Experimental Biology and Medicine

Epidemiologic evidence shows that elevated serum cholesterol, specifically low-density lipoprotein cholesterol (LDL-C), increases the risk of coronary heart disease (CHD). Moreover, large-scale intervention trials demonstrate that treatment with HMG-CoA reductase inhibitors (statins), the most effective drug class for lowering LDL-C, significantly reduces the risk of CHD events. Unfortunately, only a moderate percentage of hypercholesterolemic patients are achieving LDL-C targets specified by the National Cholesterol Education Program (NCEP), in part because clinicians are not effectively titrating medications as needed to achieve LDL-C goals. Recent evidence suggests that more aggressive LDL-C lowering may provide greater clinical benefit, even in individuals with moderately elevated serum cholesterol levels. Furthermore, recent studies suggest that statins have cardioprotective effects in many high-risk individuals, including those with baseline LDL-C <100 mg/dl. High-density lipoprotein cholesterol (HDL-C) was recognized by the NCEP-Adult Treatment Panel II (ATP II) as a negative risk factor for CHD. The NCEP-ATP III guidelines have also reaffirmed the importance of HDL-C by increasing the low HDL-C designation from <35 to <40 mg/dl as a major risk factor for CHD. Similarly, triglyceride control will play a larger role in dyslipidemia management. As more clinicians effectively treat adverse lipid and lipoprotein cardiovascular risk factors, patients will likely benefit from reductions in cardiovascular events.

Vitamin E, a dietary factor, is essential for reproduction in animals. It is an antioxidant present in all mammalian cells. Previously, we showed that ascorbic acid (AA) acted as an inhibitory neurotransmitter in the hypothalamus by scavenging nitric oxide (NO). Earlier studies have shown the antioxidant synergism between vitamin E and ascorbic acid (AA). Therefore, it was of interest to evaluate the effect of vitamin E on luteinizing hormone-releasing hormone (LHRH) and AA release. Medial basal hypothalami from adult male rats of the Sprague Dawley strain were incubated with Krebs-Ringer bicarbonate buffer or graded concentrations of a water soluble form of vitamin E, tocopheryl succinate polyethylene glycol 1000 (TPGS, 22–176 μM) for 1 hr. Subsequently, the tissues were incubated with vitamin E or combinations of vitamin. E + N-methyl-D-aspartic acid (NMDA), an excitatory amino acid for 30 min to study the effect of prior and continued exposure to vitamin E on NMDA-induced LHRH release. AA and LHRH released into the incubation media were determined by high-performance liquid chromatography and radioimmunoassay, respectively. Vitamin E stimulated both LHRH and AA release. The minimal effective concentrations were 22 and 88 μM, respectively. NMDA stimulated LHRH release as previously shown and this effect was not altered in the combined presence of vitamin E plus NMDA. However, AA release was significantly reduced in the combined presence of vitamin E plus NMDA. To evaluate the role of NO in vitamin E-induced LHRH and AA release, the tissues were incubated with vitamin E or combinations of vitamin E + N^G-monomethyl-L-arginine (NMMA), a competitive inhibitor of NO synthase. NMMA significantly suppressed vitamin E-induced LHRH and AA release indicating a role of NO in the release of both LHRH and AA. The data suggest that vitamin E plays a role in the hypothalamic control of LHRH and AA release and that the release is mediated by NO.

Cytochrome P-450 monooxygenase (epoxygenase)-derived arachidonic acid (AA) metabolites, including 11,12-epoxyeicosatrienoic acid (11,12-EET), possess anti-inflammatory and antipyretic properties. Prostaglandin E₂ (PGE₂), a cyclooxygenase (COX)-derived metabolite of AA, is a well-defined mediator of fever and inflammation. We have tested the hypothesis that 11,12-EET attenuates synthesis of PGE₂ in monocytes, which are the cells that are indispensable for induction of fever and initiation of inflammation. Monocytes isolated from freshly collected rat blood were stimulated with lipopolysaccharide (LPS; 100 ng/2 × 10⁵ cells) to induce COX-2 and stimulate generation of PGE₂. SKF-525A, an inhibitor of epoxygenases, significantly augmented the lipopolysaccharide-provoked synthesis of PGE₂ in cell culture in a concentration-dependent manner. It did not affect, however, elevation of the expression of COX-2 protein in monocytes stimulated with LPS. 11,12-EET also did not affect the induction of COX-2 in monocytes incubated with lipopolysaccharide. However, 11,12-EET suppressed, in a concentration-dependent fashion, the generation of PGE₂ in incubates. Preincubation of a murine COX-2 preparation for 0–5 min with three concentrations of 11,12-EET (1, 5, and 10 μM) inhibited the oxygenation of [¹⁴C]-labeled AA by the enzyme. The inhibitory effect of 11,12-EET on COX-2 was time-and-concentration-dependent, suggesting a mechanism-based inhibition. Based on these data, we conclude that 11,12-EET suppresses generation of PGE₂ in monocytes via modulating the activity of COX-2. These data support the hypothesis that epoxygenasederived AA metabolites constitute a negative feedback on the enhanced synthesis of prostaglandins upon inflammation.

Nucleosides are essential components of milk that are used for the nourishment of newborns. Effects of the three primary lactogenic hormones, including prolactin (PRL), insulin (I), and cortisol (H), on nucleoside uptake and incorporation into cultured mammary tissues taken from 12- to 14-day pregnant mice were determined; most experiments focused on the regulation of uridine uptake. Insulin alone, as well as PRL in the presence of insulin and cortisol, was shown to stimulate uridine uptake and incorporation into RNA in mammary explants taken from 12- to 14-day pregnant mice. The PRL effects were expressed at concentrations of 25 ng/ml and above, which are physiological plasma concentrations. In the absence of sodium, uridine uptake and incorporation were diminished, suggesting the presence of a sodium-dependent uridine transporter. In kinetic studies the apparent K _m for uridine uptake was calculated to be 312 μM, and the V _max 2.90 μmol/hr/L cell water; PRL had no effect on the K _m but increased the V _max to 5.88 μmol/hr/L cell water. When assessing uridine uptake in the presence of the other nucleosides at 0.1 mM, only cytidine competed with uridine uptake. The fact that distribution ratios of greater than 15:1 were achieved with uridine indicates that uridine uptake may be via an active transporter. These studies show that PRL enhances uridine update in mammary tissues by stimulating the activity, and probably synthesis, of a sodium-dependent, active uridine and cytosine transporter.

We previously found that transgenic mice overexpressing growth hormone (TGM) have elevated and progressively increasing free radical processes in brain that strongly correlates with reduced survivorship. Young mature TGM, however, displayed vastly enhanced learning of an eight-choice cued maze and qualitatively different learning curves than normal controls. Here we document the age-related patterns in learning ability of TGM and normal mice. Learning appeared inferior in both genotypes of very young mice but TGM were confirmed to be superior to normal mice upon maturity. Older TGM, however, showed rapid age-related loss of their exceptional learning, whereas normal mice at 1 year of age showed little change. The cognitive decline of TGM was abolished by a complex “anti-aging” dietary supplement formulated to promote membrane and mitochondrial integrity, increase insulin sensitivity, reduce reactive oxygen and nitrogen species, and ameliorate inflammation. Results are discussed in the context of reactive oxygen and nitrogen species, long-term potentiation, learning, aging and neuropathology, based on known impacts of the growth hormone axis on the brain, and characteristics of TGM.

Copper Deficiency (CuD) leads to hypertrophic cardiomyopathy in various experimental models. The morphological, electrophysiological, and molecular aspects of this hypertrophy have been under investigation for a long time. However the transition from compensated hypertrophy to decompensated heart failure has not been investigated in the study of CuD. We set out to investigate the contractile and hemodynamic parameters of the CuD mouse heart and to determine whether heart failure follows hypertrophy in the CuD heart. Dams of FVB mice were fed CuD or copper-adequate (CuA) diet starting from the third day post delivery and the weanling pups were fed the same diet for a total period of 5 weeks (pre- and postweanling). At week 4, the functional parameters of the heart were analyzed using a surgical technique for catheterizing the left ventricle. A significant decrease in left ventricle systolic pressure was observed with no significant change in heart rate, and more importantly contractility as measured by the maximal rate of left ventricular pressure rise (+dP/dt) and decline (−dP/dt) were significantly depressed in the CuD mice. However, left ventricle end diastolic pressure was elevated, and relaxation was impaired in the CuD animals; the duration of relaxation was prolonged. In addition to significant changes in the basal level of cardiac function, CuD hearts had a blunted response to the stimulation of the β-adrenergic agonist isoproterenol. Furthermore, morphological analysis revealed increased collagen accumulation in the CuD hearts along with lipid deposition. This study shows that CuD leads to systolic and diastolic dysfunction in association with histopathological changes, which are indices commonly used to diagnose congestive heart failure.

This study was undertaken to test the effect of immunization against luteinizing hormone-releasing hormone (LHRH) fusion proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Two LHRH fusion proteins, ovalbumin with seven LHRH peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups of male TRAMP mice were immunized with the cocktail. Primary immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n = 19) were compared with castrated (n = 19) and intact mice (n = 18) for testosterone concentration, tumor weight, and lifespan. Immunization against LHRH in the TRAMP mice resulted in significant production of antibodies to LHRH compared with surgically castrated and intact control mice. Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P < 0.05) in the immunized mice compared with intact control mice and was not different from that of castrated mice (P > 0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact control mice (lived the longest (227 ± 11 days), whereas immunized mice lived 206 ± 11 days and castrated mice lived 213 ± 13 days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or intact TRAMP mice. Prostate cancer is initially dependent upon androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The TRAMP mouse model immunized against LHRH may have utility in future studies and treatments of the androgen independent prostate cancer.

Epidemiological data from retrospective and case–control studies have indicated that estrogen replacement therapy can decrease the risk of developing Alzheimer’s disease. In addition, estrogen replacement therapy has been found to promote neuronal survival both in vivo and in vitro. We have shown that conjugated equine estrogens (CEE), containing 238 different molecules composed of estrogens, progestins, and androgens, exerted neurotrophic and neuroprotective effects in cultured neurons. In the current study, we sought to determine whether a steroidal formulation of nine synthetic conjugated estrogens (SCE) chemically derived from soybean and yam extracts is as effective as the complex multisteroidal formulation of CEE. Analyses of the neuroprotective efficacy indicate that SCE exhibited significant neuroprotection against beta amyloid, hydrogen peroxide, and glutamate-induced toxicity in cultured hippocampal neurons. Indices of neuroprotection included an increase in neuronal survival, a decrease in neurotoxin-induced lactate dehydrogenase release, and a reduction in neurotoxin-induced apoptotic cell death. Furthermore, SCE was found to attenuate excitotoxic glutamate-induced [Ca²⁺]_i rise. Quantitative analyses indicate that the neuroprotective efficacy of SCE was comparable to that of the multisteroidal CEE formulation. Data derived from these investigations predict that SCE could exert neuroprotective effects comparable to CEE in vivo and therefore could reduce the risk of Alzheimer’s disease in postmenopausal women.

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H₂O₂ and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H₂O₂ on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser⁴⁷³. Pretreatment with H₂O₂ concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 μM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H₂O₂ inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 μM), for 30 min did not block the inhibitory effects of H₂O₂ on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H₂O₂. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.

High-fat diets made with different fats may have distinct effects on body weight regulation and metabolism. In the present study, the metabolic effects of high-fat (HF) diets made with fish oil, palm oil, and soybean oil were compared with a low-fat diet in female Wistar rats that were either exercised (EX, swimming) or that remained sedentary as controls. Each adult rat was exposed to the same diet that their dams consumed during pregnancy and lactation. When they were 9 weeks old, rats began an EX regimen that lasted for 6 weeks. Twenty-four hours after the last EX bout, rats were sacrificed in a fasted state. It was observed that HF feeding of soybean oil induced more body weight and fat gain, as well as insulin resistance, as indicated by insulin/glucose ratios, than other oils. Female rats fed a HF diet made with fish oil had body weight and insulin sensitivity not different from that observed in low fat fed control rats. For rats fed HF diets made with soybean oil or palm oil, EX also exerted beneficial effects by reducing body fat %, blood insulin, triglyceride and leptin levels, as well as improving insulin sensitivity.

Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells. However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified. The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1. The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor. Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring. Intracellular calcium concentrations in STC-1 cells were increased by exposure to α-casein or casein sodium, but not to bovine serum albumin. Solubilized STC-1 membranes bound to α-casein and casein sodium but did not bind to bovine serum albumin. α-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium. Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated. Intracellular calcium responses to α-casein were suppressed by an L-type calcium channel blocker. These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.

Impaired endothelial cell proliferation has been proposed to be an early, critical defect contributing to the development of atherosclerosis. Recent studies show that high plasma tumor necrosis factor (TNF)-α levels and low serum ascorbic acid (AA) levels correlate with atherosclerosis severity. Additionally, AA has been reported to have potential beneficial effects in preventing atherosclerosis. Based on these studies, we investigated the role of AA (≤ 1mM) on TNF-α-mediated vascular endothelial cell growth inhibition in vitro. In accordance with previous reports, we found that TNF-α alone inhibited endothelial cell proliferation. Further studies revealed that AA alone enhanced endothelial cell proliferation and that AA blocked endothelial cell growth inhibition induced by TNF-α. By contrast, we observed no effect of AA on endothelial cell activation or nuclear entry of nuclear factor-κB in response to TNF-α. The protective effect of AA on endothelial cell proliferation was not simply the result of its antioxidant activity but did correlate with collagen IV expression by endothelial cells. AA pre-treatment of proliferating endothelial cells promoted retinoblastoma protein (Rb) phosphorylation and decreased p53 levels when compared to untreated cells. Furthermore, the addition of AA to TNF-α-treated proliferating endothelial cells blocked both the inhibition of retinoblastoma protein phosphorylation and enhanced p53 expression induced by TNF-α. Consistent with these results, we found that AA protects endothelial cells against TNF-α-induced apoptosis. These studies highlight the potential therapeutic role of AA in promoting endothelial cell proliferation during inflammatory conditions, such as atherosclerosis and cardiovascular disease.

The complete genomic nucleotide sequence (29.7kb) of a Hong Kong severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) strain HK-39 is determined. Phylogenetic analysis of the genomic sequence reveals it to be a distinct member of the Coronaviridae family. 5′ RACE assay confirms the presence of at least six subgenomic transcripts all containing the predicted intergenic sequences. Five open reading frames (ORFs), namely ORF1a, 1b, S, M, and N, are found to be homologues to other CoV members, and three more unknown ORFs (X1, X2, and X3) are unparalleled in all other known CoV species. Optimal alignment and computer analysis of the homologous ORFs has predicted the characteristic structural and functional domains on the putative genes. The overall nucleotides conservation of the homologous ORFs is low (<5%) compared with other known CoVs, implying that HK-39 is a newly emergent SARS-CoV phylogenetically distant from other known members. SimPlot analysis supports this finding, and also suggests that this novel virus is not a product of a recent recombinant from any of the known characterized CoVs. Together, these results confirm that HK-39 is a novel and distinct member of the Coronaviridae family, with unknown origin. The completion of the genomic sequence of the virus will assist in tracing its origin.