Nongenomic inhibition of catecholamine secretion by 17beta-estradiol in PC12 cells

We investigated the effects of 17beta-estradiol, an estrogen, on [(3)H]norepinephrine ([(3)H]NE) secretion in PC12 cells. Pretreatment with 17beta-estradiol reduced 70 mM K(+)-induced [(3)H]NE secretion in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) of 2 +/- 1 microM. The 70 mM K(+)-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) rise was also reduced when the cells were treated with 17beta-estradiol (IC(50) = 15 +/- 2 microM). Studies with voltage-sensitive calcium channel (VSCC) antagonists such as nifedipine and omega-conotoxin GVIA revealed that both L- and N-type VSCCs were affected by 17beta-estradiol treatment. The 17beta-estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17beta-estradiol. 17beta-Estradiol also inhibited the ATP-induced [(3)H]NE secretion and [Ca(2+)](i) rise. In PC12 cells, the ATP-induced [Ca(2+)](i) rise is known to occur through P2X(2) receptors, the P2Y(2)-mediated phospholipase C (PLC) pathway, and VSCCs. 17beta-Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP-induced [Ca(2+)](i) rise. Our results suggest that 17beta-estradiol inhibits catecholamine secretion by inhibiting L- and N-type Ca(2+) channels and P2X(2) receptors in a nongenomic manner.     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Production and characterization of two monoclonal antibodies against human myeloperoxidase

Two monoclonal antibodies against human myeloperoxidase, designated 3-2H3 and 4-2C11, were produced and characterized. Both bound to the native enzyme, but neither bound to the denatured enzyme or to its two denatured subunits. 4-2C11 bound to the three types of leukocyte myeloperoxidase, I, II, and III, as well as to the four types of myeloid leukemia HL-60 cell myeloperoxidase, IA, IB, II, and III. 3-2H3 did not bind to enzyme IB, but bound to the other types of leukocyte and HL-60 cell enzymes. On incubation with myeloperoxidase III, 4-2C11 inhibited the enzyme activity, but 3-2H3 did not. Both antibodies belong to the IgG1 subclass.     

The combination of berberine and irradiation enhances anti‐cancer effects via activation of p38 MAPK pathway and ROS generation in human hepatoma cells

Berberine, an isoquinoline plant alkaloid, has been known to generate a wide variety of biochemical and pharmacological effects. In order to elucidate the molecular mechanism for the berberine‐induced enhancement of radio‐sensitization, the human hepatoma HepG2 cells were treated with berberine combined with irradiation. The anti‐tumor effect of gamma radiation was found to be significantly enhanced by berberine. The evidences of apoptosis, such as apoptotic DNA fragmentation and annexin V staining, were observed in the cells treated with the combination of berberine and irradiation. Additionally, the levels of reactive oxygen species (ROS) and nitric oxide (NO) were apparently elevated in the combination system. The activations of p38, Bax, and caspase‐3 were also detected in the irradiated cells pretreated with berberine. The productions of ROS and annexin V staining in the cells treated with the combination of berberine and irradiation were significantly inhibited by the specific inhibitor of p38 MAPK, SB203580. The cell death induced by berberine alone or the combination of berberine and irradiation was suppressed by the anti‐oxidant, N‐acetyl cysteine (NAC). Taken together, the present results clearly indicate that the combination of berberine and gamma‐radiation enhance the anti‐cancer effects through the p38 MAPK pathway and ROS generation. J. Cell. Biochem. 107: 955–964, 2009. © 2009 Wiley‐Liss, Inc.     

Embryonic Stem Cell-Derived Extracellular Vesicles Maintain ESC Stemness by Activating FAK

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Regional distribution of the enzymes and substrates mediating the action of cAMP in the mammalian lens

Localization of adenylate cyclase activity in the outer cortical regions of the bovine lens correlates with the restriction of the Gs and Gi guanine nucleotide regulatory subunits of this enzyme to these same regions of the lens. In contrast, the major membrane substrates for cAMP-dependent protein kinase (cAMP-PK) (molecular masses of 18, 26 and 28 kDa) were identified in both the inner nuclear and the outer cortical regions of the lens. However, there were differences in the relative amounts of Pi incorporated into the 18 kDa and 28 kDa components in different lens regions. The three major membrane substrates for cAMP-PK were also phosphorylated when homogenates of lens cortex were incubated with [gamma-32P]ATP plus activators of the lens adenylate cyclase. In contrast, there was no incorporation of 32P into these substrates when homogenates of lens nucleus were used. When exogenous cAMP was added to homogenates of lens nucleus or cortex, 32P was incorporated into the membrane substrates for cAMP-PK in both regions of the lens, indicating that cAMP-PK was present in both regions. Interestingly, cAMP phosphodiesterase activity was at least 10-times greater in lens cortex than in the lens nucleus. These results indicate that while the major membrane substrates for cAMP-PK could be phosphorylated in all regions of the lens, there is a restriction of those enzymes that synthesize and degrade cAMP to the outer cortical regions of this organ.