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Human β-defensins are a family of cationic peptides that share a pattern of six conserved cysteine residues. We describe the cloning and characterization of the cDNAs of five novel β-defensin genes ( DEFB25–DEFB29 ) clustered on chromosome 20p13, which were identified using a bioinformatics approach. Expression analysis revealed the occurrence of the transcripts in only a few organs, with the highest abundance in the male genital tract. Examination of β-defensin expression in human epididymis by real-time quantitative RT-PCR revealed a distribution along the functionally different segments of the epididymal duct. In situ hybridization for one of the cDNAs shows mRNA restriction to the epithelial cell layer of the epididymis, known to secrete factors responsible for sperm maturation . We suggest that the novel peptides carry out physiological functions in the male genital tract that may not be directly related to bacterial growth inhibition in host defense.

The mammalian period (Per) genes, which are components of the circadian clock, are mainly regulated via an autoregulatory feedback loop. Here we provide evidence that human Per1 (hPER1) reporter gene activity shows circadian rhythmicity in a human neuroblastoma, but not in a astrocytoma or a hepatoma cell line. Medium change and various pharmacological stimuli differentially induce this behavior. This circadian oscillation was strongly dampened and could be followed over maximally three cycles. It was even possible to phase-shift the course of this oscillation by repeated application of stimuli.

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test. peerReviewed

Background Prion diseases are fatal neurodegenerative disorders whose pathogenesis mechanisms are not fully understood. In this context, the analysis of gene expression alterations occurring in prion-infected animals represents a powerful tool that may contribute to unravel the molecular basis of prion diseases and therefore discover novel potential targets for diagnosis and therapeutics. Here we present the first large-scale transcriptional profiling of brains from BSE-infected cynomolgus macaques, which are an excellent model for human prion disorders. Results The study was conducted using the GeneChip® Rhesus Macaque Genome Array and revealed 300 transcripts with expression changes greater than twofold. Among these, the bioinformatics analysis identified 86 genes with known functions, most of which are involved in cellular development, cell death and survival, lipid homeostasis, and acute phase response signaling. RT-qPCR was performed on selected gene transcripts in order to validate the differential expression in infected animals versus controls. The results obtained with the microarray technology were confirmed and a gene signature was identified. In brief, HBB and HBA2 were down-regulated in infected macaques, whereas TTR, APOC1 and SERPINA3 were up-regulated. Conclusions Some genes involved in oxygen or lipid transport and in innate immunity were found to be dysregulated in prion infected macaques. These genes are known to be involved in other neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Our results may facilitate the identification of potential disease biomarkers for many neurodegenerative diseases. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-434) contains supplementary material, which is available to authorized users.

Recent evidence indicates that regulatory small non-coding RNAs are not only components of eukaryotic cells and vesicles, but also reside within a number of different viruses including retroviral particles. Using ultra-deep sequencing we have comprehensively analyzed the content of simian immunodeficiency virions (SIV), which were compared to mock-control preparations. Our analysis revealed that more than 428,000 sequence reads matched the SIV mac239 genome sequence. Among these we could identify 12 virus-derived small RNAs (vsRNAs) that were highly abundant. Beside known retrovirus-enriched small RNAs, like 7SL-RNA, tRNALys3 and tRNALys isoacceptors, we also identified defined fragments derived from small ILF3/NF90-associated RNA snaR-A14, that were enriched more than 50 fold in SIV. We also found evidence that small nucleolar RNAs U2 and U12 were underrepresented in the SIV preparation, indicating that the relative number or the content of co-isolated exosomes was changed upon infection. Our comprehensive atlas of SIV-incorporated small RNAs provides a refined picture of the composition of retrovirions, which gives novel insights into viral packaging. peerReviewed

Background Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein (PrPc) into a pathogenic isoform (PrPSc). It is known that neurodegeneration is often accompanied by the disturbance of cholesterol homeostasis. We have recently identified a set of genes that were upregulated after prion infection of N2a neuronal cells (Bach et al., 2009). Results We have now used ultra-deep sequencing technology to profile all microRNAs (miRNA) that could be associated with this effect in these N2a cells. Using stringent filters and normalization strategies we identified a small set of miRNAs that were up- or downregulated upon prion infection. Using bioinformatic tools we predicted whether the downregulated miRNAs could target mRNAs that have been previously identified to enhance cholesterol synthesis in these cells. Application of this joint profiling approach revealed that nine miRNAs potentially target cholesterol-related genes. Four of those miRNAs are localized in a miRNA-dense cluster on the mouse X-chromosome. Among these, twofold downregulation of mmu-miR-351 and mmu-miR-542-5p was confirmed by qRT-PCR. The same miRNAs were predicted as putative regulators of the sterol regulatory element-binding factor 2 (Srebf2), the low-density lipoprotein receptor (Ldlr) or the IPP isomerase. Conclusions The results demonstrate that joined profiling by ultra-deep sequencing is highly valuable to identify candidate miRNAs involved in prion-induced dysregulation of cholesterol homeostasis.

Background Prion diseases such as bovine spongiform encephalopathies (BSE) are transmissible neurodegenerative diseases which are presumably caused by an infectious conformational isoform of the cellular prion protein. Previous work has provided evidence that in murine prion disease the endogenous retrovirus (ERV) expression is altered in the brain. To determine if prion-induced changes in ERV expression are a general phenomenon we used a non-human primate model for prion disease. Results Cynomolgus macaques (Macaca fasicularis) were infected intracerebrally with BSE-positive brain stem material from cattle and allowed to develop prion disease. Brain tissue from the basis pontis and vermis cerebelli of the six animals and the same regions from four healthy controls were subjected to ERV expression profiling using a retrovirus-specific microarray and quantitative real-time PCR. We could show that Class I gammaretroviruses HERV-E4-1, ERV-9, and MacERV-4 increase expression in BSE-infected macaques. In a second approach, we analysed ERV-K-(HML-2) RNA and protein expression in extracts from the same cynomolgus macaques. Here we found a significant downregulation of both, the macaque ERV-K-(HML-2) Gag protein and RNA in the frontal/parietal cortex of BSE-infected macaques. Conclusions We provide evidence that dysregulation of ERVs in response to BSE-infection can be detected on both, the RNA and the protein level. To our knowledge, this is the first report on the differential expression of ERV-derived structural proteins in prion disorders. Our findings suggest that endogenous retroviruses may induce or exacerbate the pathological consequences of prion-associated neurodegeneration.

Background The cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) are innate cellular factors that inhibit replication of a number of viruses, including HIV-1. Since antiviral activity of APOBEC3 has been mainly confirmed by in vitro data, we examined their role for disease progression in the SIV/macaque model for AIDS. Results We quantified A3G and A3F mRNA in PBMC and leukocyte subsets of uninfected and SIVmac-infected rhesus macaques. Compared with uninfected animals, we found increased A3G and A3F mRNA levels in PBMC, purified CD4+ T-cells and CD14+ monocytes as well as lymph node cells from asymptomatic SIV-infected macaques. APOBEC3 mRNA levels correlated negatively with plasma viral load, and highest amounts of APOBEC3 mRNA were detected in long term non-progressors (LTNPs). During acute viremia, A3G mRNA increased in parallel with MxA, a prototype interferon-stimulated gene indicating a common regulation by the initial interferon response. This association disappeared during the asymptomatic stage. Conclusion Our findings suggest a protective effect of APOBEC3 for HIV and SIV in vivo and indicate regulation of APOBEC3 by interferon during early infection and by contribution of other, hitherto undefined factors at later disease stages. Elucidating the regulatory mechanisms leading to increased APOBEC3 mRNA levels in LTNPs could help to develop new therapies against HIV.

The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel beta-defensin DEFB123. A peptide comprising the beta-defensin core region was synthesized and used for our analysis. Like other beta-defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)-alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS-mediated MAPK induction in these cells. Protection against LPS-mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic beta-defensin DEFB123 prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of beta-defensins may include interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.

The mammalian period (Per) genes which are components of the circadian clock are mainly regulated via an autoregulatory feedback loop. Here we show that a human PER1 (hPER1) reporter gene activity is stimulated by interleukin-6 (IL-6), a member of the large cytokine gene family and an inducer of the acute phase reaction, in human hepatoma (HuH-7) cells. Our results confirm and extend the view that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters.

The mammalian period (Per) genes are components of the circadian clock and appear to be regulated via an autoregulatory feedback loop. Here we show that the human PER1 (hPER1) gene is synergistically activated by protein kinases A and C (PKA, PKC) and cAMP responsive element binding protein. Activators and inhibitors of PKA as well as PKC modulate endogenous hPER1 expression and hPER1 promoter-driven reporter gene activity in a dose-dependent manner. Our results suggest that the hPER1 promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters. This regulation of hPER1 appears to be significant for rapid adaptation to changing environmental conditions.