Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.     

Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.     

IS THE GROWTH RATE HYPOTHESIS APPLICABLE TO MICROALGAE?1

The growth rate hypothesis (GRH) asserts, from known biochemistry, that maintaining high growth rates requires high concentrations of ribosomes. Since ribosomes are rich in phosphorus (P), the GRH predicts a positive correlation between growth rate and P content; this correlation is observed in some organisms. We consider the application of the GRH to phytoplankton and identify several key problems that require further research before the hypothesis can be accepted for these organisms. There are severe methodological problems that confound interpretation of data for testing the GRH. These problems include the measurement of protein and nucleic acids (such that ratio of these components carries a high level of uncertainty), studies of steady‐state versus dynamic systems, and the presentation of data per cell (especially as cell size varies with growth rate limitations) and the calculation of growth rates. In addition, because of the short generation times and rapid responses of these organisms to perturbations, ribosome and RNA content is expected to vary in response to (de)repression of various systems; content may increase on application of growth‐limiting stress. Finally, that most phytoplankton accumulate P when not P stressed conflicts with the GRH. In consequence, the value of the GRH for any sort of predictive role in nature appears to be severely limited. We conclude that the GRH cannot be assumed to apply to phytoplankton taxa without first performing experimental tests under transient conditions.     

The dyslexia-associated KIAA0319 protein undergoes proteolytic processing with {gamma}-secretase-independent intramembrane cleavage

The KIAA0319 gene has been associated with reading disability in several studies. It encodes a plasma membrane protein with a large, highly glycosylated, extracellular domain. This protein is proposed to function in adhesion and attachment and thought to play an important role during neuronal migration in the developing brain. We have previously proposed that endocytosis of this protein could constitute an important mechanism to regulate its function. Here we show that KIAA0319 undergoes ectodomain shedding and intramembrane cleavage. At least five different cleavage events occur, four in the extracellular domain and one within the transmembrane domain. The ectodomain shedding processing cleaves the extracellular domain, generating several small fragments, including the N-terminal region with the Cys-rich MANEC domain. It is possible that these fragments are released to the extracellular medium and trigger cellular responses. The intramembrane cleavage releases the intracellular domain from its membrane attachment. Our results suggest that this cleavage event is not carried out by γ-secretase, the enzyme complex involved in similar processing in many other type I proteins. The soluble cytoplasmic domain of KIAA0319 is able to translocate to the nucleus, accumulating in nucleoli after overexpression. This fragment has an unknown role, although it could be involved in regulation of gene expression. The absence of DNA-interacting motifs indicates that such a function would most probably be mediated through interaction with other proteins, not by direct DNA binding. These results suggest that KIAA0319 not only has a direct role in neuronal migration but may also have additional signaling functions.     

Molecular biology of Rh proteins and relevance to molecular medicine

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.     

Small but sufficient: the Rhodococcus phage RRH1 has the smallest known Siphoviridae genome at 14.2 kilobases

Bacteriophages are considered to be the most abundant biological entities on the planet. The Siphoviridae are the most commonly encountered tailed phages and contain double-stranded DNA with an average genome size of ～50 kb. This paper describes the isolation from four different activated sludge plants of the phage RRH1, which is polyvalent, lysing five Rhodococcus species. It has a capsid diameter of only ～43 nm. Whole-genome sequencing of RRH1 revealed a novel circularly permuted DNA sequence (14,270 bp) carrying 20 putative open reading frames. The genome has a modular arrangement, as reported for those of most Siphoviridae phages, but appears to encode only structural proteins and carry a single lysis gene. All genes are transcribed in the same direction. RRH1 has the smallest genome yet of any described functional Siphoviridae phage. We demonstrate that lytic phage can be recovered from transforming naked DNA into its host bacterium, thus making it a potentially useful model for studying gene function in phages.     

Mitochondrial genetic background plays a role in increasing risk to asthma

A number of studies suggest that mitochondrial dysfunction plays a role in the pathogenesis of asthma. To shed light for the first time on the role of the mitochondrial genome in the etiology of asthma we analyzed the mitochondrial tRNA genes and part of their flanking regions in patients with asthma compared with a set of healthy controls. We found a total of 10 mutations in 56 out of 76 asthmatic patients. Four of these mutations were not found in the control group, five were observed at a significantly lower frequency in controls, but none of the combinations of mutations detected in asthma patients was observed in the controls. Furthermore, we observed that 27.6% of the asthma patients (vs. 4% of the controls) belonged to the haplogroup U (Fisher test P = 0.00) and a positive significant correlation was found between the occurrence of the haplogroup U and the severity of the disease (Fisher test P = 0.02). Whereas further studies in larger cohorts are needed to confirm these observations we suggest that the mitochondrial genetic background plays a key role in asthma development.     

CCK-B receptor gene and response to cholecystokinin-tetrapeptide in healthy volunteers

Recent investigations suggest that genes that confer risk for panic disorder (PD) may moderate response to panicogenic agents in healthy volunteers. Given the potential role of the central cholecystokinin receptor (CCKBR) (CT) polymorphism alleles 26 and 27 in PD, the present study attempted to discern if these alleles moderated panicogenic sensitivity to the CCKBR agonist, CCK-tetrapeptide (CCK-4), in healthy volunteers. The study group consisted of 92 men and women with no personal or family history of psychiatric illness. Participants provided blood samples for genotyping of the CCKBR alleles and they received a 25 μg bolus injection of CCK-4. Behavioral, cardiovascular and hormonal responses to the peptide were assessed and analyzed with adjusted linear regression models. Carriers of the CCKBR alleles tended to have higher levels of pre-challenge anxiety and significantly higher levels of anxiety sensitivity and introversion than those without the alleles. However, they did not exhibit an enhanced panicogenic response to CCK-4. Overall, our findings do not demonstrate a role of these alleles in modulating CCK-4's panicogenicity. The significant association between the risk alleles and anxiety-related personality traits is intriguing and further exploration of this association is merited.     

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.