Polymorphism of human thyroxine binding serum globulin (TBG); existence of a new form of serum protein without detectable triiodothyronine binding power

Two kinds of TBG polymorphism are described in human, one found in deglycosylated TBG from individual blood donors, the other is a genetically determined polymorphism. TBG from plasma samples from a patient with toxic goiter, not autoimmune, (p)TBG, from the patient's mother (m)TBG and from individual donors (n)TBG, were labeled with [125I]T4 or [125I]T3 and submitted to isoelectric focusing (IEF), followed by autoradiography. Three faint [125I]T4 radiolabeled bands were detectable in (p)TBG while four strong [125I]T4 radiolabeled bands were detectable in (m)TBG and (n)TBG), respectively. IEF of the [125I]T3 incubated serum samples resulted in no detectable isoelectric radiolabeled band for (p)TBG while a normal pattern was found in (m)TBG and in (n)TBG, respectively. These data suggest a new intraindividual not linked to sexual chromosome X polymorphism characterized by a loss in hormone binding.     

Pathological Implications of Th1/Th2 Cytokine Genetic Variants in Behçet’s Disease: Data from a Pilot Study in a Sicilian Population

Cytokines act as pleiotropic polypeptides able to regulate inflammatory/immune responses and to provide important signals in physiological and pathological processes. Several cytokines (Th1, Th2, and Th17) seem to be involved in the pathophysiology of Behçet’s disease, a chronic immune-mediated disease characterized by oral and genital lesions and ocular inflammation. Its individual susceptibility seems to be modulated by genetic variants in genes codifying these cytokines. Th1 and Th17 seem to be involved in the disease’s active phases, and Th2 seems to affect the development or severity of the disease; however, contrasting data are reported. In this study, some genetic variants of the Th1/Th2 cytokine genes were investigated in Sicilian patients and age- and gender-matched controls. Three very significant associations with Behçet’s disease were detected, and combined genotypes associated with increased disease risk were identified. Results obtained point to the key role of Th1/Th2 cytokine genetic variants in disease susceptibility.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.     

A genomic timescale for the origin of eukaryotes

Background  

Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes.     

Wavelet change-point prediction of transmembrane proteins

MOTIVATION: A non-parametric method, based on a wavelet data-dependent threshold technique for change-point analysis, is applied to predict location and topology of helices in transmembrane proteins. A new propensity scale generated from a transmembrane helix database is proposed. RESULTS: We show that wavelet change-point performs well for smoothing hydropathy and transmembrane profiles generated using different scales. We investigate which wavelet bases and threshold functions are overall most appropriate to detect transmembrane segments. Prediction accuracy is based on the analysis of two data sets used as standard benchmarks for transmembrane prediction algorithms. The analysis of a test set of 83 proteins results in accuracy per segment equal to 98.2%; the analysis of a 48 proteins blind-test set, i.e. containing proteins not used to generate the propensity scales, results in accuracy per segment equal to 97.4%. We believe that this method can also be applied to the detection of boundaries of other patterns such as G + Cisochores and dot-plots. AVAILABILITY: The transmembrane database, TMALN and source code are available upon request from the authors.     

Evidence for simultaneous protein interactions between human Rad51 paralogs

In yeast, the Rad51-related proteins include Rad55 and Rad57, which form a heterodimer that interacts with Rad51. Five human Rad51 paralogs have been identified (XRCC2, XRCC3, Rad51B/Rad51L1, Rad51C/Rad51L2, and Rad51D/Rad51L3), and each interacts with one or more of the others. Previously we reported that HsRad51 interacts with XRCC3, and Rad51C interacts with XRCC3, Rad51B, and HsRad51. Here we report that in the yeast two-hybrid system, Rad51D interacts with XRCC2 and Rad51C. No other interactions, including self-interactions, were found, indicating that the observed interactions are specific. The yeast Rad51 interacts with human Rad51 and XRCC3, suggesting Rad51 conservation since the human yeast divergence. Data from yeast three-hybrid experiments indicate that a number of the pairs of interactions between human Rad51 paralogs can occur simultaneously. For example, Rad51B expression enhances the binding of Rad51C to XRCC3 and to HsRad51D, and Rad51C expression allows the indirect interaction of Rad51B with Rad51D. Experiments using 6xHis-tagged proteins in the baculovirus system confirm several of our yeast results, including Rad51B interaction with Rad51D only when Rad51C is simultaneously expressed and Rad51C interaction with XRCC2 only when Rad51D is present. These results suggest that these proteins may participate in one complex or multiple smaller ones.     

Protein kinase C contributes to desensitization of ANG II signaling in adult rat cardiac fibroblasts

We have studiedG_q-linked ANG II signaling [inositol phosphate (IP)accumulation, Ca²⁺ mobilization] in primary cultures ofrat cardiac fibroblasts (CFs) and have found that ANG II initiates aprotein kinase C (PKC)-mediated negative feedback loop that rapidlyterminates the ANG II response. Pharmacological inhibition of PKC bystaurosporine and GF-109203X doubled IP production over that achievedin response to ANG II alone. Inhibition of PKC also led to largerCa²⁺ transients in response to ANG II, suggesting thatCa²⁺ mobilization was proportional toG_q-phospholipase C-IP₃ activity underthe conditions studied. Depletion of cellular PKC by overnight treatment with phorbol 12-myristate 13-acetate (PMA) similarly augmented ANG II-induced IP production. Acute activation of PKC by PMAhalved IP formation, with an EC₅₀1 nM; 4-PMA wasinactive. Time course data demonstrated that ANG II-mediated IPproduction fully desensitized within 30 s; PKC inhibition reducedthe rate and extent of this desensitization. In cells desensitized toANG II, a purinergic agonist still mobilized intracellularCa²⁺, indicating that desensitization was homologous. TheANG II-induced Ca²⁺ signal was fully resensitized within 30 min. The data demonstrate that a large portion of theIP-Ca²⁺ responses of rat CFs to ANG II are short-livedbecause of rapid, PKC-mediated desensitization.