Molecular basis of protective effect by crocetin on survival and liver tissue damage following hemorrhagic shock

Hemorrhagic shock (HS) causes reduction of cellular energy stores, as measured by levels of ATP and ADP. Furthermore, energy depletion may cause mitochondrial damage, which in turn leads to cell death by apoptosis. The hypothesis of the present study is that by enhancing the recovery of cellular ATP and ADP and mitochondrial damage can be reduced, and the extent of apoptosis minimized. Crocetin, a carotenoid compound, appears to enhance the diffusion of oxygen in aqueous solution, and hence may improve energy stores both to the cell and within it. HS was produced in Sprague–Dawley rats by withdrawing blood from the carotid cannula until a mean arterial pressure of 35–40 mm Hg was reached, and then maintained by further withdrawals of blood for 30 and 60 min. Crocetin was administered 2–4 mg/kg in resuscitation fluid through venus cannula and the animals survived for 24–48 h after HS. Experiments designed to promote tissue reconstitution of ATP using crocetin indicate that these approaches are successful in increasing ATP post-hemorrhage and survival. Crocetin treatment also inhibited cellular damage as indicated by increase of Bcl-2 following decrease in cytosolic cytochrome c and caspase-3 after resuscitation. The prolonged energy deficit seen after hemorrhagic shock can produce late damage and rapid restoration of ATP levels to baseline can reduce apoptosis. In conclusions, crocetin can minimize the cellular damage as evidenced by apoptosis and increased the survival of rats. (Mol Cell Biochem 278: 139–146, 2005)     

Structural studies of the biomineralized species of calcified pancreatic stones in patients suffering from chronic pancreatitis

The pancreatic stones (Pancreatic calculi) collected from patients suffering from chronic calcific pancreatitis were studied in a view to explore the constituents involved in the calcification. The calcified stones were characterized by XRD, EPR and IR spectroscopic techniques. The detailed studies indicate that these stones consist of calcium carbonate as a major component, besides phosphates and other protein content. The presence of aragonite phases in the biomineralized stones is also discussed. The EPR spectra gave an evidence of the presence of traces of manganese in different oxidation states, which is used as one of the EPR probes in the present work. The samples were sintered at different temperatures to remove all the organic matter, and their EPR spectra have been studied to obtain detailed information regarding the changes in the symmetry of these stone samples. The X-irradiated sample was also characterized by EPR and the resonance signals are attributed to freely rotating CO(2)(-) radicals. The infrared spectrum reveals the presence of many organic bands corresponding to the protein amides.     

Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.     

LKB1, a protein kinase regulating cell proliferation and polarity

LKB1 is a serine-threonine protein kinase mutated in patients with an autosomal dominantly inherited cancer syndrome predisposing to multiple benign and malignant tumours, termed Peutz-Jeghers syndrome. Since its discovery in 1998, much research has focused on identification and characterisation of its cellular roles and analysing how LKB1 might be regulated. In this review we discuss exciting recent advances indicating that LKB1 functions as a tumour suppressor perhaps by controlling cell polarity. We also outline the current understanding of the molecular mechanisms by which LKB1 is regulated in vivo, through interaction with other proteins as well as by protein phosphorylation and prenylation.     

Heat and osmotic stress responses of probiotic Lactobacillus rhamnosus HN001 (DR20) in relation to viability after drying

The viability of lactic acid bacteria in frozen, freeze-dried, and air-dried forms is of significant commercial interest to both the dairy and food industries. In this study we observed that when prestressed with either heat (50 degrees C) or salt (0.6 M NaCl), Lactobacillus rhamnosus HN001 (also known as DR20) showed significant (P < 0.05) improvement in viability compared with the nonstressed control culture after storage at 30 degrees C in the dried form. To investigate the mechanisms underlying this stress-related viability improvement in L. rhamnosus HN001, we analyzed protein synthesis in cultures subjected to different growth stages and stress conditions, using two-dimensional gel electrophoresis and N-terminal sequencing. Several proteins were up- or down-regulated after either heat or osmotic shock treatments. Eleven proteins were positively identified, including the classical heat shock proteins GroEL and DnaK and the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, enolase, phosphoglycerate kinase, and triose phosphate isomerase, as well as tagatose 1,6-diphosphate aldolase of the tagatose pathway. The phosphocarrier protein HPr (histidine-containing proteins) was up-regulated in cultures after the log phase irrespective of the stress treatments used. The relative synthesis of an ABC transport-related protein was also up-regulated after shock treatments. Carbohydrate analysis of cytoplasmic contents showed higher levels (20 +/- 3 microg/mg of protein) in cell extracts (CFEs) derived from osmotically stressed cells than in the unstressed control (15 +/- 3 microg/mg of protein). Liquid chromatography of these crude carbohydrate extracts showed significantly different profiles. Electrospray mass spectrometry analysis of CFEs revealed, in addition to normal mono-, di-, tri-, and tetrasaccharides, the presence of saccharides modified with glycerol.     

An organism-specific method to rank predicted coding regions in Trypanosoma brucei

Genome annotation in differently evolved organisms presents challenges because the lack of sequence-based homology limits the ability to determine the function of putative coding regions. To provide an alternative to annotation by sequence homology, we developed a method that takes advantage of unusual trypanosomatid biology and skews in nucleotide composition between coding regions and upstream regions to rank putative open reading frames based on the likelihood of coding. The method is 93% accurate when tested on known genes. We have applied our method to the full complement of open reading frames on Chromosome I of Trypanosoma brucei, and we can predict with high confidence that 226 putative coding regions are likely to be functional. Methods such as the one described here for discriminating true coding regions are critical for genome annotation when other sources of evidence for function are limited.     

Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.     

Cholesterol alters the interaction of glycosphingolipid GM3 with alpha5beta1 integrin and increases integrin-mediated cell adhesion to fibronectin

Integrins bind to their ligand in the extracellular matrix (ECM), such as fibronectin (FN), through a specific interaction between the amino acid motifs in the ligand, and binding sites in the extracellular domains of the integrin molecule generated jointly by its alpha and beta subunits. It has been proposed that membrane cholesterol and glycosphingolipids (GSLs) can regulate integrin-ECM interactions and it has been demonstrated that increased membrane cholesterol leads to increased cell adhesion to FN. Here, we have shown that a specific glycosphingolipid GM3 binds directly to alpha5beta1 integrin and an increase in membrane cholesterol results in the redistribution of GM3-associated alpha5beta1 integrin molecules specifically on the surface that is in contact with the substratum. Our results suggest that GM3-associated alpha5beta1 integrins bind less avidly to FN than GM3-free integrins and that cholesterol and GM3 play an interdependent role in the distribution of alpha5beta1integrin molecules in the membrane and regulation of cell adhesion.     

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.