Hydrogen peroxide-induced phospholipase D2 activation in lymphocytic leukemic L1210 cells

Extracellular hydrogen peroxide (H2O2) has been implicated in the activation of phospholipase D (PLD). However, it was still unclear how this activation occurs and what the molecular identity of the H2O2-stimulated PLD isozyme is. This study shows that H2O2 potently increases the PLD activity in mouse lymphocytic leukemic L1210 cells, which contain exclusively PLD2. In addition, H2O2 increased PLD activity only in PLD2-transfected COS-7 cells and not in PLD1-transfected cells. This suggests that PLD2 is selectively activated by H2O2. Depletion of extracellular Ca2+ with EGTA completely blocked the H2O2-induced PLD activation, indicating that Ca2+ influx is required. Moreover, pretreatment of the cells with the protein kinase C (PKC) inhibitors GF-109203X and RO-31-8220 and down-regulation of PKCalpha by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate inhibited the H2O2-stimulated PLD2 activity, which points to the involvement of PKCalpha. Based on these new findings we suggest that PLD2 activity is specifically up-regulated by H2O2 and that the H2O2-induced PLD2 activation is mediated by Ca2+ influx and PKCalpha activation.     

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.     

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.     

Ectopic expression of a cecropin transgene in the human malaria vector mosquito Anopheles gambiae (Diptera: Culicidae): effects on susceptibility to Plasmodium

Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.     

Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons

Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 microM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed approximately 60% of the LVA ICa, whereas approximately 10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega-conotoxin GVIA (omega-CgTx), an N-type ICa blocker, suppressed approximately 65% of the whole cell HVA ICa at the concentration of 1 microM. The omega-CgTx-resistant HVA ICa was sensitive to nifedipine (10 microM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by approximately 25% of the drug-free control ( approximately 70% of the omega-CgTx-resistant ICa). The combination of 10 microM nifedipine and 1 microM omega-CgTx left approximately 13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 microM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.     

Modulation of transglutaminase expression in rat skeletal muscle by induction of atrophy and endurance training.

The persistence of muscle fiber number regardless of size reduction in muscle atrophy has not yet been fully explained. For the mechanism inherent in skeletal muscle tissues for preventing cellular death, the protective function of muscle tissue through transglutaminases has been tested, since the enzyme is responsible for structural stabilization and participates in signal transduction. In the present experiment, hindlimb suspension for two weeks caused a marked muscle atrophy in Wistar female rats. Comparison of muscle weight and histological analysis showed that suspension-induced atrophy in the hindlimb was more prominent in the soleus muscle, comprised mainly of type I fiber than that in the plantaris muscle of type II fibers. The immunohistochemical analysis with antitransglutaminase C antibody (anti TGase C Ab) showed that some atrophic bundles of soleus muscle were positively reacted with the antibody. The anti-TGase C Ab-reactive substances were observed to disappear significantly after endurance exercise, indicating their characteristic atrophy-dependency. The enzymatic analysis of transglutaminase showed the increase in activity in the atrophic soleus muscle tissue, compared with that in the normal or exercise-trained muscle tissues. From these results, the expression of TGase in the atrophic muscle is suggested to be the possible marker for muscle atrophy and its expression is probably related with the protective mechanism of the muscle tissue to prevent further cellular damage in the atrophic process.     

Regulation of DNA synthesis and DNA polymerase in rat pancreas. II. Age-dependent changes in glucocorticoid control.

The effect of glucocorticoid on DNA synthesis and DNA polymerase in the rat pancreas of stable mass has been studied in order to elucidate the mode of control of DNA metabolism in the tissue whose increase in DNA content has reached a steadystate. The DNA synthesis was assessed by the pulse-labeling method using radio-active thymidine as the precursor. The polymerase activity was assayed by measuring the amount of [³H]TTP incorporated in vitro into the acid insoluble fraction in the presence of activated calf thymus DNA. In the pancreas which has ceased to accumulate DNA, the effect of glucocorticoid is monophasic: administration of glucocorticoid inhibits DNA synthesis and DNA polymerase activities, but it does not elicit the subsequent stimulation of DNA synthesis and DNA polymerase activities such as that observed in the actively growing pancreas. The absence of the stimulatory effect in the older animals is an age-dependent alteration in the tissue-response to the steroid observed for the first time.