The eukaryote chaperonin CCT is a cold shock protein in Saccharomyces cerevisiae

The eukaryotic Hsp60 cytoplasmic chaperonin CCT (chaperonin containing the T-complex polypeptide-1) is essential for growth in budding yeast, and mutations in individual CCT subunits have been shown to affect assembly of tubulin and actin. The present research focused mainly on the expression of the CCT subunits, CCTalpha and CCTbeta, in yeast (Saccharomyces cerevisiae). Previous studies showed that, unlike most other chaperones, CCT in yeast does not undergo induction following heat shock. In this study, messenger ribonucleic acid (mRNA) and protein levels of CCT subunits following exposure to low temperatures, were examined. The Northern blot analysis indicated a 3- to 4-fold increase in mRNA levels of CCTalpha and CCTbeta genes after cold shock at 4 degrees C. Interestingly, Western blot analysis showed that cold shock induces an increase in the CCTalpha protein, which is expressed at 10 degrees C, but not at 4 degrees C. Transfer of 4 degrees C cold-shocked cells to 10 degrees C induced a 5-fold increase in the CCTalpha protein level. By means of fluorescent immunostaining and confocal microscopy, we found CCTalpha to be localized in the cortex and the cell cytoplasm of S. cerevisiae. Localization of CCTalpha was not affected at low temperatures. Co-localization of CCT and filaments of actin and tubulin was not observed by microscopy. The induction pattern of the CCTalpha protein suggests that expression of the chaperonin may be primarily important during the recovery from low temperatures and the transition to growth at higher temperatures, as found for other Hsps during the recovery phase from heat shock.     

Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5'-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25 degrees C to 2 degrees C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP-aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.     

Evaluation of a mass screening program for lysinuric protein intolerance in the northern part of Japan

Lysinuric protein intolerance (LPI:MIM 222700) is an autosomal recessive disease characterized by defective transport of the dibasic amino acids. We recently reported a local cluster of LPI in the northern part of Japan (Koizumi et al., 2000). Mutational analysis of the LPI patients in this local cluster revealed they were exclusively homozygous for the R410X mutation. The effectiveness of early intervention with citrulline therapy (200 mg/kg per day) and protein restriction (1.5 g/kg per day) was confirmed in these patients. Mass screening was conducted in 4,568 newborn babies between 1999 and 2002, which was estimated to cover 100% of almost all newborns delivered in the screened area. Forty heterozygous newborns were found (0.88%), leading to an estimated incidence of LPI of 1:51,984. The number of people that required screening to detect one case was 51,984, and the cost for mass screening was 30 cents/person (a total of dollars 15,600). This is comparable to, or even less than, the cost of currently screened diseases in Japan. Therefore, we conclude that a mass screening program for LPI can be introduced effectively and economically into an area where an LPI cluster is located as the result of a founder mutation.     

Configurations of polyunsaturated sesterterpenoids from the diatom, Haslea ostrearia

The partial configurations of C25 isoprenoid alkenes isolated from the diatom Haslea ostrearia Gaillon (Simonsen) have been established. A combination of NMR spectroscopy studies of the alkenes with chiral shift reagents in conjunction with soluble silver beta-diketonate complexes and enantioselective gas chromatography of oxidation products of the alkenes was used. Unexpected differences in highly branched isoprenoid isomer configurations were observed between different laboratory cultures of the alga.     

The Middle Subunit of Replication Protein A Contacts Growing RNA-DNA Primers in Replicating Simian Virus 40 Chromosomes

The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.     

Expression of Multiple Sexual Signals by Fathers and Sons in the East-Mediterranean Barn Swallow: Are Advertising Strategies Heritable?

The level of expression of sexually selected traits is generally determined by genes, environment and their interaction. In species that use multiple sexual signals which may be costly to produce, investing in the expression of one sexual signal may limit the expression of the other, favoring the evolution of a strategy for resource allocation among signals. As a result, even when the expression of sexual signals is condition dependent, the relative level of expression of each signal may be heritable. We tested this hypothesis in the East-Mediterranean barn swallow (Hirundo rustica transitiva), in which males have been shown to express two uncorrelated sexual signals: red-brown ventral coloration, and long tail streamers. We show that variation in both signals may partially be explained by age, as well as by paternal origin (genetic father-son regressions), but that the strongest similarity between fathers and sons is the relative allocation towards one trait or the other (relative expression index), rather than the expression of the traits themselves. These results suggest that the expression of one signal is not independent of the other, and that genetic strategies for resource allocation among sexual signals may be selected for during the evolution of multiple sexual signals.     

Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis

The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.     

A DJ-1 Based Peptide Attenuates Dopaminergic Degeneration in Mice Models of Parkinson's Disease via Enhancing Nrf2

Drugs currently used for treating Parkinson''s disease patients provide symptomatic relief without altering the neurodegenerative process. Our aim was to examine the possibility of using DJ-1 (PARK7), as a novel therapeutic target for Parkinson''s disease. We designed a short peptide, named ND-13. This peptide consists of a 13 amino acids segment of the DJ-1-protein attached to 7 amino acids derived from TAT, a cell penetrating protein. We examined the effects of ND-13 using in vitro and in vivo experimental models of Parkinson''s disease. We demonstrated that ND-13 protects cultured cells against oxidative and neurotoxic insults, reduced reactive oxygen species accumulation, activated the protective erythroid-2 related factor 2 system and increased cell survival. ND-13 robustly attenuated dopaminergic system dysfunction and in improved the behavioral outcome in the 6-hydroxydopamine mouse model of Parkinson''s disease, both in wild type and in DJ-1 knockout mice. Moreover, ND-13 restored dopamine content in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model. These findings validate DJ-1 as a promising therapeutic target in Parkinson''s disease and identify a novel peptide with clinical potential, which may be significant for a broader range of neurological diseases, possibly with an important impact for the neurosciences.     

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described. The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency against cruzipain. Further SAR in P2 portion led to the identification of compounds, such as 26, that have a unique selectivity profile against other cysteine proteases and offering new opportunities for safer treatment of Chagas disease.