Ingestion of proanthocyanidins derived from cacao inhibits diabetes-induced cataract formation in rats

Proanthocyanidins derived from cacao (CLP) have various antipathophysiological functions. We have tested whether dietary supplementation with CLP prevents cataract formation in rats with diabetes induced by streptozotocin (STZ), using histological, histochemical, and biochemical analyses. Starting at 7 days after the streptozotocin challenge, the animals were fed either a normal diet or a diet containing 0.5% w/w CLP over 10 weeks. There were no significant differences in plasma and urine glucose concentrations, plasma fructose amines, and plasma thiobarbituric reactive substances (TBARS) between the two dietary groups. Antioxidant status as assessed by measuring lipid peroxide production in plasma in response to azocompounds was lower in the STZ-rats fed control diet than in animals fed CLP. Opacity was first detected in the lenses of the control dietary group 5 weeks after STZ injection and cataracts had developed in the majority of these animals by 10 weeks. These changes were rarely seen in the STZ/CLP diet group. Histological examinations of the eyes of the STZ-treated normal diet group revealed focal hyperplasia of the lens epithelium and liquefaction of cortical fibers. There were similar but considerably less severe changes in the animals fed CLP. Hydroxynonenal (HNE), a marker of oxidative stress, was detected immunohistochemically in the lenses of the STZ-treated normal diet group, but not of those receiving CLP. Our findings suggest that CLP inhibits diabetes-induced cataract formation possibly by virtue of its antioxidative activity.     

Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety

Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied. All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure. The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog. The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity. This agreed with the result of a cross-linking assay. These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.     

Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

Production of transgenic rats by ooplasmic injection of spermatogenic cells exposed to exogenous DNA: a preliminary study

The aim of the present study was to investigate the efficiencies of producing transgenic rats by the ooplasmic injection of sperm heads (intracytoplasmic sperm injection: ICSI) and elongating spermatids (elongating spermatid injection: ELSI) exposed to the EGFP DNA solution. A slightly lower proportion of ICSI oocytes using sperm heads exposed to a concentration of 0.5 microg/ml DNA solution for 1 min developed into offspring (13.3%, 48/361) when compared to that of oocytes injected with nontreated sperm heads (19.4%, 32/165). Eight ICSI offspring were found to be EGFP-carrying transgenic rats (16.7% per offspring; 2.2% per embryo). After a 1-min exposure of the elongating spermatids to 5 microg/ml of DNA solution, 8.8% (45/511) of the ELSI oocytes developed into offspring while 12.7% (22/173) of the ELSI oocytes using nontreated spermatids developed. Six ELSI offspring carried the EGFP DNA (13.3% per offspring; 1.2% per embryo). The conventional pronuclear microinjection of 5 microg/ml of DNA solution resulted in the higher production of offspring (29.7%, 104/350) and the birth of three transgenic rats (2.9% per offspring; 0.9% per embryo). Thus, sperm heads and elongating spermatids were practically useful as the vector of exogenous DNA if the DNA-exposed spermatogenic cells were microinseminated into rat oocytes.     

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD₅₀ (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of ¹O₂. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN₃, a ¹O₂ scavenger. Based on a quenching study, the rate constant of the reaction of ¹O₂ with NaN₃ was determined to be 1.5–3.5 M^–1 s^–1, which is close to the reported value (3.8×10⁸ M^–1 s^–1). The intensity of the ¹O₂-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 s) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 s), coproporphyrin I (50 s), or coproporphyrin III (36 s). These results demonstrate the photodynamic generation of ¹O₂ by Zincphyrin.     

Asymmetric temporal properties in the receptive field of retinal transient amacrine cells

The speed of signal conduction is a factor determining the temporal properties of individual neurons and neuronal networks. We observed very different conduction velocities within the receptive field of fast-type On-Off transient amacrine cells in carp retina cells, which are tightly coupled to each other via gap junctions. The fastest speeds were found in the dorsal area of the receptive fields, on average five times faster than those detected within the ventral area. The asymmetry was similar in the On- and Off-part of the responses, thus being independent of the pathway, pointing to the existence of a functional mechanism within the recorded cells themselves. Nonetheless, the spatial decay of the graded-voltage photoresponse within the receptive field was found to be symmetrical, with the amplitude center of the receptive field being displaced to the faster side from the minimum-latency location. A sample of the orientation of varicosity-laden polyaxons in neurobiotin-injected cells supported the model, revealing that approximately 75% of these processes were directed dorsally from the origin cells. Based on these results, we modeled the velocity asymmetry and the displacement of amplitude center by adding a contribution of an asymmetric polyaxonal inhibition to the network. Due to the asymmetry in the conduction velocity, the time delay of a light response is proposed to depend on the origin of the photostimulus movement, a potentially important mechanism underlying direction selectivity within the inner retina.     

Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 micro g per 10(4) cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 x 10(6) PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.     

Redox properties of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Paracoccus denitrificans produces two primary enzymes for the amine oxidation, tryptophan-tryptophylquinone (TTQ)-containing methylamine dehydrogenase (MADH) and quinohemoprotein amine dehydrogenase (QH-AmDH). QH-AmDH has a novel cofactor, cysteine tryptophylquinone (CTQ) and two hemes c. In this work, the redox potentials of three redox centers in QH-AmDH were determined by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique. Kinetics of the electron transfer from QH-AmDH to three kinds of metalloproteins, amicyanin, cytochrome c(550), and horse heart cytochrome c were examined on the basis of the theory of mediated-bioelectrocatalysis. All these metalloproteins work as a good electron acceptor of QH-AmDH and donate the electron to the terminal oxidase of P. denitrificans, which was revealed by reconstitution of the respiratory chain. These properties are in marked contrast with those of MADH, which shows high specificity to amicyanin. These electron transfer kinetics are discussed in terms of thermodynamics and structural property.     

L-cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation

Recent studies showed that the function of some amino acids is not only nutritional but also pharmacological. However, the effects of amino acids on liver fibrosis and hepatic stellate cell (HSC) remain unclear. In this research, as a result of screening of amino acids using liver fibrosis induced by DMN administration, L-cysteine was selected as a suppressor of liver fibrosis. Furthermore, the number of activated HSCs, which increased in the fibrotic liver after DMN administration, was decreased in L-cysteine-fed rats. Treatment of freshly isolated HSCs with L-cysteine resulted in inhibition of the increase in smooth muscle alpha-actin (alphaSMA) expression by HSCs and BrdU incorporation into the activated HSCs. These findings suggest that L-cysteine is effective against liver fibrosis. The mechanism of inhibition of fibrosis in the liver is surmized to be direct inhibition of activated HSC proliferation and HSC transformation by L-cysteine.     

Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.