Combined Kinetin and Spermidine Treatments Ameliorate Growth and Photosynthetic Inhibition in Vigna angularis by Up-Regulating Antioxidant and Nitrogen Metabolism under Cadmium Stress.

Pot experiments were conducted to investigate the probable beneficial role of the individual as well as combined application of kinetin (50 µM Kn) and spermidine (200 µM Spd) on Vigna angularis under cadmium (Cd) stress. Cd treatment reduced growth by declining the content of chlorophylls and carotenoids, photosynthesis, and gas exchange parameters. Exogenously, Kn and Spd application enhanced the photosynthetic parameters and up-regulated the antioxidant system by improving the activities of antioxidant enzymes and the content of non-enzymatic components. In addition, the application of Kn and Spd resulted in significant improvement in the content of sugars, proline, and glycine betaine, ameliorating the decline in relative water content. Oxidative stress parameters including hydrogen peroxide, superoxide, lipid peroxidation, lipoxygenase activity, and electrolyte leakage increased due to Cd stress; however, the application of Kn and Spd imparted a significant decline in all these parameters. Further, reduced Cd uptake was also observed due to Kn and Spd application. Total phenols and flavonoids also increased due to Kn and Spd treatments under normal as well as Cd stress conditions, which may have further helped with the elimination of reactive oxygen species. Reduction in the activity of nitrate reductase and the content of nitrogen was ameliorated due to the exogenous application of Kn and Spd. Therefore, the exogenous application of Kn and Spd benefited Vigna angularis counteracting the damaging effects of Cd stress by up-regulating the tolerance mechanisms, including antioxidant and osmolyte metabolism.

Long-term oral kinetin does not protect against α-synuclein-induced neurodegeneration in rodent models of Parkinson's disease.

Mutations in the mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) cause Parkinson's disease (PD), likely by disrupting PINK1's kinase activity. Although the mechanism(s) underlying how this loss of activity causes degeneration remains unclear, increasing PINK1 activity may therapeutically benefit some forms of PD. However, we must first learn whether restoring PINK1 function prevents degeneration in patients harboring PINK1 mutations, or whether boosting PINK1 function can offer protection in more common causes of PD. To test these hypotheses in preclinical rodent models of PD, we used kinetin triphosphate, a small-molecule that activates both wild-type and mutant forms of PINK1, which affects mitochondrial function and protects neural cells in culture. We chronically fed kinetin, the precursor of kinetin triphosphate, to PINK1-null rats in which PINK1 was reintroduced into their midbrain, and also to rodent models overexpressing α-synuclein. The highest tolerated dose of oral kinetin increased brain levels of kinetin for up to 6 months, without adversely affecting the survival of nigrostriatal dopamine neurons. However, there was no degeneration of midbrain dopamine neurons lacking PINK1, which precluded an assessment of neuroprotection and raised questions about the robustness of the PINK1 KO rat model of PD. In two rodent models of α-synuclein-induced toxicity, boosting PINK1 activity with oral kinetin provided no protective effects. Our results suggest that oral kinetin is unlikely to protect against α-synuclein toxicity, and thus fail to provide evidence that kinetin will protect in sporadic models of PD. Kinetin may protect in cases of PINK1 deficiency, but this possibility requires a more robust PINK1 KO model that can be validated by proof-of-principle genetic correction in adult animals.

Evaluating the Transport Kinetics of a Model Compound Released From Cellulosic Coacervate Compositions Into Artificial Sebum.

Complex coacervates of cationic polymers and anionic surfactants, which are produced spontaneously during the use of rinse-off formulations, represent an important delivery vehicle for topical agents to the skin surface and appendages. In this study, an artificial sebum-loaded cell culture insert method for determining the sebum diffusion properties of topical agents was optimized for in vitro release testing. This method was subsequently used to evaluate the transport kinetics of a model compound, kinetin, released from semi-solid coacervate formulations into sebum. Coacervate compositions were prepared with cationic-hydroxyethyl cellulose dodecyl sulfate (cat-HECDS), sodium dodecyl sulfate (NaDS), and water. Tested compositions ranged from 90 to 50 wt% water and had a cat-HECDS to NaDS wt% ratio of 2:1, 1:1, or 1:2, mimicking the in vivo hydration range and relative excess surfactant content expected from commercial rinse-off formulations. Steady-state flux of the model compound from each coacervate composition was found to vary with water content of the composition. When flux was plotted versus [(cat-HECDS:NaDS) × (1 - weight fraction water)]-1, a strong linear correlation (R2 = 0.89) emerged. The in vitro release testing method proved capable of discriminating between clinically relevant differences in transport kinetics from different coacervate formulations using a practical sample size.

Over-the-counter treatments for acne and rosacea.

Acne and rosacea are common inflammatory processes historically classified in the same disease category, but evolving understanding of their disparate pathophysiology and exacerbating factors have generated an enormous armamentarium of therapeutic possibilities. Patients seek over-the-counter therapies first when managing cutaneous disease; therefore, this review defines ingredients considered to be effective over-the-counter acne and rosacea products, their mechanisms, and safe formulations, including botanical components, oral supplements, and other anecdotal options in this vast skin care domain.

The effect of cyclin-dependent kinases inhibitor treatment on experimental herpes simplex encephalitis mice.

Herpes simplex encephalitis(HSE) is the most common and serious viral encephalitis in humans. There is a lack of effective medication to date for HSE. A better understanding of the mediators of tissue damage is essential for finding new targets for therapeutic intervention. In this project, we explored the effect of cyclin-dependent kinases inhibitor olomoucine treatment on experimental HSE mice. The following results were obtained: (1) olomoucine increased survival in HSE mice; (2) olomoucine inhibited microglial activation and reduced HSV-1-induced cytokines release; (3) olomoucine prevented neural cells apoptosis and attenuated brain tissue pathological changes following HSV-1 infection; (4) olomoucine reduced brain edema and improved neurological function in HSE. Overall, olomoucine can induce a blunted inflammatory response, maintain the blood vessel wall intact, improve neurological function and increase survival in HSE mice.

Down-regulation of BNIP3 by olomoucine, a CDK inhibitor, reduces LPS- and NO-induced cell death in BV2 microglial cells.

Proinflammatory responses eliciting the microglial production of cytokines and nitric oxide (NO) have been reported to play a crucial role in the acute and chronic pathogenic effects of neurodegeneration. Chemical inhibitors of cyclin-dependent kinases (CDKs) may prevent the progression of neurodegeneration by both limiting cell proliferation and reducing cell death. However, the mechanism underlying the protective effect of CDK inhibitors on microglia remains unexplored. In this study, we found that olomoucine, a CDK inhibitor, alleviated lipopolysaccharide (LPS)-induced BV2 microglial cell death by reducing the generation of NO and inhibiting the gene expression of proinflammatory cytokines. In addition, olomoucine reduced inducible NO synthase promoter activity and alleviated NF-κB- and E2F-mediated transcriptional activation. NO-induced cell death involved mitochondrial disruptions such as cytochrome c release and loss of mitochondrial membrane potential, and pretreatment with olomoucine prior to NO exposure reduced these disruptions. Microarray analysis revealed that olomoucine treatment induced prominent down-regulation of Bcl2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a pro-apoptotic Bcl-2 family protein that is involved in mitochondrial disruption. As BNIP3 knock-down significantly increased the viability of LPS- and NO-treated BV2 cells, we conclude that olomoucine may protect cells by limiting proinflammatory responses, thereby reducing NO generation. Simultaneously, down-regulation of BNIP3 prevents NO stimulation from inducing mitochondrial disruption.

Development and Evaluation of Solid Lipid Nanoparticles of N-6-Furfuryl Adenine for Prevention of Photoaging.

N-6-furfuryl adenine (N6FA) also known as "kinetin" is a biologically active natural phytochemical. It belongs to the category of cytokinins, the natural plant growth hormones that promote cell division and play role in cell differentiation. Overall, N6FA aids in increasing the plant's life span. Human cells also contain.small quantities of N6FA. Scientists are trying to understand its function in humans. N6FA is being investigated for its properties such as antiplatelet, antioxidant, antiproliferative and anti-aging effects on human cells. The aim of the present investigation was to prepare solid lipid nanoparticle (SLN) based topical formulations of N6FA and to evaluate its efficacy against ultraviolet (UV) radiation induced skin photodamage. SLNs were prepared by hot microemulsion technique and optimized for the type and concentration of lipid and surfactant(s). The optimized SLN formulation was characterized in terms of particle size, drug entrapment efficiency, zeta potential and pH; evaluated for stability, spreadability, ex-vivo skin permeation and photoprotective effects against UV induced skin damage. The cumulative amount of drug permeated through mice skin using SLNs was 3 folds higher than from conventional cream base. The results of biochemical and histopathological investigations of skin treated with N6FA loaded SLNs clearly demonstrated the efficacy of optimized formulation in preventing photodamage (lesions, ulcers and changes in skin integrity) due to chronic UV exposure. The effects were comparable with widely used marketed formulation, Garnier wrinkle lift anti-aging cream. Results suggested that N6FA incorporated into SLNs may provide therapeutic as well as cosmeceutical benefits.

Efficacy and safety of 0.1% kinetin cream in the treatment of photoaging skin.

BACKGROUND: Kinetin is a plant-derived compound, which is reported to possess antiaging properties. It has been used in a topical cream to manage facial photo-damage and aging. Although studies elsewhere have shown its benefits, not many studies of the effects of kinetin in Asian skin are available. OBJECTIVES: To assess the efficacy and tolerability of 0.1% kinetin cream in the treatment of facial photo-aging. METHODS: The study was designed to be open-label and single-blinded, without a control group. One hundred Thai female and male subjects with mild, moderate or severe facial photo-damage were enrolled. They were asked to apply 0.1% kinetin cream twice daily for 12 weeks and follow up at 4, 8, and 12 weeks. Subjective patient self-assessment and physician assessment of facial skin photo-damage were accompanied by digital photographic analysis using the VISIA® (Canfield Scientific Inc, Fairfield, NJ) imaging system. RESULTS: At baseline, most patients reported moderate skin changes related to photo-damage, skin texture, skin color and wrinkles. After 12 weeks, physician and patient assessments showed slight but statistically significant improvements in overall skin condition, skin texture, color, and wrinkles. Findings were similar with the digital photographic system analysis, especially in relation to skin color. Facial ultraviolet spots and redness also showed statistically significant improvements after 12 weeks. The treatment was generally well tolerated. LIMITATIONS: The study was designed to be pragmatic and hence no randomization was carried out; there were also no intrapatient or interpatient control observations, and no comparison arm. CONCLUSION: Kinetin (0.1%) cream was found to slightly improve cutaneous facial photo-damage after 12 weeks of use in a group of Thai patients.

Production of flavonoids and isoflavonoids in jasmonic acid-induced red clover suspension cultures.

Effect of exogenously applied jasmonic acid (JA) in combination with calcium and verapamil (a calcium channels blocker) on the production of flavonoids and isoflavonoids in suspension cultures of Trifolium pratense L. was investigated. The culture was cultivated in Gamborg medium with an addition of 2 mg.l(-1) of 2,4-dichlorophenoxyacetic acid and 2 mg.l(-1) of 6-benzylaminopurine, at the temperature of 25 °C, 16-hr light/8-hr dark period. The best effect of jasmonic acid on the production of flavonoids and isoflavonoids was manifested after a 24-hour application of the 50 µmol.l(-1) concentration. The maximum production of JA-induced suspension culture was observed when cells were treated with a high level of calcium (10 mmol.l(-1)). The addition of all concentrations of verapamil to JA-induced suspension culture decreased production of flavonoids and isoflavonoids.

Light and nitrogen nutrition regulate apical control in Rosa hybrida L.

Apical control is defined as the inhibition of basal axillary bud outgrowth by an upper actively growing axillary axis, whose regulation is poorly understood yet differs markedly from the better-known apical dominance. We studied the regulation of apical control by environmental factors in decapitated Rosa hybrida in order to remove the apical hormonal influence and nutrient sink. In this plant model, all the buds along the main axis have a similar morphology and are able to burst in vitro. We concentrated on the involvement of light intensity and nitrate nutrition on bud break and axillary bud elongation in the primary axis pruned above the fifth leaf of each rose bush. We observed that apical control took place in low light (92 µmol m(-2)s(-1)), where only the 2-apical buds grew out, both in low (0.25 mM) and high (12.25 mM) nitrate. In contrast, in high light (453 µmol m(-2)s(-1)), the apical control only operates in low nitrate while all the buds along the stem grew out when the plant was supplied with a high level of nitrate. We found a decreasing photosynthetic activity from the top to the base of the plant concomitant with a light gradient along the stem. The quantity of sucrose, fructose, glucose and starch are higher in high light conditions in leaves and stem. The expression of the sucrose transporter RhSUC2 was higher in internodes and buds in this lighting condition, suggesting an increased capacity for sucrose transport. We propose that light intensity and nitrogen availability both contribute to the establishment of apical control.

Extracts from black carrot tissue culture as potent anticancer agents.

Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38-46% at 6.25 µg/ml concentration for all calli and natural extracts. However, a significantly high IC50 value of 170.13 µg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells.

Specific correction of a splice defect in brain by nutritional supplementation.

Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.

Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia.

Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-κ-B kinase complex-associated protein/elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase WT IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine whether oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/d for 28 d. An increase in WT IKBKAP mRNA expression in leukocytes was noted after 8 d in six of eight individuals; after 28 d, the mean increase compared with baseline was significant (p = 0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients but also that effect seems to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine whether kinetin will prove therapeutic in FD patients.

Cytochalasin D, LY294002 and olomoucine synergize in promoting death of melanoma cells through activation of caspase-3 and apoptosis.

Many of the current anticancer therapies rely on the induction of apoptosis, and several mechanisms that protect cells against apoptosis may be upregulated in tumors. A growing body of evidence suggests that single drugs with a clearly defined intracellular target may be less efficient in arresting tumor growth and induction of apoptosis than multitargeted strategies. To prove that this is also the case for melanoma, we studied five cell lines, which represent different stages of tumor progression. We tested cell viability, terminal dUTP nick-end labeling and activation of caspase-3 upon exposure to cytochalasin D, LY294002 and olomoucine, added either alone or in various combinations. The obtained data were compared with effects caused by staurosporine. The results show that whereas staurosporine efficiently induced apoptosis in all tested melanoma cell lines, the other drugs had only moderate effects when administered alone. In contrast, the combinations of drugs were more effective in inducing caspase-3 activity and reducing cell viability. In particular, the triple combination of cytochalasin D+LY294002+olomoucine was almost as effective as staurosporine in inducing caspase-3 activity and apoptosis. These results prove that it is possible to design new pharmacological strategies that will effectively induce caspase-3 activity and apoptosis in melanoma. The possible explanations of the observed synergy between the tested drugs are also discussed.

Rat focal cerebral ischemia induced astrocyte proliferation and delayed neuronal death are attenuated by cyclin-dependent kinase inhibition.

Astroglial proliferation and delayed neuronal death are two common pathological processes in the ischemic brain. However, it is not clear if astrogliosis causes delayed neuronal death. In this study, we addressed this potential linkage by examining the relationship between attenuated astrocyte proliferation, induced by cyclin-dependent kinase (CDK) inhibition, and delayed neuronal death in rat ischemic hippocampus. Our results show that following middle cerebral artery occlusion (MCAO), astrocyte hypertrophy and proliferation were closely associated with delayed neuronal death. Importantly, administration of olomoucine, a selective CDK inhibitor, not only suppressed astroglial proliferation and glial scar formation, but also decreased neuronal cell death in the ischemic boundary zone and hippocampal CA1 region at days 1 and 30 after MCAO. These results indicate that reactive astrogliosis and delayed neuronal death, at least in rat hippocampus, are sequential pathological events following MCAO. Therefore, suppressing astroglial cell cycle progression in acute focal cerebral ischemia may be beneficial to neuronal survival. Our study also implies that cell cycle regulation should be considered as a promising future therapeutic intervention in treating those neurological diseases characterized by an excessive astrocyte proliferation.

The clinical anti-aging effects of topical kinetin and niacinamide in Asians: a randomized, double-blind, placebo-controlled, split-face comparative trial.

BACKGROUND: Kinetin and niacinamide are used in the cosmetic industry as anti-aging agents. Neither the interactive/additive effects of these compounds nor the anti-aging efficacy on Asian skin has been studied. Objective To assess the clinical anti-aging effects and efficacy differences between kinetin plus niacinamide and niacinamide alone vs. vehicle placebo in an Asian cohort. METHODS: Fifty-two Taiwanese subjects were enrolled in a randomized, double-blind, placebo-controlled, split-face comparative study. Group 1 subjects were treated with kinetin 0.03% plus niacinamide 4%, whereas group 2 subjects received niacinamide 4%. The treatment formulation was applied on one side of the face, whereas a placebo was applied on the other for a period of 12 weeks. We used noninvasive biometrological instruments to evaluate a variety of skin parameters at baseline and at weeks 4, 8, and 12. RESULTS: Persistent and significant reductions in spot, pore, wrinkle, and evenness counts were found at weeks 8 and 12 in group 1. A significant increase in corneal hydration status was also evident at week 12, whereas persistent decreases in erythema index were apparent at 8 and 12 weeks. In group 2, significant reductions in pore and evenness counts at week 8 and wrinkle counts at week 12 were noted. CONCLUSION: We found kinetin and niacinamide exert a synergistic anti-aging effect. Our data suggest that these compounds have multiactive, multifunctional, and pluripotent effects on skin. They are also both promising to be included in the cutaneous anti-aging cosmeceuticals in the future.