Resveratrol inhibits AGEs-induced proliferation and collagen synthesis activity in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats

Advanced glycation end-products (AGEs) of plasma proteins and/or matrix proteins are candidate mediators for various vascular complications such as atherosclerosis. We previously reported a significantly larger accumulation of AGEs of the aorta in stroke-prone spontaneously hypertensive rats (SHRSP) than in age-matched Wistar-Kyoto rats (WKY). In this study, we examined the effects of AGEs on vascular smooth muscle cells (VSMC) from SHRSP and WKY rats. We also studied the in vitro effects of resveratrol (3, 4',5-trihydroxystilbene), a natural phytestrogen, on VSMC proliferation, DNA synthesis, and collagen synthesis activity in SHRSP-VSMC. AGEs accelerated the proliferation of SHRSP- or WKY-VSMC in a time- and dose-dependent manner. VSMC from SHRSP were more sensitive to AGEs than VSMC from normotensive WKY. AGEs also significantly increased DNA synthesis and prolyl hydroxylase activity, a marker for collagen synthesis, in SHRSP-VSMC. AGEs-induced increases in TGF-beta1 mRNA in SHRSP-VSMC were significantly greater than in WKY-VSMC. Resveratrol inhibited AGEs-stimulated proliferation, DNA synthesis, and prolyl hydroxylase activity in SHRSP-VSMC in a dose-dependent manner. ICI 182780, a specific estrogen receptor antagonist, partly blocked the inhibitory effects of resveratrol on AGEs-stimulated proliferation, DNA synthesis, and prolyl hydroxylase activity. Resveratrol significantly inhibited AGEs-induced TGF-beta1 mRNA increases in a dose-dependent manner. Thus, resveratrol may confer protective effects on the cardiovascular system by attenuating vascular remodeling and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease.     

Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1

Autotaxin (ATX) is a tumor cell motility-stimulating factor originally isolated from melanoma cell supernatant that has been implicated in regulation of invasive and metastatic properties of cancer cells. Recently, we showed that ATX is identical to lysophospholipase D, which converts lysophosphatidylcholine to a potent bioactive phospholipid mediator, lysophosphatidic acid (LPA), raising the possibility that autocrine or paracrine production of LPA by ATX contributes to tumor cell motility. Here we demonstrate that LPA and ATX mediate cell motility-stimulating activity through the LPA receptor, LPA(1). In fibroblasts isolated from lpa(1)(-/-) mice, but not from wild-type or lpa(2)(-/-), cell motility stimulated with LPA and ATX was completely absent. In the lpa(1)(-/-) cells, LPA-stimulated lamellipodia formation was markedly diminished with a concomitant decrease in Rac1 activation. LPA stimulated the motility of multiple human cancer cell lines expressing LPA(1), and the motility was attenuated by an LPA(1)-selective antagonist, Ki16425. The present study suggests that ATX and LPA(1) represent potential targets for cancer therapy.     

Nucleotide sequence variation is frequent in the mitochondrial DNA displacement loop region of individual human tumor cells

The mitochondrial DNA (mtDNA) displacement loop (D-loop) regions of 76 various tumor cell lines were examined to investigate the existence of a specific relationship between a somatic mtDNA sequence and initiation and/or progression of a tumor. Based on molecular cloning-sequencing analysis, a nucleotide sequence in the D-loop region in each cell line was found to be homoplasmic. Several site-specific nucleotide variations were found in stomach and liver tumor cell lines more frequently than those in other tumor cell lines. Subsequently, 20 pairs of noncancerous and cancerous parts from stomach and liver tumor tissues were examined. In the liver tumor tissue, 80% of the noncancerous parts exhibited slightly higher heterogeneity than the corresponding cancerous parts. Several site-specific nucleotide variations found in 76 tumor cell lines were also detected in noncancerous or cancerous parts of stomach and liver tumor tissues. However, it remains unclear why the mtDNA D-loop sequence is homoplasmic in each tumor cell line. The data indicate that mtDNA exhibits heterogeneity even in the noncancerous part and a slight decrease in heterogeneity during tumorigenesis and/or tumor progression. Homoplasmy of the mtDNA population in the tumor cell line would be acquired in the cloning process of establishing a cell line. Site-specific nucleotide substitutions might not be directly involved in the tumorigenesis process.     

Congenital abnormality of pituitary-thyroid axis in spontaneously hypertensive rats (SHR) and stroke-prone rats (SPR).

In an attempt to study whether TSH abnormality was genetically determined in SHR and SPR, plasma T4, T3, TSH and prolactin concentrations were measured in the animals with intervals of 1 to 3 months. Hypertension was found in 6-month-old SHR and SPR, but it was not found in younger animals. In contrast, a decrease of plasma T3 and an increase of plasma TSH were found in 15-day-old SHR. Also, an increase of TSH was found in 1-month-old SPR in spite of normal plasma T3 concentration. These abnormalities in SHR and SPR increased progressively with age. It is suggested that thyroid-pituitary abnormality was genetically determined in SHR and SPR.     

Production of prostacyclin-like substance in stroke-prone and stroke-resistant spontaneously hypertensive rats.

Activities of aortae to produce prostaglandin (PG) I2-like substance in stroke-prone spontaneously hypertensive rats (SHRSP), stroke-resistant SHR (SHRSR) and normotensive control rats from the Wistar-Kyoto (WK) colony were compared. PGI2-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and the amount produced was estimated by comparison of its anti-aggregatory activity with that produced by known amounts of the sodium salt of synthetic PGI2. Before the development of stroke, amounts of this substance generated in SHRSP and SHRSR were significantly higher than those in WK rats (p less than 0.01 and p less than 0.02, respectively). Remarkably reduced capacity to generate PGI2-like substance was observed in some SHRSP after the development of stroke.     

The Solar Action Spectrum of Photosystem II Damage

The production of oxygen and the supply of energy for life on earth rely on the process of photosynthesis using sunlight. Paradoxically, sunlight damages the photosynthetic machinery, primarily photosystem II (PSII), leading to photoinhibition and loss of plant performance. However, there is uncertainty about which wavelengths are most damaging to PSII under sunlight. In this work we examined this in a simple experiment where Arabidopsis (Arabidopsis thaliana) leaves were exposed to different wavelengths of sunlight by dispersing the solar radiation across the surface of the leaf via a prism. To isolate only the process of photodamage, the repair of photodamaged PSII was inhibited by infiltration of chloramphenicol into the exposed leaves. The extent of photodamage was then measured as the decrease in the maximum quantum yield of PSII using an imaging pulse amplitude modulation fluorometer. Under the experimental light conditions, photodamage to PSII occurred most strongly in regions exposed to ultraviolet (UV) or yellow light. The extent of UV photodamage under incident sunlight would be greater than we observed when one corrects for the optical efficiency of our system. Our results suggest that photodamage to PSII under sunlight is primarily associated with UV rather than photosynthetically active light wavelengths.Plants absorb sunlight to power the productive photochemical reactions of photosynthesis. Absorption of sunlight may also lead to deleterious photochemistry that damages the photosynthetic machinery. The PSII protein complex is important in this regard as it seems to be most susceptible to photodamage that results in photoinhibition and ultimately suppresses photosynthetic CO₂ assimilation, growth, and productivity (Long et al., 1994; Takahashi and Murata, 2008). Although plants have photoprotection mechanisms (Niyogi, 1999) and can effectively repair photodamaged PSII through the PSII repair cycle (Aro et al., 1993), photoinhibition still occurs under stressful environmental conditions (Nishiyama et al., 2006; Murata et al., 2007; Takahashi and Murata, 2008).The onset of photoinhibition is strongly correlated with the absorption of excessive excitation energy for photosynthesis. Therefore, photodamage to PSII was most readily assumed to be attributed to the excess light absorbed by photosynthetic pigments (Melis, 1999). However, the extent of photodamage that is measured under conditions where the repair of photodamaged PSII is prevented by inhibiting chloroplast protein synthesis (i.e. lincomycin or chloramphenicol) is directly proportional to the intensity of light (Mattoo et al., 1984; Tyystjärvi and Aro, 1996; Nishiyama et al., 2001, 2004; Allakhverdiev and Murata, 2004; Chow et al., 2005). Furthermore, recent studies have demonstrated that interruption of the Calvin cycle (Hakala et al., 2005; Takahashi and Murata, 2005; Takahashi et al., 2007) and inhibition of electron transfer between Q_A and Q_B (Jegerschöld et al., 1990; Kirilovsky et al., 1994; Allakhverdiev et al., 2005) have no effect on the rate of photodamage to PSII, but in fact cause inhibition of the repair of photodamaged PSII due to suppression of the de novo synthesis of PSII proteins (Allakhverdiev et al., 2005; Takahashi and Murata, 2005, 2006). Thus, photodamage to PSII is paradoxically not associated with the excess light absorbed by photosynthetic pigments (Nishiyama et al., 2006; Murata et al., 2007; Takahashi and Murata, 2008).Studies of the effect of monochromatic light on the photodamage process have suggested that photodamage to PSII primarily occurs at the manganese cluster of the oxygen-evolving complex (OEC) through a direct photoexcitation of manganese (Hakala et al., 2005; Ohnishi et al., 2005). Release of manganese ions (Mn²⁺) from thylakoid membranes is accompanied by photodamage to PSII (Hakala et al., 2005; Zsiros et al., 2006), suggesting that disruption of the manganese cluster upon absorption of light might be a primary event in photodamage. It is likely that the reaction center of PSII is secondarily damaged by light absorbed by photosynthetic pigments after inactivation of the OEC (Hakala et al., 2005; Ohnishi et al., 2005), if an alternative electron transfer donor from lumenal ascorbate is not available (Mano et al., 2004; Tóth et al., 2009). These findings have lead to a recent photodamage model called the manganese (or two-step; Ohnishi et al., 2005) mechanism of photoinhibition (Tyystjärvi, 2008).Studies of the action spectrum of photodamage to PSII have shown that UV damages PSII more effectively than visible light (Jones and Kok, 1966; Jung and Kim, 1990; Hakala et al., 2005; Ohnishi et al., 2005). Thus, under identical light intensity, UV is the most damaging wavelength to PSII. However, inferring damage under natural sunlight is not straight forward as there is a need to account for the spectral distribution and intensity of sunlight. It is unclear which wavelengths of sunlight are most damaging to PSII and we cannot discount the premise that significant primary photodamage to PSII is caused by light absorbed by photosynthetic pigments (Vass and Cser, 2009). To identify which wavelengths of sunlight are most damaging to PSII, sunlight was spectrally dispersed via a prism onto an Arabidopsis (Arabidopsis thaliana) leaf infiltrated with chloramphenicol and decrease in the maximum quantum yield of PSII (F_v/F_m) was measured using an imaging pulse amplitude modulation (PAM) fluorometer. This simple but powerful approach revealed the in vivo spectral dependence of photodamage that had two peaks at UV and yellow wavelengths. Since the spectral efficiency of our optical system decreased below 400 nm, we calculated photodamage to PSII under incident sunlight. Our results show that photodamage to PSII was primarily associated with UV wavelengths and secondarily with yellow light wavelengths. This finding indicates that photodamage to PSII is less associated with light absorbed by photosynthetic pigments under sunlight and suggest that most of photodamage to PSII is potentially avoidable during photosynthesis.     

Effect of cyproterone acetate on active and inactive renin secretion in patients with precocious puberty and genetic short stature.

To evaluate the effect of cyproterone acetate (CA) on the renin-angiotensin-aldosterone axis, we measured the plasma active, inactive and total renin concentrations (PARC, PIRC and PTRC) during and after CA treatment in patients with precocious puberty and genetic short stature. CA was administered at a daily dose of 150-170 mg/m2 in all subjects. PARC and PTRC were measured by immunoradiometric assays. During CA treatment, PARC, PIRC, PTRC and the PARC/PTRC ratio were significantly decreased. The plasma renin activity, measured by enzymatic assay, and the plasma aldosterone concentration were also decreased. After CA discontinuation, all of these were increased immediately along normal ranges. PARC closely correlated with plasma renin activity. These results suggest that CA produces mineralocorticoid action and suppresses the production and activation of renin.     

Taurine in health and diseases: consistent evidence from experimental and epidemiological studies

Taurine (T) was first noted as beneficial for stroke and cardiovascular diseases (CVD) prevention in genetic rat models, stroke-prone spontaneously hypertensive rats (SHRSP). The preventive mechanisms of T were ascribed to sympathetic modulation for reducing blood pressure (BP) and anti-inflammatory action. Recent epidemiological surveys revealed the involvement of inflammatory mediators in the pathogenesis of stroke and also atherosclerosis for which T was proven to be effective experimentally. Arterio-lipidosis prone rats, a substrain of SHRSP selectively bred for higher reactive hypercholesterolemia, quickly develop not only arterial fat deposition but also fatty liver which could be attenuated by dietary T supplementation. CARDIAC (CVD and Alimentary Comparison) Study was a WHO-coordinated multi-center epidemiological survey on diets and CVD risks and mortalities in 61 populations. Twenty-four-hour urinary (24U) T was inversely related significantly with coronary heart disease mortality. Higher 24U-T excreters had significantly lower body mass index, systolic and diastolic BP, total cholesterol (T-Cho), and atherogenic index (AI: T-Cho/high density lipoprotein-cholesterol) than lower T excreters. T effects on CVD risks were intensified in individuals whose 24U-T and -magnesium (M) excretions were higher. Furthermore, higher Na excreters with higher heart rate whose BP were significantly higher than those with lower heart rate were divided into two groups by the mean of 24U-T, high and low T excreters. Since the former showed significantly lower BP than the latter, T may beneficially affect salt-sensitive BP rise. Included among the typical 61 populations, were Guiyang, China or St. John’s, Newfoundland, Canada where in which the means of both 24U-T and -M were high or low, respectively. The former and the latter had low and high CVD risks, respectively. Australian Aboriginals living at the coastal area in Victoria were supposed to eat T- and M-rich bush and sea foods and be free from CVD 200 years ago, but they presently have nearly the highest CVD risks indicating that T- and/or M-containing seafood, vegetables, fruits, nuts, milk, etc, similar to prehistoric hunters’ and gatherers’ food should be good for CVD prevention. The preventive effects of T, good for health and longevity, first noted experimentally, were also proven epidemiologically in humans.     

The rate-limiting step for CO(2) assimilation at different temperatures is influenced by the leaf nitrogen content in several C(3) crop species

Effects of nitrogen (N) supply on the limiting step of CO(2) assimilation rate (A) at 380 μmol mol(-1) CO(2) concentration (A(380) ) at several leaf temperatures were studied in several crops, since N nutrition alters N allocation between photosynthetic components. Contents of leaf N, ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) increased with increasing N supply, but the cyt f/Rubisco ratio decreased. Large leaf N content was linked to a high stomatal (g(s) ) and mesophyll conductance (g(m) ), but resulted in a lower intercellular (C(i) ) and chloroplast CO(2) concentration (C(c) ) because the increase in g(s) and g(m) was insufficient to compensate for change in A(380) . The A-C(c) response was used to estimate the maximum rate of RuBP carboxylation (V(cmax) ) and chloroplast electron transport (J(max) ). The J(max) /V(cmax) ratio decreased with reductions in leaf N content, which was consistent with the results of the cyt f/Rubisco ratio. Analysis using the C(3) photosynthesis model indicated that A(380) tended to be limited by RuBP carboxylation in plants grown at low N concentration, whereas it was limited by RuBP regeneration in plants grown at high N concentration. We conclude that the limiting step of A(380) depends on leaf N content and is mainly determined by N partitioning between Rubisco and electron transport components.     

Effects of 5-aminolevulinic acid on growth and amylase activity in the radish taproot

5-Aminolevulinic acid (ALA) promotes the growth of plants by enhancing their photosynthetic activities, but there is little information on how ALA influences the metabolism of sugars produced by photosynthesis. Here, we report the effects of ALA on tissue growth, sugar content, and amylase activity in the radish taproot. 5-Aminolevulinic acid was applied with a foliar spray (5.3–13,500 μM), and application at concentrations of 53, 530, and 2,700 μM enhanced the fresh weight of the taproot. Glucose is a major soluble sugar of the radish taproot. 5-Aminolevulinic acid slightly increased the glucose content but did not influence the fructose, sucrose, or starch contents. Radishes have β-amylase (RsBAMY1), which is expressed in the taproot. 5-Aminolevulinic acid enhanced both the amylase activity and the RsBAMY1 protein accumulation. These results suggest that ALA may control starch accumulation by increasing the RsBAMY1 expression in the radish taproot. The relationship between taproot growth and free sugar accumulation by ALA is also discussed.