Cyclooxygenase 2 inhibition promotes IFN-gamma-dependent enhancement of antitumor responses

In previous studies, we demonstrated an immune suppressive network in non-small cell lung cancer that is due to overexpression of tumor cyclooxygenase 2 (COX-2). In this study, we assessed the vaccination response to tumor challenge following either pharmacological or genetic inhibition of COX-2 in a murine lung cancer model. Treatment of naive mice with the COX-2 inhibitor, SC-58236, skewed splenocytes toward a type 1 cytokine response, inducing IFN-gamma, IL-12, and IFN-gamma-inducible protein 10, whereas the type 2 cytokines IL-4, IL-5, and IL-10 remained unaltered. Fifty percent of mice receiving SC-58236 and an irradiated tumor cell vaccine completely rejected tumors upon challenge. Those mice that did form tumors following challenge demonstrated a reduced tumor growth. In contrast, all mice either vaccinated with irradiated tumor cells alone or receiving SC-58236 alone showed progressive tumor growth. Studies performed in CD4 and CD8 knockout mice revealed a requirement for the CD4 T lymphocyte subset for the complete rejection of tumors. To determine the role of host COX-2 expression on the vaccination responses, studies were performed in COX-2 gene knockout mice. Compared with control littermates, COX-2(-/-) mice showed a significant tumor growth reduction, whereas heterozygous COX-2(-/+) mice had an intermediate tumor growth reduction following vaccination. In vivo depletion of IFN-gamma abrogated the COX-2 inhibitor-mediated enhancement of the vaccination effect. These findings provide a strong rationale for additional evaluation of the capacity of COX-2 inhibitors to enhance vaccination responses against cancer.     

Iodine and IFN-gamma synergistically enhance intercellular adhesion molecule 1 expression on NOD.H2h4 mouse thyrocytes

NOD.H2(h4) mice spontaneously develop autoimmune lymphocytic thyroiditis that mimics human Hashimoto's thyroiditis, a disease where iodine, IFN-gamma, and adhesion molecules have all been implicated in the pathogenesis. To study how iodine and IFN-gamma modulate the expression of ICAM-1, we analyzed NOD.H2(h4) thyrocytes in baseline conditions (day 0) and at several time points following supplementation of iodine in the drinking water. On day 0, a small percentage ( approximately 10%) of thyrocytes constitutively expressed ICAM-1. The expression gradually increased to 13, 25, and 41% on days 7, 14 and 28, respectively, returning to baseline (9%) on day 35. The initial ICAM-1 kinetics was paralleled by thyroidal infiltration of CD45(+) hemopoietic cells, which increased from an average of 4% on day 0 to an average of 13, 21, and 24% on days 14, 28, and 35, respectively. To distinguish whether the observed ICAM-1 increase was a direct effect of iodine or a consequence of the immune infiltrate, we treated mouse primary thyrocyte cultures with 0.01 mM sodium iodine and showed a 3-fold increased ICAM-1 expression. To assess interaction between IFN-gamma and iodine, we analyzed CD45 and ICAM-1expression on thyrocytes from NOD.H2(h4) wild-type and NOD.H2(h4) thyr-IFN-gamma transgenic littermates. Strikingly, IFN-gamma interacted synergistically with iodine to enhance ICAM-1 expression on thyrocytes. These findings suggest that iodine and IFN-gamma cooperate to promote thyroidal expression of ICAM-1 in this mouse model of thyroiditis, highlighting the complex interplay present in the pathogenesis of Hashimoto's thyroiditis.     

Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice

Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA(-/-) and IgA(+/+) animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA(-/-) animals, with the exception of IgA. Lung sections of challenged IgA(-/-) mice showed more extensive immunopathology than corresponding IgA(+/+) animals. Real-time PCR analysis demonstrated significantly greater IFN-gamma and TGF-beta mRNA expression in IgA(-/-) as compared to IgA(+/+) animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA(-/-) mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inflammation and maintenance of mucosal homeostasis.     

Heterosis studies for yield and its components in bread wheat over environments

A set of diallel crosses involving 10 parents was made to have information on the extent of heterosis over mid-parent and better parent and inbreeding depression for yield and yield contributing characters under three different environments. Marked heterobeltiosis for grain yield and its important components were observed. For grain yield, 83 crosses showed significant positive heterobeltiosis in all the three sowing dates, however, twenty crosses showed significant consistent heterobeltiosis for grain yield per plant over all the three environments. The maximum heterobeltiosis for grain yield per plant observed was 50.94% (Raj 3765 x HD 2285), 121.08% (PBW 373 x HD 2329) and 93.96% (PBW 373 x HD 2329) under early, normal and late sowing conditions, respectively. Cross PBW 373 x HD 2329 in both early and normal plantings and cross Raj 3765 x HD 2285 under late planting were observed most heterotic for grain yield. The crosses showing heterosis for grain yield were not heterotic for all the characters. Heterosis for grain yield per spike followed by tillers per plant and 1000-grain weight was independently associated with heterosis for grain yield in early and normal plantings. However, heterosis for grain yield per spike, dwarf plant height and tillers per plant contributed maximum towards yield heterosis. Significant inbreeding depression was recorded frequently for yield and yield contributing traits, however, in a few traits it was observed significant negative indicated that F(2) was superior to F(1) considered desirable combination for trait(s). The study reveals good scope for commercial exploitation of heterosis as well as isolation of pure lines among the progenies of heterotic F(1) for improvement of yield levels in bread wheat.     

Effects of alpha-1 adrenergic receptor antagonist, terazosin, on cardiovascular functions in anaesthetised dogs

Initially a dose-response curve of phenylephrine was constructed at dose strengths of 1-16 microg/kg in a cumulative manner. Phenylephrine caused a significant rise in the mean arterial pressure, left ventricular systolic pressure, left ventricular contractility, stroke volume and a significant decline in the heart rate. Terazosin was administered in three selected doses of 10, 100 and 300 microg/kg. Following each dose of terazosin, dose-response curve of phenylephrine was constructed. Terazosin, per se, decreased the basal mean arterial pressure, left ventricular systolic pressure, left ventricular contractility and stroke volume significantly in a dose dependent manner with an increase in the heart rate with no significant change in the cardiac output. The baroreflex sensitivity at all the three doses remained unchanged. In conclusion, the present findings support the view that terazosin reduces the blood pressure in a physiologically more favorable manner by maintaining the neural integrity of the cardiovascular system.     

Comparison of methylprednisolone with dexamethasone in treatment of acute spinal injury in rats

Effect of methylprednisolone sodium succinate (MPSS) and its comparison with dexamethasone in experimentally induced acute spinal cord compression in adult rats was studied. The rats were divided into group A (control) and group B, which was subdivided into B1, B2, B3 where MPSS was given after 1, 8 and 24 hr and B4 where dexamethasone was given after 1 hr of cord injury respectively. Proper neurological evaluation was done with mobility, running and climbing score. Recovery index was evaluated for 7 days. After sacrificing the rats, spinal cord was observed histopathologically. Mean recovery index and microscopic findings based on hemorrhage in gray and white matter, neuronal degeneration, hematomyelia and edema in white matter were recorded. The results suggested that MPSS was effective in promoting post-traumatic clinical and histological recovery and to a greater extent, when given 1 hr after trauma. MPSS is more effective than dexamethasone in reducing edema when both are given after interval of 1 hr.     

Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators

DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.     

Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.     

Acetoxy drug: protein transacetylase of buffalo liver-characterization and mass spectrometry of the acetylated protein product

The purification and characterization of the buffalo liver microsomal transacetylase (TAase) catalyzing the transfer of acetyl groups from a model acetoxy drug: 7,8-diacetoxy-4-methylcoumarin (DAMC) to GST3-3 has been described here. The enzyme was routinely assayed using DAMC and cytosolic GST as the substrates and was partially purified from microsomes of the buffalo liver. The enzyme was found to have approximate molecular of weight 65 kDa. The action of TAase and DAMC on liver cytosolic GST resulted in the formation of monoacetoxymonohydroxy-4-methylcoumarin (MAMHC) and 7,8-dihydroxy-4-methylcoumarin (DHMC), although the former was the major metabolite. The buffalo liver microsomal TAase exhibited hyperbolic kinetics and yielded K(m) (1667 microM) and V(max) (192 units) when the concentration of DAMC was varied keeping the concentration of GST constant. After having characterized the nature of the substrates and a product of the TAase-catalyzed reaction, we set out to identify the acetylated protein which is another product of the reaction. GST3-3 was used as a model protein substrate for the action of TAase using DAMC as the acetyl donor. The subunit of control and modified GST3-3 were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and digested with trypsin. The tryptic peptides were extracted from the gel pieces and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOFMS). The data search for calibrated and labeled mass peaks of peptides was performed on the Matrix Science Server using the search engine Mascot. The peptide maps so obtained covered 97% of the GST3-3 sequence. On comparison of MALDI peptide maps of modified and control GST, seven new peaks were recognized corresponding to the potentially acetylated peptides in peptide map. The mass value of each of them was 42 Da higher than the theoretical mass of a non-modified GST3-3 tryptic peptide, strongly suggesting acetylation. By examining the fragmentation patterns and by comparing experimental and predicted values for MS/MS daughter ions, the identity of the seven acetylated GST tryptic peptides could be confirmed by the application of LC/MS/MS. In the modified GST, N-terminal proline and six lysines (Lys(51), Lys(82), Lys(123), Lsy(181), Lys(191) and Lys(210)) were found to be acetylated. The structure of acetylated GST revealed that the lysines that underwent acetylation were peripheral in positions.     

AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin

Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.