Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/ modification of nuclear proteins.     

Optimising surgical management of elderly cancer patients

BACKGROUND: Elderly population is on rise. It is an ethical dilemma how aggressive one should be when it comes to treat cancer in elderly. Presumed fear of increased postoperative morbidity and mortality has resulted in delivery of sub-optimal cancer surgery. METHODS: In this review article we visit physiology of the aged, tools available to assess surgical risks in oncogeriatric patients, and current practice in the management of common cancers encountered in surgical oncology, with the view of increasing awareness on optimising surgical management of senior patients with cancer. A pubmed search for cancer, surgery, elderly, was carried out. RESULTS: Cancer is on rise with increasing age predominantly affecting breast, gastrointestinal tract and lung. Increasingly more surgeons are offering surgery to elderly cancer patient but selection bias is prevalent. Available data reflect short and long-term outcome of cancer surgery in elderly is not greatly different to that of younger patient. Declining physiological reserve along with inability to respond adequately to physiological stress are salient age related changes. Comprehensive Geriatric Assessment (CGA) is not tested in surgical patient. There is need for a tool to define individualised operative risk. Preoperative assessment of cancer in elderly is designed to offer this information based on functional status of an individual utilising currently available tools of risk assessment. CONCLUSION: All elderly cancer patients should be offered optimal treatment depending on their functional status not on chronological age. Oncogeriatric patient would benefit from dedicated multidisciplinary approach. Recruitment of elderly cancer patients to more clinical trials is needed to enhance our knowledge and to offer optimum treatment to this unique subgroup.     

Microvascular PO2 during extreme hemodilution with hemoglobin site specifically PEGylated at Cys-93(beta) in hamster window chamber

The oxygen transport capacity of nonhypertensive polyethylene glycol (PEG)-conjugated hemoglobin solutions were investigated in the hamster chamber window model. Microvascular measurements were made to determine oxygen delivery in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Two isovolemic hemodilution steps were performed with a 6% Dextran 70 (70-kDa molecular mass) plasma expander until Hct was 35% of control. Isovolemic blood volume exchange was continued using two surface-modified PEGylated hemoglobins (P5K2, P(50) = 8.6, and P10K2, P(50) = 8.3; P(50) is the hemoglobin Po(2) corresponding to its 50% oxygen saturation) until Hct was 11%. P5K2 and P10K2 are PEG-conjugated hemoglobins that maintain most of the hemoglobin allosteric properties and have a cooperativity index of n = 2.2. The effects of these molecular solutions were compared with those obtained in a previous study using MP4, a PEG-modified hemoglobin whose P(50) was 5.4 and cooperativity was 1.2 (Tsai et al., Am J Physiol Heart Circ Physiol 285: H1411-H1419, 2003). Tissue oxygen levels were higher after P5K2 (7.0 +/- 2.5 mmHg) and P10K2 (6.3 +/- 2.3 mmHg) versus MP4 (1.7 +/- 0.5 mmHg) or the nonoxygen carrier Dextran 70 (1.3 +/- 1.2 mmHg). Microvascular oxygen delivery was higher after P5K2 and P10K2 (2.22 and 2.34 ml O(2)/dl blood) compared with MP4 (1.41 ml O(2)/dl blood) or Dextran 70 (0.90 ml O(2)/dl blood); however, all these values were lower than control (7.42 ml O(2)/dl blood). The total hemoglobin in blood was similar in all cases; therefore, the improvement in tissue Po(2) and oxygen delivery appears to be due to the increased cooperativity of the new molecules.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Selective amidation of carboxyl groups of the intermolecular contact regions of hemoglobin S: Structural aspects

Carboxyl groups of HbS are readily activated by water-soluble carbodiimide atpH 6.0 and room temperature. These o-acylurea intermediates (activated carboxyl) are accessible for nucleophilic attack by amines. With glycine ethyl ester, the amidation is very selective for the -carboxyl of Glu-43() and more than 65% of the glycine ethyl ester incorporated is on this carboxyl group. In contrast, glucosamine derivatizes the -carboxyl group of Glu-22() as well as that of Glu-43() to nearly the same degree. However, the total amidation of HbS by glucosamine is lower than that with glycine ethyl ester. The differential selectivity of the two amines is apparently related to the differences in the microenvironment of the -carboxyl groups of Glu-22() and Glu-43(), which either facilitates or refracts the aminolysis of the activated carboxyl with the two amines to different degrees. The carboxyl groups of isolated -chain exhibit a higher reactivity for amidation with glycine ethyl ester than does the tetramer. The carboxyl groups of Glu-22() and Glu-43() and that of Asp-47() are all activated by carbodiimide suggesting that the higherpK_a of these carboxyl groups (facilitating the activation) is a property of tertiary interaction of the polypeptide chain. The interaction of the -chain with -chain, i.e., generation of the quaternary interactions, reduces overall reactivity of the carboxyl groups of the protein. The higher selectivity of hemoglobin S for amidation at Glu-43() with glycine ethyl ester compared with that of isolated -chain appears to be primarily a consequence of decreased amidation at sites other than at Glu-43().     

Stimulation of glycine catabolism in isolated perfused rat liver by calcium mobilizing hormones and in isolated rat liver mitochondria by submicromolar concentrations of calcium.

Glucagon stimulates flux through the glycine cleavage system (GCS) in isolated rat hepatocytes (Jois, M., Hall, B., Fewer, K., and Brosnan, J. T. (1989) J. Biol. Chem. 264, 3347-3351. In the present study, flux through GCS was measured in isolated rat liver perfused with 100 nM glucagon, 1 microM epinephrine, 1 microM norepinephrine, 10 microM phenylephrine, or 100 nM vasopressin. These hormones increased flux through GCS in perfused rat liver by 100-200% above the basal rate. The possibility that the stimulation of flux by adrenergic agonists and vasopressin is mediated by increases in cytoplasmic Ca2+ which in turn could regulate mitochondrial glycine catabolism was examined by measuring flux through GCS in isolated mitochondria in the presence of 0.04-2.88 microM free Ca2+. Flux through GCS in isolated mitochondria was exquisitely sensitive to free Ca2+ in the medium; half-maximal stimulation occurred at about 0.4 microM free Ca2+ and maximal stimulation (7-fold) was reached when the free Ca2+ in the medium was 1 microM. The Vmax (nanomoles/mg protein/min) and Km (millimolar) values for the flux through GCS in intact mitochondria were 0.67 +/- 0.16 and 20.66 +/- 4.82 in the presence of 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and 3.28 +/- 0.76 and 10.98 +/- 1.91 in presence of 0.5 microM free Ca2+, respectively. The results show that the flux through GCS is sensitive to concentrations of calcium which would be achieved in the cytoplasm of hepatocytes stimulated by calcium-mobilizing hormones.     

Standardised Extract of Bacopa monniera (CDRI-08) Improves Contextual Fear Memory by Differentially Regulating the Activity of Histone Acetylation and Protein Phosphatases (PP1α, PP2A) in Hippocampus

Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT_3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15–29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus.     

Proteomic analysis of human osteoarthritis synovial fluid

Background

Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.

Results

We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.

Conclusions

We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.     

In vitro culture methods in sorghum with shoot tip as the explant material

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.