Proinflammatory interleukin-1 cytokines increase mesangial cell hexokinase activity and hexokinase II isoform abundance

Mesangial cell hexokinase (HK) activity is increased by a diverse array of factors that share both an association with pathological conditions and a common requirement for classic MAPK pathway activation. To better understand the relationship between glucose (Glc) metabolism and injury and to indirectly test the hypothesis that these changes constitute a general adaptive response to insult, we have sought to identify and characterize injury-associated factors that couple to mesangial cell HK regulation. Proinflammatory interleukin-1 (IL-1) cytokines activate the MAPK pathway and have known salutary effects in this cell type. We therefore examined their ability to influence mesangial cell HK activity, Glc utilization, MAPK pathway activation, and individual HK isoform abundance. IL-1beta increased HK activity in both a time- and concentration-dependent manner: activity increased maximally by approximately 50% between 12 and 24 h with an apparent EC(50) of 3 pM. IL-1alpha mimicked, but did not augment, the effects of IL-1beta. Specific IL-1 receptor antagonism and selective MAPK/ERK kinase or upstream Ras inhibition prevented these increases, whereas PKC inhibition did not. Changes in HK activity were associated with both increased Glc metabolism and selective increases in HKII isoform abundance. We conclude that IL-1 cytokines can regulate cellular Glc phosphorylating capacity via an IL-1 receptor-, Ras-, and classic MAPK pathway-mediated increase in HKII abundance. These findings suggest a novel, previously undescribed mechanism whereby metabolism may be coupled to inflammation and injury.     

Ankyrin peptide blocks falcipain-2-mediated malaria parasite release from red blood cells

Falcipain-2 (FP-2) is a dual-function protease that cleaves hemoglobin at the early trophozoite stage and erythrocyte membrane ankyrin and protein 4.1 at the late stages of parasite development. FP-2-mediated cleavage of ankyrin and protein 4.1 is postulated to cause membrane instability facilitating parasite release in vivo. To test this hypothesis, here we have determined the precise peptide sequence at the hydrolysis site of ankyrin to develop specific inhibitor(s) of FP-2. Mass spectrometric analysis of the hydrolysis products showed that FP-2-mediated cleavage of ankyrin occurred immediately after arginine 1,210. A 10-mer peptide (ankyrin peptide, AnkP) containing the cleavage site completely inhibited the FP-2 enzyme activity in vitro and abolished all of the known functions of FP-2. To determine the effect of this peptide on the growth and development of P. falciparum, the peptide was delivered into intact parasite-infected red blood cells (RBCs) via the Antennapedia homeoprotein internalization domain. Growth and maturation of trophozoites and schizonts was markedly inhibited in the presence of the fused AnkP peptide. <10% of new ring-stage parasites were detected compared with the control sample. Together, our results identify a specific peptide derived from the spectrin-binding domain of ankyrin that blocks late-stage malaria parasite development in RBCs. Confocal microscopy with FP-2-specific antibodies demonstrated the proximity of the enzyme in apposition with the RBC membrane, further corroborating the proposed function of FP-2 in the cleavage of RBC skeletal proteins.     

HLA-DRB1*0402 (DW10) transgene protects collagen-induced arthritis-susceptible H2Aq and DRB1*0401 (DW4) transgenic mice from arthritis

To investigate the role of HLA-DR4 in predisposition to arthritis, we generated transgenic mice carrying DRB1*0401 and DRB1*0402 genes. We have previously shown that DRB1*0401 molecule renders B10.RQB3 (H2A(q)) mice susceptible to porcine and human type II collagen-induced arthritis. We report that the introduction of DRB1*0402 transgene does not lead to development of arthritis in mice when they are immunized with porcine and human type II collagen. In addition, DRB1*0402 protects B10.RQB3 mice against developing arthritis with bovine type II collagen. These data show that DRB1 can modulate the disease mediated by A(q). In vivo depletion of DRB1*0402 did not lead to induction of collagen-induced arthritis in transgenic mice. In vitro cytokine analysis shows that mice protected from collagen-induced arthritis produce lower amounts of Th1 and higher levels of Th2 type cytokines upon immunization with type II collagen. Protection of mice was also related to higher apoptosis in DW10 mice as indicated by higher amounts of BclII in response to type II collagen. On the basis of our observations in HLA transgenic mice, we hypothesize that DRB1 polymorphism can modulate disease by shaping the T cell repertoire in thymus and select autoreactive T cells.     

A multidisciplinary approach to the management of hormone-refractory prostate cancer

The patient with hormone-refractory prostate cancer (HRPC) presents unique management challenges for both the urologist and the medical oncologist. Because of a lack of effective treatment options, the management of patients with HRPC has historically been palliative. Over the past 10 years, the advent of relatively efficacious chemotherapeutic regimens, particularly taxane-based chemotherapy, has resulted in a desire to treat patients with HRPC more aggressively. The complex needs of these patients have made a multidisciplinary approach, inclusive of specialists with expertise in disease processes directly affecting the patient, the optimal means of treating HRPC. An understanding of the natural history and complications of HRPC, combined with a systemic evaluative process, can allow the multidisciplinary team to comprehensively address the needs of the individual patient with HRPC.     

Antioxidants prevent UV-induced apoptosis by inhibiting mitochondrial cytochrome c release and caspase activation in Spodoptera frugiperda (Sf9) cells

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV-induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV-induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf-caspase-1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced-glutathione (GSH) on ultra violet B (UVB)-induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf-caspase-1 activation was inhibited by pre-treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.     

Effect of amino acids and vitamins on laccase production by the bird's nest fungus Cyathus bulleri

Various amino acids, their analogues and vitamins have shown stimulatory as well as inhibitory effects on laccase production by Cyathus bulleri. DL-methionine, DL-tryptophan, glycine and DL-valine stimulated laccase production, while L-cysteine monohydrochloride completely inhibited the enzyme production. Among vitamins tested biotin, riboflavin and pyridoxine hydrochloride were found to induce laccase production.     

CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis

To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8.CD4(-/-) mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8.CD8(-/-) mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8.CD8(-/-) and DQ8 mice produced rheumatoid factor. In addition, DQ8.CD8(-/-) mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4(-/-) mice. An increased frequency of CD3(+) double-negative (DN) T cells was found in DQ8.CD8(-/-) compared with DQ8.CD4(-/-) and DQ8 mice. These CD3(+) DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3(+) and CD3(+) DN T cells in DQ8.CD8(-/-) mice compared with DQ8.CD4(-/-) and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8(+) T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.     

Assessment of copper and zinc status in hair and urine of young women descendants of NIDDM parents

The concentration of copper (Cu) and zinc (Zn) in hair and urine were studied in young nonpregnant healthy women whose both parents were diagnosed for noninsulin-dependent diabetes mellitus (NIDDM descendants) and were compared with those of young healthy nonpregnant females with no family history of NIDDM or hypertension (non-NIDDM descendants) and NIDDM patients. The concentration of Zn in hair in NIDDM descendants was significantly higher than that of non-NIDDM descendants (p < 0.001) and insignificantly higher than that of NIDDM patients. The hair Cu concentrations in NIDDM descendant and patients were significantly lower than that of non-NIDDM descendants (p < 0.001). Hyperzincuria was detected in some NIDDM patients and hypocuperuria in all NIDDM descendants and patients. The data suggest that the young healthy NIDDM descendants possess high-Zn and low-Cu reserves in their bodies, and the observed perturbation appears to be associated with Cu-Zn antagonism.     

HLA-DR/DQ Transgenic, class II deficient mice as a novel model to select for HSP T cell epitopes with immunotherapeutic or preventative vaccine potential

Protective immunity against mycobacteria is dependent on antigen/MHC class II specific, CD4⁺ Th1 cells. HLA-DR3-restricted Th1 cells respond to a subset of mycobacterial antigens, including the immunodominant hsp65, and recognize a single epitope in hsp65, notably p1-20. Altered peptide ligands (APL) of p1-20 can inhibit p1-20/hsp65-induced proliferation of DR3-restricted T cells in an allele specific mannerin vitro. In order to develop a preclinical model in which p1-20 APL can be testedin vivo in the context of HLA, we have used murine class II deficient, HLA transgenic (Ab⁰) mice, in which all CD4⁺ T cells are restricted by the tg HLA molecule. BCG-immunized DR3.Ab⁰ and DQ8.Ab⁰ mice both responded well to hsp65. Furthermore, DR3.Ab⁰ mice recognized precisely the same p1-20 epitope as DR3-restricted human T cells, whereas DQ8.Ab⁰ mice responded to a different set of hsp65 peptides. This shows that (i) the same immunodominant protein and peptide epitope are recognized by T cells from DR3.Ab⁰ mice and DR3⁺ humans and (ii) indicates the major role of HLA-polymorphism in controlling the human T cell response to mycobacterial antigens. Thus, HLA-transgenic, Ab⁰ mice provide a novel, preclinical model system to analyze APL and vaccines in the context of HLA polymorphism.     

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.