The b iosynthesis of histidine: imidazoleglycerol phosphate, imidazoleacteol phosphate, and histidinol phosphate

This is a report on the isolation and characterization of D-erythro-imidazoleglycerol phosphate (IGP), imidazoleacetol phosphate (IAP), and L-histidinol phosphate, which are accumulated in the mycelia of several of these mutants

Prevention of Mutation, Cancer, and Other Age-Associated Diseases by Optimizing Micronutrient Intake

I review three of our research efforts which suggest that optimizing micronutrient intake will in turn optimize metabolism, resulting in decreased DNA damage and less cancer as well as other degenerative diseases of aging. (1) Research on delay of the mitochondrial decay of aging, including release of mutagenic oxidants, by supplementing rats with lipoic acid and acetyl carnitine. (2) The triage theory, which posits that modest micronutrient deficiencies (common in much of the population) accelerate molecular aging, including DNA damage, mitochondrial decay, and supportive evidence for the theory, including an in-depth analysis of vitamin K that suggests the importance of achieving optimal micronutrient intake for longevity. (3) The finding that decreased enzyme binding constants (increased Km) for coenzymes (or substrates) can result from protein deformation and loss of function due to an age-related decline in membrane fluidity, or to polymorphisms or mutation. The loss of enzyme function can be compensated by a high dietary intake of any of the B vitamins, which increases the level of the vitamin-derived coenzyme. This dietary remediation illustrates the importance of understanding the effects of age and polymorphisms on optimal micronutrient requirements. Optimizing micronutrient intake could have a major effect on the prevention of cancer and other degenerative diseases of aging

Mitochondrial decay in aging

AbstractSeveral mitochondrial functions decline with age. The contributing factors include, the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging

(R)-␣-Lipoic Acid Protects Retinal Pigment Epithelial Cells from Oxidative Damage

PURPOSE. To determine whether (R)-␣-lipoic acid (LA) protects cultured human fetal retinal pigment epithelial (hfRPE) cells against oxidative injury and identify the pathways that may mediate protection. METHODS. Cultured hfRPE cells were pretreated with various concentrations of LA for 14 to 16 hours followed by treatment with a chemical oxidant, tert-butylhydroperoxide (t-BuOOH; 0.8 mM, 3 hours). Reactive oxygen species (ROS) production and cell viability were measured using H 2 DCF and MTT assays, respectively. RPE cells were evaluated with fluorescent dyes (SYTOX Orange and SYTO Green; Molecular Probes, Eugene, OR), which differentiate between live and dead cells. Apoptosis was visualized by using the TUNEL assay. Changes in mitochondrial membrane potential were detected by JC-1 dye. Intracellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by HPLC. Regulation of ␥-glutamylcysteine ligase (GCL), the rate-controlling enzyme of GSH production, was assayed by RT-PCR. RESULTS. Pretreatment of hfRPE cells with LA, 0.2 mM and 0.5 mM, significantly reduced the levels of t-BuOOH-induced intracellular ROS, by 23% and 49%, respectively. LA (0.5 mM) prevented oxidant-induced cell death and apoptosis and also increased the viability of oxidant-treated hfRPE cells from 38% to 90% of control. LA upregulated the mRNA expression of GCL, and was protective against t-BuOOH-induced decreases in both mitochondrial membrane potential and intracellular levels of GSH and GSH/GSSG. CONCLUSIONS. The present study suggests that the protective effect of LA involves multiple pathways and that LA could be effective against age-associated increase in oxidative stress and mitochondrial dysfunction in RPE cells. (Invest Ophthalmol Vis Sci. 2005;46:4302-4310) DOI:10.1167/iovs.04-1098 A ge-related macular degeneration (AMD) is the major cause of severe visual impairment for elderly individuals in developed countries. 1-3 Although the underlying pathogenic mechanisms and risk factors are not well understood, 1-9 epidemiologic studies suggest that environmental factors associated with oxidative stress-for example, cigarette smoking, sunlight exposure, and low dietary intake of antioxidants-are contributing factors in the development of AMD. 4 -7 Recent results have shown that antioxidants and zinc protect against the progression of the disease. 8 There is good evidence to suggest that the retinal pigment epithelium (RPE), choriocapillaris, and distal retina are all involved in this process, but the pathology associated with each can be difficult to distinguish, given their close anatomic, physiologic, and metabolic relationships. 7,15 A significant oxidative load is associated with the RPE phagocytosis of photoreceptor outer segments, 16 Cellular membranes with high polyunsaturated fatty acids are particularly susceptible to radical-induced chain reactions of lipid peroxidation. 17 Age-associated decreases in antioxidant defense mechanisms throughout the body can further increase the levels of RPE exposure to oxidants. For example, the levels of plasma glutathione (GSH), one of the major water-soluble antioxidants, decrease with age. 19 -21 Oxidantinduced mitochondrial dysfunction and death of RPE cells may contribute to the onset of AMD. 27-29 ␣-Lipoic acid is a potent intracellular antioxidant that can induce all three cellular protective mechanisms. 36 t-BuOOH is a relatively stable alkyl peroxide that readily penetrates cell membranes From th

γ-Tocopherol Attenuates Ozone-induced Exacerbation of Allergic Rhinosinusitis in Rats

Compared to healthy subjects, individuals with allergic airway disease (e.g., asthma, allergic rhinitis) have enhanced inflammatory responses to inhaled ozone. We created a rodent model of ozone-enhanced allergic nasal responses in Brown Norway rats to test the therapeutic effects of the dietary supplement g-tocopherol (γT). Ovalbumin (OVA)-sensitized rats were intranasally challenged with 0% or 0.5% OVA (in saline) on Days 1 and 2, and then exposed to 0 or 1 ppm ozone (eight hours/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg γT (p.o., in corn oil) on days 2 through 5, beginning twelve hours after the last OVA challenge. On Day 6, nasal tissues were collected for histological evaluation and mor-phometric analyses of intraepithelial mucosubstances (IM) and eosinophilic inflammation. Nasal septal tissue was microdissected and analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for mucin glycoprotein 5AC (MUC5AC) expression levels. Histological analysis revealed mild to moderate eosinophil influx in the mucosa lining the nasal airways and maxillary sinus of OVA-challenged rats (eosinophilic rhinosinusitis). Ozone exposure of allergic rats further increased eosinophils in the maxillary sinus (400%), nasolacrimal duct (250%), and proximal midseptum (150%). Storage of intraepithelial mucosubstances (IM) was not significantly affected by OVA challenge in filtered air-exposed rats, but it was increased by ozone in the septum (45%) and maxillary sinus (55%) of allergic compared to control rats. Treatment with γT attenuated the ozone/ OVA-induced synergistic increases in IM and mucosal eosinophils in both nasal and paranasal airways. γ-Tocopherol also blocked OVA and ozone-induced MUC5AC gene expression. Together, these data describe a unique model of ozone enhancement of allergic rhinosinusitis and the novel therapeutic efficacy of a common supplement, γT, to inhibit ozone exacerbation of allergic airway responses

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells

Eotaxin-3 (CCL-26), a potent chemokine for eosinophil recruitment and contributing significantly to the pathogenesis of asthma, is secreted by lung epithelial cells in response to T helper 2 cytokines including interleukin 13 (IL-13). Here we showed that vitamin E forms, but not their metabolites, differentially inhibited IL-13-stimulated generation of eotaxin-3 in human lung epithelial A549 cells. The relative inhibitory potency was γ-tocotrienol (γ-TE) (IC50 ~15 μM) > γ-tocopherol, δ-tocopherol (IC50 ~25-50 μM) > α-tocopherol. Consistent with suppression of eotaxin, γ-TE treatment impaired IL-13-induced phosphorylation of STAT6, the key transcription factor for activation of eotaxin expression, and consequently blocked IL-13 stimulated DNA-binding activity of STAT6. In search of the upstream target of γTE by using inhibitor and siRNA approaches, we discovered that the atypical protein kinase C (aPKC) signaling, instead of classical PKC, p38 MAPK, JNK or ERK, played a critical role in IL-13-stimulated eotaxin generation and STAT6 activation. While showing no obvious effect on aPKC expression or phosphorylation, γ-TE treatment resulted in increased expression of PAR4, an endogenous negative regulator of aPKCs. Importantly, γ-TE treatment led to enhanced formation of aPKC/PAR4 complex that is known to reduce aPKC activity via protein-protein crosstalk. Our study demonstrated that γ-TE inhibited IL-13/STAT6-activated eotaxin secretion via up-regulation of PAR4 expression and enhancement of aPKC-PAR-4 complex formation. These results support the notion that specific vitamin E forms may be useful anti-asthmatic agents

Ozone enhancement of lower airway allergic inflammation is prevented by γ-tocopherol

Ozone is a commonly encountered environmental oxidant which has been linked to asthma exacerbation in epidemiological studies. Ozone induces airway inflammation and enhances response to inhaled allergen. It has been suggested that antioxidant therapy may minimize the adverse effects of ozone in asthma. We have previously shown that the antioxidant gamma-tocopherol (γT), an isoform of vitamin E, also has anti-inflammatory effects. We employed a Brown Norway rat model of ozone-enhanced allergic responses to test the therapeutic effects of γT on O3-induced airway inflammation. Ovalbumin (OVA) -sensitized rats were intranasally challenged with 0 or 0.5% OVA on Days 1 and 2, and exposed to 0 or 1 ppm ozone (8h/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg γT on Days 2 through 5. Pulmonary tissue and bronchoalveolar lavage fluid (BALF) were collected on Day 6. OVA challenge caused increased total cells (267% increase) and eosinophils (4000%) in BALF that was unaffected by ozone exposure. Morphometric evaluation of lung tissue revealed increases in intraepithelial mucosubstances (IM) (300%) and subepithelial eosinophils (400%) in main axial airways. Ozone exposure of allergic rats enhanced IM increases in proximal axial airways (200%), induced cys-leukotrienes, MCP-1 and IL-6 production in BALF, and upregulated expression of IL-5 and IL-13 mRNA. γT treatment had no effect on IM increases by allergen, but blocked enhancement by ozone. γT attenuated both OVA- or ozone –stimulated eosinophilic infiltration, and increases of BALF cys-leukotrienes, MCP-1 and IL-6, as well as IL-5 and IL-13 mRNA. These data demonstrate broad anti-inflammatory effects of a γT and suggest it may be an effective therapy of allergic airway inflammation