS) mRNA expression, as verified by RT-PCR. These findings suggest that HIV and MTB-induced iNOS expression of astroglial cells may be involved in the neuronal damage associated with HIV infection, particularly in the presence of opportunistic infections such as tuberculosis.

Tuberculosis (TB) is still a major health problem, both as a single disease entity and as a cofactor in AIDS. The interaction between macrophage and Mycobacterium tuberculosis (MTB) is a critical step in the establishment of an early chronic infection. This study analyses the capacity of MTB to induce apoptosis in cells obtained by broncho-alveolar lavage (BAL) from patients with reactive pulmonary tuberculosis and from AIDS patients with disseminated pulmonary tuberculosis. Apoptosis was increased three-fold in BAL cells obtained from patients with pulmonary tuberculosis and even more markedly in alveolar macrophages of MTB-infected AIDS patients, compared with controls. Apoptosis was analysed and characterized by propidium iodide (PI) incorporation, terminal deoxy transferase (TDT)-mediated dUTP-biotin nick end labelling (TUNEL), and tissue transglutaminase (tTG) expression. The MTB–macrophage interaction was also investigated in vitro by infecting monocyte-derived macrophages (MDM) with MTB (virulent strain H37Rv). The induction of apoptosis by MTB required viable bacteria, was dose-dependent, and was restricted to H37Rv. Infection with either Mycobacterium avium complex (MAC) or HIV-1 and treatment with heat-killed MTB failed to induce apoptosis. © 1997 by John Wiley & Sons, Ltd.

This study investigates the effects of cysteamine alone and in association with zidovudine or didanosine on the replication of human immunodeficiency virus type 1 (HIV-1). More than 90% viral inhibition was obtained by 200 microM cysteamine in lymphocytes and 100 microM cysteamine in macrophages against 4 primary isolates and 2 laboratory strains of HIV-1. Polymerase chain reaction analysis demonstrated that cysteamine interferes with early steps of HIV-1 replication, before proviral DNA formation. The use of cysteamine in conjunction with zidovudine or didanosine brought about an additive antiviral effect without concomitant increases in toxicity. The concentrations of cysteamine that are effective against HIV-1 in vitro have been well tolerated over long periods by patients under treatment for cystinosis, an inherited disorder. These observations suggest that cysteamine alone or in combination with zidovudine or didanosine could be a new potential treatment of HIV-1 infection.

In sub-Saharan Africa, newborns and children continue to suffer from insufficient access to early diagnosis and antiretroviral (ARV) treatments. A survey had been conducted in Burkina Faso, Ghana and Ivory Coast, from January 2010 to February 2011 to identify the major challenges regarding HIV prophylaxis and treatment of children in western Africa. The results of this survey highlight that only a small proportion of HIV-exposed newborns receive ARV prophylaxis. However, this problem is often not perceived at the national level. The problem could be faced by improving the communication process between the peripheral health services and the national procurement system. Moreover, supporting the development of local pharmaceutical industries could facilitate the availability of child-sized drugs, contextualized to the socio-cultural needs of such area, adequate not only in terms of efficacy, safety and tolerability, but also in terms of palatability, storage, distribution and cost.

The frequencies of HLA-A, HLA-B and HLA-DRB1 alleles in 118 unrelated Libyans from Benghazi (Cyrenaica) were analysed using high resolution typing and compared with other populations. Their relatedness has been tested by correspondence analyses and principal component analysis. The most frequent HLA-A alleles were A ∗ 02:01:01:01 (15.7%), A ∗ 01:01:01:01 (11.4%) and A ∗ 03:01:01:01 (9.3%). For the HLA-B locus, the commonest allele was HLA-B ∗ 50:01:01 (14.4%) followed by B ∗ 51:01:01 (9.8%) and B ∗ 08:01:01 (6.4%). For the HLA-DRB1 locus, the commonest was HLA-DRB1 ∗ 07:01:01:01 (16.9%) followed by DRB1 ∗ 03:01:01:01 (13.6%) and DRB1 ∗ 13:02:01 (9.3%). The most frequent two-locus haplotypes were HLA-A ∗ 02:01:01:01-B ∗ 07:02:01 (3.0%) and HLA-B ∗ 50:01:01-DRB1 ∗ 07:01:01:01 (9.6%), and three-locus haplotypes were HLA-A ∗ 02:01:01:01-B ∗ 50:01:01-DRB1 ∗ 07:01:01:01 (4.2%) and HLA-A ∗ 11:01:01-B ∗ 52:01:01:01-DRB1 ∗ 15:02:01 (2.5%). This study is the first on the HLA status of a Libyan population. The results, when compared to similar HLA data obtained previously from African and Mediterranean populations, indicate genetic influences from several ethnic groups. Moreover, the differences in the HLA allele frequencies between the Libyan population and others reveals that significant admixture has occurred between the original Berber inhabitants and neighbouring and more distant populations, even though a strong genetic Berber substratum remains. These data will be of value to future anthropological and disease association studies involving the Libyan population.

Vγ9Vδ2 T lymphocytes have been shown to respond to a variety of non-peptide antigens including alkylamines and phosphoantigens. Recently, aminobisphosphonates have also been shown to stimulate this subset of γδ+ T cells. In this study we analyzed the proliferative responses of freshly isolated γδ T lymphocytes obtained from human cord blood when challenged with pyrophosphomonoesters or aminobisphosphonates. Nitrogen-containing aminobisphopsphonates, in contrast to phoshoantigens, readily stimulated expansion of Vδ2Vγ9 cells in human cord blood. Expanded cells displayed an activated mature phenotype, and were capable of producing TNFα and IFNγ but not perforin following secondary stimulation, consistent with the development of a regulatory, as opposed to cytotoxic, phenotype. This approach may provide a useful strategy for a new approach to the treatment of neonatal pathologies.

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4(+) T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be gamma delta T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well gamma delta T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of gamma delta T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.

Mitotic chromosomes of the European water frogs Rana ridibunda and Rana lessonae, the parental species of Rana esculenta, differ significantly in their centromeric regions: when C-banded or when made fluorescent, the centromeres of R. ridibunda (and of the ridibunda chromosomes in R. esculenta) are visible as a conspicuous dark granule or as a conspicuous fluorescent spot; the centromeres of R. lessonae (and of the lessonae chromosomes in R. esculenta) are inconspicuous or not fluorescent. Lampbrush chromosomes of these three taxa are described in detail for the first time; those of R. ridibunda and R. lessonae differ significantly in morphostructural characters such as conspicuousness of centromeres and number, form, and location of giant loops as well as in chiasma frequency. Chromosomes of the two parental species can thus be distinguished when present in lampbrush complements of hybrids. Reproduction in both sexes of natural R. esculenta lineages is hemiclonal: only the unrecombined genome of one parental species, usually R. ridibunda, is transmitted to haploid gametes (hybridogenesis). In 18 hybrids from natural populations of Poland, somatic tissues had allodiploid complements with chromosomes from each parental species. In contrast, spermatocytes I of five males and oocytes I of seven of eight females (221 of 222 oocytes) were autodiploid and contained only R. ridibunda chromosomes that formed n bivalents. These 12 hybrids thus were hybridogenetic. A single female hybrid had oocytes I (33 of 34) with genomes of both parental species; they showed various disturbances including tetraploidy, reduced number of chiasmata, and incomplete synapsis resulting in univalents. This individual thus was not hybridogenetic. The irregular lampbrush patterns indicate that such hybrids will have severely reduced fertility and most of their successful gametes will result in allotriploid progeny.

Atopic dermatitis (AD), a chronic inflammatory skin disease, frequently associated with respiratory allergy, is one of the most common skin disorders observed in children. The prevalence of AD and other allergic diseases is increasing in industrialized countries, representing a major burden on health care cost. AD has been proposed as an "entry point" for subsequent allergic diseases, suggesting the possibility that effective management of AD could prevent the development of respiratory allergy or at least reduce the severity of asthma and allergic rhinitis. AD and asthma share a common genetic and pathogenic basis, and several longitudinal studies provided evidence for the atopic march from AD to allergic rhinitis and asthma. However, because only a few prospective studies starting at children's births and having a sufficiently long follow-up have been developed, little is known about the natural course of AD and the potential succession of atopic phenotypes in childhood. Finally, recent genetic and epidemiological data raised the question whether AD may either develop to asthma or be part of a syndrome consisting of both diseases.

European water frog hybrids Rana esculenta (R. ridibunda×R. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.

Germline cell fate decisions are primarily controlled at the post-transcriptional level with DEAD-box RNA helicases playing a crucial role in germline development. In this study, we report the identification of two DEAD-box vasa/PL10 orthologues (RlVlg and RlPL10) in a species complex of the genus Rana, characterized by hybridogenetic reproduction, an enigmatic process that involves the exclusion of an individual genome, and endoreduplication events. Both genes were expressed during the early stages of gametogenesis of R. ridibunda, R. lessonae, and their natural hybrid R. esculenta. RlVlg expression was germline specific. On the other hand, RlPL10 was also expressed in somatic tissues, although only at low levels. The two genes were expressed in different phases of mitotic and meiotic spermatogenetic divisions as demonstrated by immunostaining with an anti-H3 phosphohistone antibody. The data indicate that RlVlg and RlPL10 may represent useful markers for dissecting the molecular aspects of genome exclusion and endoreduplication of the hybridogenetic gametogenesis.

Mycobacterium tuberculosis (MTB) is a monocyte/macrophage (M/M) parasite, which has developed several mechanisms to survive and multiply intracellularly. On the other hand, infected cells are engaged in the effort to reduce mycobacterial viability. On this ground, we report that MTB infection predisposes M/M to a pro-apoptotic ATP-based signalling, which is aimed at decreasing MTB replication. In fact, we show that mycobacterial infection leads to an increased expression of P2X(7) purinergic receptors, which is paralleled by intracellular accumulation and subsequent extracellular release of ATP by infected macrophages. Activation of this signal is conceived to induce apoptosis in MTB-infected cells, since blocking P2X(7) receptor by means of oxidized ATP (oATP) prevents MTB induced cell death. Finally, we show that an ATP stimulation of MTB-infected M/M, besides increasing cellular apoptosis, strongly enhances intracellular MTB killing, as evaluated through Colony Forming Unit assay, and such effect is subverted through oATP pulsing of infected cells. Taken together, our data indicate a role of P2X(7) purinergic receptors in MTB-induced M/M apoptosis, suggesting the existence of an autocrine/paracrine loop leading to apoptosis of infected M/M and the feasible protective role of ATP-triggered cell death in tuberculosis.

While fetal liver is a major hematopoietic organ, normal adult liver provides a suitable microenvironment for a variety of immune cells and, in several pathological conditions, may become a site of extramedullary hematopoiesis. The direct influence of hepatocytes on hematopoietic cell differentiation is poorly understood. We have previously reported that the Met murine hepatocyte (MMH) untransformed hepatocytic lines retain several morphological and functional features of hepatocytes in vivo and are able to support the survival, self-renewal, and differentiation of hematopoietic precursors in a cell-cell contact system. Here we report the effects of soluble factors released by MMH lines on bone marrow-derived cells. Lymphohematopoietic cells were cultured in two different cell contact-free systems: transwell inserts on MMH feeder layers, and MMH conditioned medium (MMH-CM). Both culture systems were able to promote a substantial expansion of bone marrow-derived cells and their differentiation to natural killer (NK) cells that express the NK1.1 and U5A2-13 markers. Purified hematopoietic stem cells (Sca-1+Lin-), either plated as a bulk population or as single cells, were also able to differentiate into NK cells, when cultured in MMH-CM; thus, soluble factors secreted by MMH lines promote the expansion and differentiation of NK precursor cells. MMH-CM-derived NK cells are functionally active; stimulation by interleukin (IL)-12 together with IL-18 was required to induce interferon-gamma (IFNgamma) expression and to enhance their cytotoxic activity. In conclusion, our findings may imply a direct role of hepatocytes in NK cell development, and the system we have used may provide a tool for studying the molecular mechanisms of NK cell differentiation.

In the green frog hybrids there exists an exclusive cytogenetic reproductive mechanism, referred to as hybridogenesis, whose evolutionary value is still under debate. As a contribution to the knowledge of the possible link between reproductive strategies and phylogenetic relationships, the analysis of repetitive DNA elements in the genomes of the Balkan green frogs, Rana epeirotica and R. shqiperica was carried out to compare their molecular DNA organization with those already known for R. ridibunda and R. lessonae. The Balkan species were chosen, because they give rise to spontaneous, although infrequent and not hybridogenetic hybrids with R. ridibunda. The elements of the RrSl family, a centromeric satellite DNA previously isolated in R. ridibunda, are clustered at centromeric regions also in both Balkan species, although the molecular organization of the RrSl sequences proved to be most similar in R. ridibunda and R. epeirotica. Furthermore, a repetitive DNA family (R1BamHI) is interspersed th...

In humans, the circulating pool of mycobacteria-reactive Vγ9Vδ2+ T cells is expanded with age and may contribute to Mycobacterium tuberculosis immunosurveillance. We observed that two subsets of Vγ9Vδ2+ T cells could be identified on the basis of CD27 expression in immunocompetent adults, showing that functionally differentiated γδ T cells have lost CD27 expression. In contrast, the CD27−CD45RA−Vγ9Vδ2+ T cell subset of effector cells was absent in cord blood cells from healthy newborns and lacking in the peripheral blood from HIV-infected patients. Moreover, circulating Vγ9Vδ2+ T cell effectors were significantly reduced in patients with acute pulmonary tuberculosis, resulting in a reduced frequency of IFN-γ-producing cells after stimulation with nonpeptidic mycobacterial ligands. These observations indicate that monitoring and boosting γδ T cell effectors could be clinically relevant both in immunocompromised hosts and during active tuberculosis disease.

The localization of the TATA-binding protein (TBP) associated factor II70 (TAFII70) in the germinal vesicle (GV) of newt oocytes was investigated. In spreads of GV content, anti-hTAFII70 monoclonal antibody (mAb) stained Cajal bodies (CBs) that were either attached to specific sites on the lampbrush chromosomes or free in the nucleoplasm. To confirm this localization the PwTAFII70 cDNA was cloned and myc-tagged transcripts injected into the oocyte cytoplasm. Newly translated PwTAFII70 protein was detected a few hours later in the Cajal bodies. These data support the hypothesis that Cajal bodies are the assembly sites of the transcription machinery of the oocyte nucleus. TAFII70 protein can play a role in lampbrush transcription; alternatively TAFII70 can be considered a component in the subset of TFIID complexes that do not function during oogenesis, but are accumulated in the oocyte for later use during early development.Key words: TAFII70, Cajal body, lampbrush chromosomes, RNA transcription and processing, newts, Pleurodeles.

The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.

Better understanding of the mechanisms of proinflammatory cytokine production during human immunodeficiency virus (HIV) type 1 infection is of pivotal importance. The effect of HIV-1 infection on recombinant CD40 ligand (CD40L)-induced interleukin (IL)-1beta and IL-6 production by human macrophages was analyzed. ELISA and cytofluorometric analysis demonstrated that CD40L stimulation of HIV-1-infected macrophages resulted in substantial production of IL-1beta and IL-6. In contrast, no cytokine response was observed in uninfected cells. No modulation of the receptor for CD40 was found to account for the enhanced response to CD40L. The CD40L effect was not due to lipopolysaccharide contamination and was completely abrogated by preincubation with a monoclonal anti-CD40L antibody. mRNA studies indicated that the priming effect of HIV-1 on the macrophage response to CD40L was regulated at the transcriptional level. Finally, the effect of HIV-1 on the cytokine response could not be abolished by the HIV-1 protease inhibitor U75875 at concentrations that completely suppressed HIV-1 replication.

Vgamma9Vdelta2 T lymphocytes are broadly reactive against various intracellular pathogens and display both lytic and proliferative responses to human immunodeficiency virus (HIV)-infected cells. HIV infection of peripheral blood mononuclear cell cultures led to absolute increases in Vgamma9Vdelta2 T cells accompanied by decreased p24 levels. Strong gammadelta T cell activation with nonpeptidic mycobacterial phosphoantigens (TUBAg1 extract or synthetic isopentenyl pyrophosphate) resulted in potent inhibition of HIV replication through soluble released factors. Subsequent analyses showed that phosphoantigen-activated gammadelta T cells produced substantial amounts of beta-chemokines (macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, and regulated-on-activation, normal T-cell-expressed and -secreted beta-chemokine [RANTES]), which represent the natural ligand for the CCR5 HIV coreceptor. Accordingly, anti-beta-chemokine antibodies neutralized the inhibition of monocytotropic HIV strains by gammadelta T cell-released factors. Moreover, a T-tropic HIV strain using the CXCR4 coreceptor for virus entry was potently inhibited. Together, these data reveal that phosphoantigen-activated gammadelta T cells are an important source of CC chemokines and may suppress HIV replication through cell-released antiviral factors.

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.

Historically, water quality studies have been directed toward obtaining physical and chemical measurements on toxicants occurring in the aquatic environment. At present, bioassays are increasingly used as sensitive indicators of pollutant toxicity, since they are rapid, inexpensive, applicable to a variety of toxicants and allow several acute and chronic endpoints to be assessed simultaneously. The analysis of the potential toxicity of heavy metals was conducted on two aquatic model systems, an amphibian, Pleurodeles waltl, and a freshwater planarian, Dugesia etrusca. Aluminum, chromium and cadmium were chosen since human activity has led to a sharp increase of these metals in the environment. Environmentally realistic and supra-environmental concentrations were assayed in short-term toxicity tests. The effects of these metal ions were evaluated by the analysis of both malformations and lethality in amphibian embryos and alterations to planarian regeneration. Aluminum (0.15, 0.75 and 1.5 mmol 1-1) and Cr (0.75 and 1.5 mmol 1-1) were toxic to P. waltl development and both the ions affected planarian regeneration at concentrations ranging from 0.25 to 1.5 mmol 1-1. Cadmium was highly toxic to amphibian embryos at concentrations ranging from 0.18 to 50 μmol 1-1. This metal ion was also highly toxic to planarians, but it did not significantly affect the regeneration process. The results indicate that bioassays with these two organisms are useful to assess the toxic potential of aquatic pollutants.

Objectives: In order to evaluate the role played by cytokine profile as a co-factor involved in the resistance to HIV infection in couples serodiscordant for HIV, we studied HIV-seronegative subjects with multiple unprotected sexual exposures. Design and methods: Twenty-one HIV-exposed seronegative subjects (HEPS), their 21 HIV-seropositive partners and 10 HIV-seronegative unexposed individuals were studied for T helper (Th) types 1-2 cell pattern and CCR5 receptor. Results: Twelve out of 21 HIV-seropositive partners of HEPS showed a CD4 cell count below 200 lymphocytes/mu l. HIV strains were isolated from peripheral blood mononuclear cells (PBMC) in 17 patients (81%): seven subjects with syncytium-inducing strains and 10 with non-syncytium-inducing isolates. Low Th1 cytokine production and high levels of IL-4 were detected in HIV-seropositive subjects. A significant reduction of IL-2 and IFN-gamma expression in the CD4 and CD8 cells of HEPS was found compared with HIV-seronegative unexposed individuals. Similar levels of low IL-4 were present in both HEPS and controls. The partial deletion of a single allele (wild type/Delta 32) of CCR5 was found in only one HEPS. Conclusion: The downregulated Th1 profile we observed in HEPS could be related to a cellular anergy state with a protective role in the transmission rate of HIV. Low levels of IL-2 and IFN-gamma could be involved in a low-grade activation state of CD4 lymphocytes. A decrease of IFN-gamma levels could render macrophage cells incapable of antigen presentation, thus resulting in a reduction of the cell-to-cell spread of Introduction infection. (C) 1999 Lippincott Williams & Wilkins.

Nuclei of amphibian oocytes contain spheres, either attached at specific loci of lampbrush chromosomes or free in the nucleo‐plasm, the structure and function of which have been investigated for long time. These spheres were initially shown to contain components involved in pre‐mRNA splicing, pre‐rRNA processing and histone pre‐mRNA 3’ end formation. Later, the spheres were labelled by B24/3 monoclonal antibody, which recognizes a maternal protein of 104 kDa. Recently, a cDNA clone encoding protein B24 has been isolated in Triturus carnifex and analysis of the nu‐cleotide sequence has shown that B24 protein belongs to the Mcm/P1 family, which is thought to play an important and conserved role in initiation of DNA replication. To test the function of B24 protein microinjections of B24/3 mAb were made into one blastomere of two and four‐cell stage embryos. The injected cell degenerated and only hemi‐embryos or underdeveloped embryos were obtained, because B24 protein plays an important role in cell...

Abundant natural interspecies hybrids between the European water frog Rana ridibunda and at least three other taxa reproduce hemiclonally, by hybridogenesis: the non-ridi- bunda genome is excluded in the germ line before meiosis, and the unrecombined ridibunda ge- nome is transmitted to haploid gametes. In contrast, natural hybrids between Rana ridibunda and either of two Balkan species (Rana shqiperica and Rana epeirotica) do not show such genome exclusion. This plausibly results from failure of Balkan Rana ridibunda genomes to "induce" such exclusion in the germ line of hybrids, from "resistance" of Rana shqiperica and Rana epeirotica genomes to such exclusion in hybrids with an "inducing" Rana ridibunda genome, or both. We tested the second hypothesis by examining lampbrush chromosome patterns in oocytes of hybrids that in the soma contain one "inducing" ridibunda genome and one genome of either of the two Balkan species. Several lampbrush chromosome markers (e.g., presence and location of certain giant loops and conspicuousness and width of centromeres) discriminate sets of Rana ridibunda chromosomes from those of Rana shqiperica and Rana epeirotica. Based on such markers, nine diploid female hybrids between Rana ridibunda or Rana esculenta from natural hybridogenetic lineages (Rana ridibunda ◊ Rana lessonae, making ridibunda gametes) from central Poland and either Rana shqiperica or Rana epeirotica each contained both parental genomes in primary oo- cytes; the bivalents showed reduced numbers of chiasmata compared with parental species. It follows that none of these hybrids was hybridogenetic. This conclusion is confirmed, for two hy- brids between Rana epeirotica and either Rana ridibunda or Rana esculenta, by protein electro- phoretic comparison of somatic tissues with primary oocytes, all of which evidenced allelic markers of both parental species. Because Rana ridibunda genomes that are known to induce germ line genome exclusion when combined in hybrids with Rana lessonae genomes were used, these data provide the first compelling evidence for resistance of Rana shqiperica as well as Rana epeirotica genomes to such exclusion. J. Exp. Zool. 279:163-176, 1997. © 1997 Wiley-Liss, Inc.

Several phenotypic and functional changes of monocytes (M phi) have been described in HIV-1+ subjects and AIDS patients. Some of these changes that are pertinent for immunopathogenesis of the disease may be induced by HIV-1 envelope glycoprotein 120 (gp120). In the present study the effect of recombinant full length gp120 (FLgp120) and its two fragments: rp120cd (aa 410-511) and rp120 (aa 446-511) on the expression of the surface molecules of M phi cultured in vitro was determined. The FLgp120 and rp120cd caused upregulation of CD14 and CD44. The rp120cd peptide significantly increased the expression of CD16 (Fc gamma receptor type III) and TNF receptor type II. In contrast, the rp120 downregulated HLA-DR, CD64 (Fc gamma RI), interferon gamma receptor and induced IL-10 production by M phi. This study indicates that gp120 molecule and its fragments may induce several phenotypic changes of M phi in particular the increased proportion of CD14+CD16+ cells that is observed in the blood of AIDS patients. These results provide further evidence for variable response of M phi to gp120 which may explain the variability of phenotypic changes and heterogeneity of M phi subsets seen in HIV-1 disease.

Hybrid water frogs Rana esculenta reproduce by hybridogenesis: one parental genome (of Rana lessonae) is excluded in the germ line, the other (of Rana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher in Rana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed in Rana ridibunda genomes by the restriction endonuclease Stul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1-5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes all Rana ridibunda chromosomes. RrS1 represents about 2.5% of the genome of Rana ridibunda; it may represent as little as 0.2% of the genome of Rana lessonae, and cannot be detected in Xenopus laevis frogs or Salamandra salamandra and Triturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.