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Introduction: Non-alcoholic fatty liver disease (NAFLD) is an increasing cause of chronic liver disease that accompanies obesity and the metabolic syndrome. Excess fructose consumption can initiate or exacerbate NAFLD in part due to a consequence of impaired hepatic fructose metabolism. Preclinical data emphasized that fructose-induced altered gut microbiome, increased gut permeability, and endotoxemia play an important role in NAFLD, but human studies are sparse. The present study aimed to determine if two weeks of excess fructose consumption significantly alters gut microbiota or permeability in humans. Methods: We performed a pilot double-blind, cross-over, metabolic unit study in 10 subjects with obesity (body mass index [BMI] 30–40 mg/kg/m2). Each arm provided 75 grams of either fructose or glucose added to subjects’ individual diets for 14 days, substituted isocalorically for complex carbohydrates, with a 19-day wash-out period between arms. Total fructose intake provided in the fructose arm of the study totaled a mean of 20.1% of calories. Outcome measures included fecal microbiota distribution, fecal metabolites, intestinal permeability, markers of endotoxemia, and plasma metabolites. Results: Routine blood, uric acid, liver function, and lipid measurements were unaffected by the fructose intervention. The fecal microbiome (including Akkermansia muciniphilia), fecal metabolites, gut permeability, indices of endotoxemia, gut damage or inflammation, and plasma metabolites were essentially unchanged by either intervention. Conclusions: In contrast to rodent preclinical findings, excess fructose did not cause changes in the gut microbiome, metabolome, and permeability as well as endotoxemia in humans with obesity fed fructose for 14 days in amounts known to enhance NAFLD.

Background and AimsNon-alcoholic fatty liver disease (NAFLD) is an increasing cause of chronic liver disease that accompanies obesity and the metabolic syndrome. Excess fructose consumption can initiate or exacerbate NAFLD due in part to a consequence of impaired hepatic fructose metabolism. Pre-clinical data have emphasized that fructose-induced altered gut microbiome, increased gut permeability, and endotoxemia play an important role in NAFLD, but human studies are sparse. The present study aimed to explore the relevance of these pre-clinical studies to observations in humans.MethodsWe performed a classical double-blind metabolic unit study in 10 obese subjects (BMI 30-40 mg/kg/m2) providing 75gms. of either fructose or glucose in their individual diets substituted isocalorically for complex carbohydrates in a cross-over study. Excess fructose intake was provided in the fructose arm of the study and totaled a mean of 22.7% of calories.ResultsRoutine blood, uric acid, liver function and lipid measurements were unaffected by the fructose intervention. The fecal microbiome (including Akkermansia muciniphilia), fecal metabolites, gut permeability, indices of endotoxemia, gut damage or inflammation and plasma metabolites were essentially unchanged by either intervention.ConclusionsAlthough pre-clinical rodent studies have shown that excess fructose causes pronounced changes in the gut microbiome, metabolome, and permeability as well as endotoxemia, this did not occur in obese individuals fed fructose in amounts known to enhance NAFLD. Therapeutic efforts to improve NAFLD through changes in the gut microbiome and gut homeostasis may not be beneficial.

Background Acute alcohol consumption and chronic alcohol consumption increase the burden placed on emergency departments (EDs) by contributing to injury and disease. Whether the prevalence of alcohol-related ED visits in the United States has changed in recent years is unknown. The purpose of this study was to examine trends in ED visits involving acute and chronic alcohol consumption in the United States by age and sex between 2006 and 2014. Methods Data from the Nationwide Emergency Department Sample (NEDS), the largest all-payer ED database in the United States involving 945 hospitals in 33 states and Washington, DC, were analyzed to assess changes in prevalence and rates of ED visits involving acute and chronic alcohol consumption by age and sex over time among persons aged ≥12 between 2006 and 2014. Results Between 2006 and 2014, the number of ED visits involving alcohol consumption increased 61.6%, from 3,080,214 to 4,976,136. The rate increased 47% from 1,223 to 1,802 per 100,000 population and the total cost of such visits increased 272% from $4.1 billion to $15.3 billion. The number of acute alcohol-related ED visits increased 51.5% from 1,801,006 to 2,728,313 and the rate increased 40% from 720.9 to 1,009.6 per 100,000 population. The number chronic alcohol-related visits increased 75.7% from 1,279,208 to 2,247,823 and the rate increased 57.9% from 502.2 to 792.9 per 100,000. The annual percentage change in rates of all alcohol-related ED visits was larger for females than for males (5.3% vs. 4.0%). Other drug involvement increased the likelihood of admission for inpatient treatment. Conclusions Alcohol consumption contributed to an increasing number of ED visits in the United States between 2006 and 2014, especially among females. Increased utilization of evidence-based interventions is needed.

Background: Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D [25(OH)D] concentration

Background The association of antiseizure medication (ASM) and bone density abnormalities has long been recognized; however, there remains a lack of consensus on efficacy and optimal vitamin D dosing in patients receiving enzyme inducing and non-inducing ASMs. The objective was to explore the relationship between ASMs and vitamin D supplementation requirements in a population of adult patients with epilepsy. Methods Patients with a diagnosis of epilepsy receiving supplemental vitamin D were included in this retrospective chart review. All instances of 25-hydroxyvitamin D3 (25−OHD) measured among those patients were compared between patients taking an enzyme inducing antiseizure medication (EIASM) to patients receiving ASM regimens only containing non-enzyme inducing antiseizure medications (NIASM). ASM use, prescription and over the counter (OTC) vitamin D use, 25-OHD plasma concentration, presence of chronic kidney disease (CKD), age, gender, and ethnicity were collected. Multiple linear regression was used to adjust for potentially confounding variables; the model included a cluster by participant term to account for repeated patients in the dataset. Results There were 542 vitamin D levels evaluated from 172 unique patients. There was an 11.5 % higher absolute percent increase in patients who achieved a 25-OHD level over 30 ng/mL in the NIASM (p = 0.012). Patients on EIASMs were supplemented with an additional 508 units of vitamin D daily (95 %CI 136–878, p = 0.007). When adjusted for CKD, OTC vitamin D use, OTC multivitamin use, age, gender, and ethnicity, patients on EIASMs were supplemented with an additional 445 units of vitamin D (95 %CI -69 to 960, p = 0.089) compared to NIASM use. Conclusions Patients taking EIASMs had an increase in vitamin D deficiency and vitamin D supplementation suggesting that EIASMs impact vitamin D metabolism. Closer monitoring of vitamin D status in patients with epilepsy, especially those on EIASMs, is warranted. This evaluation suggests that for patients taking ASM, use of a lower dose OTC requires closer monitoring of vitamin D status in patients with epilepsy, especially those on EIASMs, is warranted. vitamin D agent may not be adequate.

Significance Nucleotides are biosynthesized from a ribose pyrophosphate species, with enzymes and magnesium ions as catalysts. Magnesium ions alone can perform this process with synthetic ribose pyrophosphates, by binding to the pyrophosphate groups and linking cyclic nitrogen bases to the ribose. In a kinase mimic ATP transfers the terminal phosphate to receptors catalyzed by magnesium ions, binding to pyrophosphate groups, but magnesium ion at low concentrations inhibits the process, and only at higher concentrations is it a catalyst. This affords great insight into the mechanism of ATP phosphorylation, a fundamental biological process. Our enzyme mimics could well operate on prebiotic earth before enzymes were present. This work strengthens the picture of how important components of life could have been formed prebiotically.

Note from the Editor: August Kekule's dream and his theory of the structure of benzene (1865). Julius Thomsen's logic that benzene contains equivalent electrons between its carbon atoms (ca. 1900). Richard Willstatter's synthesis and observation that 1,3,5,7-cyclooctatetraene is not aromatic (early 1900s). Sir Robert Robinson's proposal of the ‘aromatic sextet’ (1925). Erich Huckel's molecular orbital treatment of benzene and other unsaturated compounds that separated sigma and pi electrons (early 1930s). Tetsuo Nozoe and Michael Dewar's independent proposal of a new aromatic structure for a cycloheptatrieneone (mid-1940s). Ronald Breslow's demonstration that cyclopropenyl cation is aromatic (1958). Breslow's proposal of ‘antiaromaticity’ (1965). I offer several comments. First, in the field of aromaticity, Breslow is in mighty exceptional company. Second, his research has extended well beyond aromaticity. Yes, Breslow is applying the concepts of antiaromaticity toward the development of highly conductive organic materials. But his research has encompassed enzyme mimics, remote functionalization reactions, unnatural DNA analogues and cancer chemotherapy. Indeed, Breslow is the co-discoverer of a highly successful drug, Vorinostat, which is FDA-approved for the treatment of cutaneous T-cell lymphoma. We thank Professor Breslow for joining our project honoring his and our friend, Tetsuo Nozoe. Tetsuo Nozoe would have would have been deeply touched. —Jeffrey I. Seeman Guest Editor University of Richmond Richmond, Virginia 23173, USA E-mail: jseeman@richmond.edu Ronald Breslow with Koji Nakanishi (left) and Tetsuo Nozoe (center), Sendai, 1970. Abstract We contributed to the field of non-benzenoid aromatic compounds by creating the cyclopropenyl cation and various of its derivatives, including cyclopropenone; it was the first aromatic system with other than six pi electrons in a single ring, and the simplest aromatic system. The pioneering work of Tetsuo Nozoe in tropolone chemistry was celebrated with the founding of ISNA, the International Symposium on Non-Benzenoid Aromatic Compounds, where I described our work in the field. It fit the prediction that aromaticity would be found in systems with 4n + 2 pi electrons, where n is an integer. I was also concerned with the properties of monocyclic systems with 4n cyclically conjugated pi electrons. They were expected not to be aromatic, but the interesting question was whether they were actually antiaromatic, especially destabilized by the cyclic conjugation in such 4n species as the cyclopropenyl anion, cyclobutadiene, and cyclopentadienyl cation. The evidence supports antiaromaticity in these cases. We also examined compounds where 4n cyclic pi systems were fused with aromatic systems, and most interestingly systems in which two 4n pi systems were fused. In these cases the periphery of the molecules had 4n + 2 pi electrons, for aromaticity, but the components were antiaromatic. Recently we have studied electrical conductivities in aromatic molecules such as thiophene and saw that aromaticity added resistance to the systems, so non-aromatic compounds are better conductors and antiaromatic compounds are predicted to be the best of all.

Vitamin D deficiency, defined by the total serum 25-hydroxyvitamin D [25(OH)D] level, is common and more prevalent among Blacks than whites. Vitamin D-binding protein (DBP) levels vary with race and may modulate "bioavailable" levels of 25(OH)D.To determine the effect of DBP levels on the functional response to vitamin D.A randomized, placebo-controlled trial of vitamin D repletion for 2 mo, which took place at an outpatient research unit. Participants included 150 vitamin D-deficient (25(OH)D20 ng/mL) adults. Participants were randomly assigned to receive either 50,000 IU of vitamin D3 or placebo weekly for 8 weeks. This is a post-hoc analysis using DBP, 25(OH)D, PTH, and calcium levels.Blacks had lower total 25(OH)D (12 vs 15 ng/mL, P.001) and DBP levels (119 vs 234 μg/mL, P.001) than non-Blacks. DBP levels were similar before and after vitamin D3 or placebo treatment (r = 0.98, P.001). Baseline total 25(OH)D levels were a significant determinant of baseline PTH levels (P.001). The change in total 25(OH)D was associated with the change in PTH (P0.001) and calcium levels (P.05). In contrast, DBP levels were not a determinant of baseline PTH (P = .57) nor significantly related to changes in either PTH (P = .53) or calcium levels (P = .88).DBP levels are stable in Blacks and non-Blacks, and do not change with correction of vitamin D deficiency. Even for individuals with total 25(OH)D levels20 ng/mL, Blacks have significantly lower DBP levels than non-Blacks. However, within this range of total 25(OH)D, DBP levels do not influence the effect of vitamin D repletion on PTH or calcium levels.

We describe the syntheses of thiophene, cyclopentadienone and dimethylketalcyclopentadienone derivatives and their single molecule electrical conductivities. The ketone had the lowest conductivity of the three, although it has some antiaromaticity. The carbonyl group is electron attracting and its ionization potential outweighs any conductivity increase that the antiaromaticity effect might bring.

The synthesis of an excess of D-glyceraldehyde by coupling glycolaldehyde with formaldehyde under prebiotic conditions is catalyzed by L amino acids having primary amino groups at acidic pH's, but at neutral or higher pH's they preferentially form L-glyceraldehyde. L Amino acids having secondary amino groups, such as proline, have the reverse preferences, affording excess L-glyceraldehyde at low pH but excess D-glyceraldehyde at higher pHs. Detailed mechanistic proposals make these preferences understandable. The relevance of these findings to the origin of D sugars on prebiotic Earth is described.

Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs.

Objective Obesity and diabetes are particularly high in indigenous populations exposed to a Western diet and lifestyle. The prevalence of obesity, diabetes, hyperglycemia, dyslipidemia, and hypertension in one such population, the Micronesian island of Kosrae was described. Design and Methods Longitudinal screenings for metabolic traits were conducted on adult Kosraens ≥ 20 years of age in 1994 and again in 2001. Data was obtained on 3,106 Kosraens, comprising ∼80% of the adult population. Diabetes was diagnosed using World Health Organization guidelines. Prevalences of obesity, hyperglycemia, dyslipidemia, and hypertension were assessed. Results The overall age-adjusted prevalence of diabetes increased from 14 to 21%. The most significant change observed in the population was increases in obesity and hyperglycemia, especially among young Kosraens and women. Obesity age-adjusted prevalence increased from 45 to 62%, and hyperglycemia age-adjusted prevalence increased from 19 to 44%. Of note, Kosraens as a group had unusually low high density lipoprotein (HDL) levels with 80% classified as low HDL by NCEP-ATPIII criteria, despite lacking the usually accompanying increase in triglycerides. Comparison to reports from other populations shows that Kosrae experiences one of the highest rates of obesity, hyperglycemia, and low HDL globally while maintaining relatively healthy levels of triglycerides. Conclusion Our study shows a dramatic increase in obesity and hyperglycemia in Kosrae in just 7 years and forebodes significantly increased health risks for this part of the world.

Vitamin D deficiency is common in the general population and even more prevalent in patients with chronic kidney disease (CKD). Low 25-hydroxyvitamin D [25(OH)D] levels have been associated with cardiovascular disease, though a definitive mechanistic link has not been established. Further, it is unclear if repleting vitamin D mitigates the excess risk observed in epidemiologic studies. Because vitamin D may regulate innate immunity and gut epithelial differentiation, we hypothesized that oral cholecalciferol (D3) would result in decreased blood endotoxin activity, a potential risk factor for cardiovascular disease. STUDY DESIGN, SETTINGPARTICIPANTS, INTERVENTION: We studied 12 stable outpatients with CKD stage 3 and 25(OH)D deficiency, who received D3 30,000 units weekly for 8 weeks. The primary endpoint was the change in blood endotoxin activity.Baseline endotoxin activity correlated with 25(OH)D levels (r = -0.60, p = 0.04). Endotoxin activity decreased by 25% from baseline (p = 0.03). Despite the decrease in endotoxin activity, there was no change in intestinal permeability.The results of this study suggest that vitamin D repletion therapy may have an effect on endotoxin activity in early CKD. Further intervention studies using vitamin D in the CKD population are required.

Background HDL cholesterol levels are inversely correlated with coronary heart disease risk in humans, and in animal studies, HDL elevation decreases formation and progression of foam-cell lesions. The potential for HDL to affect preexisting advanced atherosclerotic lesions is not known. To approach this issue, we used a novel mouse aortic transplantation model. Methods and Results ApoE-deficient (EKO) mice were fed a Western-type diet for 6 months, and thoracic aortic segments containing advanced lesions replaced segments of the abdominal aorta of 4-month-old EKO syngeneic mice not expressing (plasma HDL cholesterol ≈26 mg/dL) or expressing (HDL ≈64 mg/dL) a human apoAI (hAI) transgene. Both types of recipients had comparable non-HDL cholesterol levels. Five months after transplantation, mice were killed and grafts analyzed. Compared with lesion area in pretransplant mice (0.14±0.04 mm 2 , mean±SEM), there was progression in the EKO recipients (0.39±0.06 mm 2 , P 2 , P 80% ( P 300% ( P Conclusions Increasing HDL cholesterol levels in EKO mice retards progression of advanced atherosclerotic lesions and remodels them to a more stable-appearing phenotype.

Molecular electrical conductivities between gold electrodes have been measured at the single molecule level through a variety of systems. The results show that the aromaticity of molecules is partly disrupted by passage of electricity, leading to increased resistance. The possibility of the opposite effect resulting from the disruption of antiaromaticity was explored in biphenylene derivatives. When gold atoms terminate benzene or polymethylene molecules they incorporate into the electrodes, greatly diminishing the resistance from other terminating atoms. In paracyclophanes with two, three, and four benzenes in stacks the gold electrodes can directly contact them, and thus measure conductivity directly through the stacks.

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2(Akita)Ldlr⁻/⁻). Ins2(Akita)Ldlr⁻/⁻ mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr⁻/⁻ mice, 20-week-old Ins2(Akita)Ldlr⁻/⁻ mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P0.001) in males and 30% (P0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2(Akita)Ldlr⁻/⁻ mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2(Akita)Ldlr⁻/⁻ mice. Thus, the Ins2(Akita)Ldlr⁻/⁻ mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.

Charge transport across metal-molecule interfaces has an important role in organic electronics. Typically, chemical link groups such as thiols or amines are used to bind organic molecules to metal electrodes in single-molecule circuits, with these groups controlling both the physical structure and the electronic coupling at the interface. Direct metal-carbon coupling has been shown through C60, benzene and π-stacked benzene, but ideally the carbon backbone of the molecule should be covalently bonded to the electrode without intervening link groups. Here, we demonstrate a method to create junctions with such contacts. Trimethyl tin (SnMe(3))-terminated polymethylene chains are used to form single-molecule junctions with a break-junction technique. Gold atoms at the electrode displace the SnMe(3) linkers, leading to the formation of direct Au-C bonded single-molecule junctions with a conductance that is ∼100 times larger than analogous alkanes with most other terminations. The conductance of these Au-C bonded alkanes decreases exponentially with molecular length, with a decay constant of 0.97 per methylene, consistent with a non-resonant transport mechanism. Control experiments and ab initio calculations show that high conductances are achieved because a covalent Au-C sigma (σ) bond is formed. This offers a new method for making reproducible and highly conducting metal-organic contacts.

Steroidogenic acute regulatory protein (StAR)D4 is a member of the StAR related lipid transfer family. Homology comes from the approximately 210 amino acid lipid binding domain implicated in intracellular transport, cell signaling, and lipid metabolism. StARD4 was identified as a gene downregulated 2-fold by dietary cholesterol (Soccio, R. E., R. M. Adams, K. N. Maxwell, and J. L. Breslow. 2005. Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. J. Biol. Chem. 280: 19410-19418). A mouse knockout was created to investigate StARD4's functionality and role in lipid metabolism. Homozygous knockout mice exhibited normal Mendelian mating genetics, but weighed less than wild-type littermates, an effect not accounted for by energy metabolism or food intake. Body composition as analyzed by DEXA scan showed no significant difference. No significant alterations in plasma or liver lipid content were observed on a chow diet, but female knockout mice showed a decrease in gallbladder bile cholesterol and phospholipid concentration. When challenged with a 0.2% lova-statin diet, StARD4 homozygous mice exhibited no changes. However, when challenged with a 0.5% cholesterol diet, female StARD4 homozygous mice showed a moderate decrease in total cholesterol, LDL, and cholesterol ester concentrations. Microarray analysis of liver RNA found few changes. However, NPC1's expression, a gene not on the microarray, was decreased approximately 2.5-fold in knockouts. These observations suggest that StARD4's role can largely be compensated for by other intracellular cholesterol transporters.

Rationale : The complications of atherosclerosis are a major cause of death and disability in type 2 diabetes. Defective clearance of apoptotic cells by macrophages (efferocytosis) is thought to lead to increased necrotic core formation and inflammation in atherosclerotic lesions. Objective : To determine whether there is defective efferocytosis in a mouse model of obesity and atherosclerosis. Methods and Results : We quantified efferocytosis in peritoneal macrophages and in atherosclerotic lesions of obese ob/ob or ob/ob;Ldlr −/− mice and littermate controls. Peritoneal macrophages from ob/ob and ob/ob;Ldlr −/− mice showed impaired efferocytosis, reflecting defective phosphatidylinositol 3-kinase activation during uptake of apoptotic cells. Membrane lipid composition of ob/ob and ob/ob;Ldlr −/− macrophages showed an increased content of saturated fatty acids (FAs) and decreased ω-3 FAs (eicosapentaenoic acid and docosahexaenoic acid) compared to controls. A similar defect in efferocytosis was induced by treating control macrophages with saturated free FA/BSA complexes, whereas the defect in ob/ob macrophages was reversed by treatment with eicosapentaenoic acid/BSA or by feeding ob/ob mice a fish oil diet rich in ω-3 FAs. There was also defective macrophage efferocytosis in atherosclerotic lesions of ob/ob;Ldlr −/− mice and this was reversed by a fish oil–rich diet. Conclusions : The findings suggest that in obesity and type 2 diabetes elevated levels of saturated FAs and/or decreased levels of ω-3 FAs contribute to decreased macrophage efferocytosis. Beneficial effects of fish oil diets in atherosclerotic cardiovascular disease may involve improvements in macrophage function related to reversal of defective efferocytosis and could be particularly important in type 2 diabetes and obesity.

The nuclear hormone receptor pregnane X receptor (PXR; also called SXR) functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Although many clinically relevant PXR ligands have been shown to affect cholesterol levels, the role of PXR in cholesterol homeostasis and atherosclerosis has not been thoroughly investigated. Here, we report that activation of PXR by feeding the PXR agonist pregnenolone 16α-carbonitrile (0.02%) for 2 weeks to wild-type (WT) mice significantly increased total cholesterol levels and atherogenic lipoproteins VLDL and LDL levels, but had no effect in PXR knockout (PXR−/−) mice. Chronic PXR activation in atherosclerosis prone apolipoprotein E deficient (ApoE−/−) mice was found to decrease HDL levels and increase atherosclerotic cross-sectional lesion area at both the aortic root and in the brachiocephalic artery by 54% (P < 0.001) and 116% (P < 0.01), respectively. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV, and CYP39A1, in both WT and ApoE−/− mice. Furthermore, PXR activation can increase CD36 expression and lipid accumulation in peritoneal macrophages of ApoE−/− mice. In summary, PXR activation in WT mice increases levels of the atherogenic lipoproteins VLDL and LDL, whereas in ApoE−/− mice, PXR increases atherosclerosis, perhaps by diminishing levels of the antiatherogenic ApoA-IV and increasing lipid accumulation in macrophages.—Zhou, C., N. King, K. Y. Chen, and J. L. Breslow. Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice.

The crystal structure of bovine neurophysin-II in its liganded state (Chen et al. [1991] Proc. Natl. Acad. Sci. USA 88, 4240-4244) indicates that the 1-6 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the monomer-monomer interface. Cleavage of the 1-6 sequence by Staphylococcus protease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 1-6 sequence in the unliganded state were obtained by NMR and related biophysical comparisons of the native and des-1-6 proteins. NMR spectra demonstrated that the environment and/or conformation of residues in the 1-6 sequence differed in liganded and unliganded states. Additionally, the unliganded des-1-6 protein exhibited a dimerization constant four to five times that of the native protein, potentially accounting for the observation that its peptide affinity was also increased. NMR studies further indicated that the increased dimerization constant of the des-1-6 protein correlated with the presence in the native protein of two isoenergetic forms of the monomer, in contrast to only a single form in the des-1-6 protein, as evidenced by signals from an internal dimerization-sensitive alpha-proton. Thus, the 1-6 sequence reduces the dimerization constant by stabilization of an alternative monomer conformation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des-1-6 protein with an internal clip after binding site residue Glu-47, the clip presumably breaking the short 3,10 helix that most directly connects the interface to the interface to the binding site. This product, although unable to bind peptide, retained the dimerization constant of the des-1-6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the effects of the 1-6 sequence. A model is proposed in which the 1-6 sequence stabilizes the second conformation of the unliganded monomer via interactions affecting the loop region that separates the two neurophysin domains and which has been shown to influence neurophysin self-association.

Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr-35, a residue that provides a potential probe of the monomer-monomer interface and of allosteric interrelationships between this region and the binding site. Mesotocin-associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel-filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr-35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non-denaturing conditions. Reaction of Tyr-35 of the mesotocin-associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr-35 to an unusual 237 nm band in the mesotocin-associated protein. Small shifts in the 350 nm CD band of nitrated Tyr-35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr-35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin-associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.

Chiral polyamines can be utilized for a variety of potential applications, ranging from asymmetric catalysis to nonviral gene delivery systems for DNA and RNA. They can also be utilized to solubilize carbon nanotubes. Thus, methods for the straightforward synthesis of chiral polyamines are needed. We present herein two synthetic strategies for accessing chiral polyamines. The potential of these chiral amines to catalyze two organic reactions with a high degree of chiral induction was also explored.Text: Chiral polyamines have been utilized for a variety of applications. First, polyamines are polycationic at neutral pH; as such, they interact strongly with both DNA and RNA.1 They can therefore be utilized as effective nonviral gene delivery agents.2 Second, chiral polyamines are efficient catalysts for various organic transformations.3 Polyamines have also been used to solubilize carbon nanotubes.4 Finally, chiral polyamines are excellent ligands for many transition metals.5 Due to their numerous applications, high-yielding synthetic strategies for their preparation are in great demand. We present herein two synthetic strategies for accessing chiral polyamines, and the potential of these chiral amines to catalyze two organic reactions.

PAMAM dendrimers have been constructed with a pyridoxamine core, and chiral capping amino groups. Transamination to form phenylalanine and alanine from their related ketoacids produced enantioselectivities induced by the formally remote chiral caps, supporting computer models that indicate folding of the dendrimer chains back into the core region.

Objective: The aim of this study is to identify risk factors for the loss of measurable plasma C-peptide in newly diagnosed 15- to 35-year-old diabetic subjects. Methods: This Swedish study included 778 subjects. C-peptide levels were obtained each year for 6 years after diagnosis. Loss of measurable C-peptide was defined as a level at or below the lower detection limit of the local assay (0.13 nmol/1). In addition to C-peptide, other baseline covariates included gender, age, body mass index, HLA genotype, and autoantibody levels. Results: Compared with autoantibody-negative subjects, autoantibody-positive subjects had lower median baseline C-peptide (0.27 vs. 0.50, P

In our quest to understand why dimethyl sulfoxide (DMSO) can cause growth arrest and terminal differentiation of transformed cells, we followed a path that led us to discover suberoylanilide hydroxamic acid (SAHA; vorinostat (Zolinza)), which is a histone deacetylase inhibitor. SAHA reacts with and blocks the catalytic site of these enzymes. Extensive structure-activity studies were done along the path from DMSO to SAHA. SAHA can cause growth arrest and death of a broad variety of transformed cells both in vitro and in tumor-bearing animals at concentrations not toxic to normal cells. SAHA has many protein targets whose structure and function are altered by acetylation, including chromatin-associated histones, nonhistone gene transcription factors and proteins involved in regulation of cell proliferation, migration and death. In clinical trials, SAHA has shown significant anticancer activity against both hematologic and solid tumors at doses well tolerated by patients. A new drug application was approved by the US Food and Drug Administration for vorinostat for treatment of cutaneous T-cell lymphoma. More potent analogs of SAHA have shown unacceptable toxicity.

Significance We discovered an improved hydroxamic acid-based small-molecule N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)methyl)benzamide)] that selectively inhibits histone deacetylase 6 catalytic activity in vivo and in vitro. This can have important anticancer activity.

Background Rapid drug desensitization (RDD) is used to address hypersensitivity reactions to chemotherapeutics and monoclonal antibodies, allowing patients to be treated with optimal pharmacological agents. RDD protocols are tailored to each individual patient's reaction and needs, and protect against anaphylaxis, but overall risks, costs, and benefits have not been determined. Objective We investigated the safety, efficacy, costs, and life expectancy of patients in a large population undergoing RDD. Methods We analyzed 2177 RDD procedures performed in 370 patients with cancer, vasculitis, and hematological and connective tissue diseases who presented 402 reactions. A subgroup of carboplatin allergic patients with ovarian cancer treated with RDD was analyzed for costs and life expectancy and compared with a nonallergic control group. Results RDD allowed all patients to receive safely the full dose of the medication to which they were reactive. A gradual increase in the fraction of outpatient desensitizations from 81% to 98% was achieved through risk stratification. Of the 2177 desensitizations, 93% had no or mild reactions whereas 7% had moderate to severe reactions, which did not preclude the completion of the treatment, and there were no deaths. Overall health costs in the carboplatin allergic group were not higher than those in the nonallergic group treated with standard of care. Administration of carboplatin through RDD was as effective as standard administration with a nonsignificant increase in life expectancy in desensitized patients as compared with nonallergic, nondesensitized controls. Conclusions RDD is cost effective and safe for allergic patients with cancer and chronic disease to remain on first line therapy.

An intercross between C57BL/6J and CASA/Rk mice was used to study the genetics of biliary bile acid composition. In parental strains, male C57BL/6J mice had significantly higher cholic acid (CA; 14%) and lower beta-muricholic acid (betaMC; 27%) than CASA/Rk mice, whereas females did not differ. However, quantitative trait locus analysis of F2 mice revealed no significant chromosome 9 loci in males but loci in females on chromosome 9 for percentage CA (%CA) at 72 centimorgan (cM) [logarithm of the odds (LOD) 5.89] and %betaMC at 54 cM (LOD 4.09). Chromosome 9 congenic and subcongenic strains representing CASA/Rk intervals 38-73 cM (9KK) and 68-73 cM (9DKK) on the C57BL/6J background were made. In 9KK and 9DKK males, %CA was increased and %betaMC was unchanged, whereas in 9KK but not 9DKK females, %CA was increased and %betaMC was decreased. Sterol 12alpha-hydroxylase (Cyp8b1) channels bile acid precursors into CA and maps at chromosome 9 (73 cM). However, there was no significant difference in Cyp8b1 mRNA or enzymatic activity between parental mice, parental-congenic-subcongenic mice, or high-low biliary %CA F2 mice. In summary, two chromosome 9 loci control sexually dimorphic effects on biliary bile acid composition: a distal (68-73 cM) major determinant in males, and a more proximal (38-68 cM) major determinant in females. In this intercross, Cyp8b1, a strong candidate, does not appear to be responsible.

Molecular electronics is often limited by the poorly defined nature of the contact between the molecules and the metal surface. We describe a method to wire molecules into gaps in single-walled carbon nanotubes (SWNTs). Precise oxidative cutting of a SWNT produces carboxylic acid–terminated electrodes separated by gaps of ≤10 nanometers. These point contacts react with molecules derivatized with amines to form molecular bridges held in place by amide linkages. These chemical contacts are robust and allow a wide variety of molecules to be tested electrically. In addition to testing molecular wires, we show how to install functionality in the molecular backbone that allows the conductance of the single-molecule bridges to switch with pH.

Associations between alcohol drinking and cardiovascular disease mortality could be confounded by diet if alcohol drinking and diet are related. Depending on the alcohol measure, alcohol-diet relations may or may not be observed. The authors examined associations between alcohol and diet quality (Healthy Eating Index (HEI) scores) using cross-sectional, nationally representative data from the 1999-2000 National Health and Nutrition Examination Survey. Weighted analyses included 3,729 participants agedor =20 years. In adjusted analyses among current alcohol drinkers, as quantity increased from 1 toor =3 drinks/drinking day, the mean HEI score decreased from 65.3 (95% confidence interval (CI): 63.4, 67.1) to 61.9 (95% CI: 60.5, 63.2). As frequency increased from the lowest quartile to the highest, the mean HEI score increased from 60.9 (95% CI: 58.7, 63.2) to 64.9 (95% CI: 63.4, 66.4). As average volume ((quantity x frequency)/365.25) increased from1 drink/day toor =3 drinks/day, the mean HEI score increased from 62.9 (95% CI: 61.2, 64.5) to 65.2 (95% CI: 62.7, 67.8). In stratified analyses, the lowest HEI score, 58.5 (95% CI: 55.5, 61.5), occurred among drinkers who consumed the highest quantity at the lowest frequency. Average volume of alcohol consumed is driven by and masks the contributions of its components. These results suggest the importance of measuring drinking patterns (quantity, frequency, and stratified combinations) in epidemiologic alcohol-diet studies.

Amine boranes bearing hydrophobic substituents were used to reduce aryl ketones in competition with a methyl ketone. Their high stability in protic solvents combined with their ease of preparation made amine boranes useful compounds in the study of hydrophobically directed selective reductions. Several characteristics of the reducing agent were found to be important in determining the reaction selectivity, including available hydrocarbon surface area, degree of fluorination, and proximity of the hydrophobic group to the active hydrides.

In November 2013, the American Heart Association (AHA)/American College of Cardiology (ACC) released a new Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults.1 Recommendations were made for several groups of patients. This perspective will focus only on the recommendation for those with no history of atherosclerotic cardiovascular disease (ASCVD) but with a 10-year ASCVD risk of ≥7.5% and a low-density lipoprotein (LDL) cholesterol level of 70 to 189 mg/dL and who are between the ages of 40 to 75 years. The guideline recommends such individuals receive moderate-intensity statin therapy to reduce LDL cholesterol between 30% and 49%. The guideline specifies that the 10-year risk of ASCVD, defined as nonfatal myocardial infarction (MI), coronary heart disease death, and nonfatal and fatal stroke, be calculated using the pooled cohort equations. The pooled cohort equations represent a new algorithm for estimating risk, and the spreadsheet for risk calculation can be downloaded from http://my.americanheart.org/professional/StatementsGuidelines/PreventionGuidelines/Prevention-Guidelines\_UCM\_457698_SubHomePage.jsp. Unfortunately, this algorithm was announced simultaneously with the new guideline without a prior period for external scientific evaluation. In fact, the algorithm has already been severely criticized.2 Ridker and Cook “calculated predicted 10-year risks of the same atherosclerotic events using the new ACC/AHA risk prediction algorithm and compared these estimates with observed event rates in 3 large-scale primary prevention cohorts, the Women’s Health Study, the Physicians’ Health Study, and the Women’s Health Initiative Observational Study.” They found “in all 3 of these primary prevention cohorts that the new ACC/AHA risk prediction algorithm systematically overestimated observed risks by 75% to 150%, roughly doubling the actual observed risk.” Ridker and Cook also found a similar overestimation of risk in 2 additional cohorts, prompting them to observe “that as many as 40% to 50% of the 33 million middle-aged Americans targeted by the new …

We have measured the conductance of single-molecule junctions created with three different molecular wires using the scanning tunneling microscope-based break-junction technique. Each wire contains one of three different cyclic five-membered rings: cyclopentadiene, furan, or thiophene. We find that the single-molecule conductance of these three wires correlates negatively with the resonance energy of the five-membered ring; the nonaromatic cyclopentadiene derivative has the highest conductance, while the most aromatic of this series, thiophene, has the lowest. Furthermore, we show for another wire structure that the conductance of furan-based wires is consistently higher than for analogous thiophene systems, indicating that the negative correlation between conductance and aromaticity is robust. The best conductance would be for a quinoid structure that diminishes aromaticity. The energy penalty for partly adopting the quinoid structure is less with compounds having lower initial aromatic stabilization. An additional effect may reflect the lower HOMOs of aromatic compounds.

The first functional catalytic mimic of the enzyme dialkylglycine decarboxylase is described. This system utilizes a hydrophobically modified polyethylenimine polymer, a pyridoxamine cofactor, and a 2-aryl-2-alkylglycine sacrificial amine source to convert alpha-keto acids to alpha-amino acids at biologically relevant temperatures with multiple turnovers of the pyridoxamine catalyst. The effects of hydrophobic and electronic factors in the 2,2-disubstituted sacrificial amine source and the pyridoxamine catalyst on turnover frequency and turnover number are explored.

Fractalkine (CX3CL1) is of particular interest in atherogenesis because it can serve as an adhesion molecule and a chemokine. Fractalkine and its receptor CX3CR1 are expressed in atherosclerotic lesions of humans and mice. However, the effect of fractalkine deficiency on atherosclerosis susceptibility is unknown. Fractalkine-deficient mice on the C57BL/6 (B6) background were bred to the atherosclerosis-sensitizing B6.ApoE -/- and B6.LDLR -/- backgrounds. Compared with controls, aortic-root lesion area was unchanged in fractalkine-deficient male and female B6.ApoE -/- mice at 16 weeks of age and males at 12 weeks of age, but it was mildly reduced (30%, P = 0.005) in females at 12 weeks of age. In contrast, lesion area at the brachiocephalic artery (BCA) was reduced dramatically by ≈85% in fractalkine-deficient females [42,251 ± 26,136 μm 2 ( n = 15) vs. 6,538 ± 11,320 μm 2 ;( n = 24), P < 0.0001] and males [36,911 ± 32,504 μm 2 ( n = 24) vs. 6,768 ± 8,595 μm 2 ( n = 14); P = 0.001] at 16 weeks of age. Fractalkine-deficient B6.ApoE -/- mice were comparable with controls in body weight, plasma cholesterol, plasma high-density lipoprotein cholesterol and white blood cell counts. On the B6.LDLR -/- background, lesion areas were reduced by 35% at the aortic root ( P < 0.01) and by 50% at the BCA ( P < 0.05) in fractalkine-deficient females at 16 weeks of age. Lesions in fractalkine-deficient mice on the B6.ApoE -/- and B6.LDLR -/- backgrounds were less complex and contained significantly fewer macrophages than controls. In conclusion, the major reduction of atherosclerosis in fractalkine-deficient mice appears to be at the BCA rather than the aortic root.

Screening of 932 adults on the Pacific island of Kosrae for plasma plant sterol levels disclosed three subjects, two of them asymptomatic, with phytosterolemia. Sequencing the ATP binding cassette subfamily G member 8 (ABCG8) gene revealed a novel exon 2 mutation that causes a change in codon 24 from glutamine to histidine and a frame shift followed by a premature stop codon, precluding the formation of a functional ABCG8 protein. Genotyping of 1,090 Kosraens revealed 150 as carriers, a 13.8% carrier rate. DNA sequencing of 67 carriers revealed the same mutation as in the probands. In carriers, plasma campesterol and sitosterol levels were 55% and 30% higher, respectively, than in noncarriers. Moreover, compared with noncarriers, carriers showed 21% lower plasma levels of lathosterol, a surrogate marker for cholesterol biosynthesis. There was no difference between the groups in plasma total cholesterol, triglycerides, apolipoprotein B, or apolipoprotein A-I levels. In summary, on the island of Kosrae, a strong founder effect of a mutant ABCG8 allele results in a large number of carriers with increased plasma plant sterol levels and decreased lathosterol levels. The latter finding suggests that heterozygosity for a mutated ABCG8 allele results in a modest increase in dietary cholesterol absorption and a decrease in cholesterol biosynthesis.

Intracellular cholesterol transport is essential for the maintenance of cholesterol homeostasis. Many aspects of cholesterol metabolism are well-known, including its synthesis in the endoplasmic reticulum, its extracellular transport in plasma lipoproteins, its uptake by the low-density lipoprotein receptor, and its regulation of SREBP and LXR transcription factors. These fundamental pathways in cholesterol metabolism all rely on its proper intracellular distribution among subcellular organelles and the plasma membrane. Transport involving the ER and endosomes is essential for cholesterol synthesis, uptake, and esterification, whereas cholesterol catabolism by enzymes in mitochondria and ER generates steroids, bile acids, and oxysterols. Cholesterol is a highly hydrophobic lipid that requires specialized transport in the aqueous cytosol, involving either vesicles or nonvesicular mechanisms. The latter includes hydrophobic cavity transporters such as StAR-related lipid transfer (START) proteins. Molecular understanding of intracellular cholesterol trafficking has lagged somewhat behind other aspects of cholesterol metabolism, but recent advances have defined some transport pathways and candidate proteins. In this review, we discuss cholesterol transport among specific intracellular compartments, emphasizing the relevance of these pathways to cholesterol homeostasis.

These studies were aimed at an initial characterization of the human vasopressin precursor and the evaluation of factors leading to misfolding by the pathological 87STOP mutation. This mutation deletes the precursor's glycosylated copeptin segment, which has been considered unnecessary for folding, and the last seven neurophysin residues. We investigated the role in folding of the last seven neurophysin residues by comparing the properties of the 87STOP precursor and its derivative neurophysin with those of the corresponding wild-type proteins from which copeptin had been deleted, leading to the following conclusions. First, despite modulating effects on several protein properties, the last seven neurophysin residues do not make a significant net thermodynamic contribution to precursor folding; stabilities of the mutant and wild-type precursors to both guanidine denaturation and redox buffer unfolding are similar, as are in vitro folding rates. Second, the monomeric forms of both precursors are unstable and predicted to fold inefficiently at physiological pH and temperature, as evidenced by precursor behavior in redox buffers and by thermodynamic calculations. Third, both precursors are significantly less stable than the bovine oxytocin precursor. These results, together with earlier studies elsewhere of vasopressin precursor behavior within rat neurons, are shown to represent a self-consistent argument for a role for glycosylated copeptin in vasopressin precursor folding in vivo, copeptin most probably assisting refolding by facilitating interaction of misfolded monomers with the calnexin/calreticulin system. This hypothesis provides an explanation for the absence of copeptin in the more stable oxytocin precursor and suggests that the loss of copeptin contributes to 87STOP pathogenicity. Reported cell culture studies of rat precursor folding are also discussed in this context. Most generally, the results emphasize the significance of monomer stability in the folding pathways of oligomeric proteins.

The quantitative effect of cosolvents on the water solubility of hydrophobic substrates can be correlated with the effect on reaction rates to determine the geometries of transition states for Diels-Alder reactions, the benzoin condensation, and alkylations of phenoxide ions and aniline. Some of these reactions have transition states with packing of hydrophobic surfaces and some do not. Methods were devised to sort out the effect of the cosolvents on solvation of hydrophobic surfaces and the effect on solvation of polar groups. The result is a set of geometries for these reactions that is consistent with theory.

Dietary cholesterol regulation of cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classical pathway of bile acid synthesis, has been implicated in plasma cholesterol responsiveness. In the current study, the effects of 0.0% and 0.5% cholesterol diets were examined in Cyp7a1 knockout (KO), heterozygous Cyp7a1 KO (Het), and human Cyp7a1 transgenic mice on the mouse Cyp7a1 KO background (Tg+KO). We confirmed previous findings that dietary cholesterol increased mouse Cyp7a1 activity in Het mice but decreased human Cyp7a1 activity in Tg+KO mice. However, in both Het and Tg+KO mice, dietary cholesterol increased bile acid pool size (36% and 72%, respectively) and fecal bile acid excretion (2.2- and 3.6-fold, respectively). The expression of cholesterol 27-hydroxylase (Cyp27), the major enzyme of the alternative pathway of bile acid synthesis, was not significantly different in cholesterol-fed KO, Het, or Tg+KO mice. Furthermore, dietary cholesterol had comparable effects on total plasma cholesterol and non-high-density lipoprotein cholesterol in KO, Het, and Tg+KO mice. Thus, in Tg+KO mice, dietary cholesterol regulates bile acid pool size, fecal bile acid excretion, and plasma cholesterol independently of Cyp7a1 activity. These results challenge the notion that dietary cholesterol regulation of Cyp7a1 is a major determinant of plasma cholesterol responsiveness.

During the past decade a number of investigators have attempted to develop mouse models of diabetic macrovascular disease. Hyperglycemia might increase vascular damage because it increases oxidant stress. For this reason we studied animals that were deficient in HDL; HDL is widely believed to protect against oxidant stress. An inbred line of mice doubly deficient in LDL receptor and apoAI was made diabetic with streptozotocin (STZ); control mice had an average glucose of 7.2+/-2mmol/l and STZ-treated mice had an average glucose of 19.4+/-6.5mmol/l. The animals were fed a high cholesterol but low fat diet leading to plasma cholesterol levels of 9.4+/-1.6mmol/l in control animals and 10.1+/-1.8mmol/l in STZ-treated mice. The control and STZ-treated animals had similar plasma lipoprotein profiles. Atherosclerosis assessed at 23 weeks averaged 38154microm(2) in control and 32962microm(2) in STZ-treated mice. Therefore STZ-induced diabetes does not alter plasma lipoproteins or atherosclerosis in HDL deficient mice.

Pyridoxamine was linked to a series of polyethylenimines (PEIs) with Mn=600, 1800, 10,000, and 60,000, both simply permethylated and with additional attached dodecyl chains. They were examined in the transamination of pyruvic acid and of phenylpyruvic acid, and showed MichaelisMenten behavior. The values of k2 and of KM determined showed only small variations with polymer size. Thus, the previously reported strong advantage of pyridoxamines attached to the Mn=60,000 PEI, relative to simple pyridoxamine alone, is seen to almost the same extent with the smaller PEIs.

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.

Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol. In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse.

beta-cyclodextrin-based receptors were synthesized and tested for their ability to induce a helical fold in peptides bearing hydrophobic amino acid residues in the i, i+11- or i, i+14-positions. Circular dichroism experiments revealed that a dimeric beta-cylodextrin receptor synthesized from a [1,1'-biphenyl]-4,4'-dithiol core demonstrated an ability to fold a designed peptide bearing the artificial amino acid L-p-t-butylphenylalanine in the i, i+11-positions, while other dimeric and monomeric receptors failed to do so. Titration studies were performed using both circular dichroism and calorimetry, the analysis of which yielded an apparent K(a) on the order of 10(4)-10(5) M(-1). However, no evidence could be obtained for helical folding with a peptide carrying tryptophan residues in place of the p-t-butylphenylalanine units. Our studies suggest that receptors of this type may be useful in molecular recognition of hydrophobic, already alpha-helical peptides in aqueous solution.

Intestinal reclamation of bile salts is mediated in large part by the apical sodium-dependent bile acid transporter (ASBT). The bile acid responsiveness of ASBT is controversial. Bile acid feeding in mice results in decreased expression of ASBT protein and mRNA. Mouse but not rat ASBT promoter activity was repressed in Caco-2, but not IEC-6, cells by chenodeoxycholic acid. A potential liver receptor homologue-1 (LRH-1) cis-acting element was identified in the bile acid-responsive region of the mouse but not rat promoter. The mouse, but not rat, promoter was activated by LRH-1, and this correlated with nuclear protein binding to the mouse but not rat LRH-1 element. The short heterodimer partner diminished the activity of the mouse promoter and could partially offset its activation by LRH-1. Interconversion of the potential LRH-1 cis-elements between the mouse and rat ASBT promoters was associated with an interconversion of LRH-1 and bile acid responsiveness. LRH-1 protein was found in Caco-2 cells and mouse ileum, but not IEC-6 cells or rat ileum. Bile acid response was mediated by the farnesoid X receptor, as shown by the fact that overexpression of a dominant-negative farnesoid X-receptor eliminated the bile acid mediated down-regulation of ASBT. In addition, ASBT expression in farnesoid X receptor null mice was unresponsive to bile acid feeding. In summary cell line- and species-specific negative feedback regulation of ASBT by bile acids is mediated by farnesoid X receptor via small heterodimer partner-dependent repression of LRH-1 activation of the ASBT promoter.

Note from the Editor: August Kekule's dream and his theory of the structure of benzene (1865). Julius Thomsen's logic that benzene contains equivalent electrons between its carbon atoms (ca. 1900). Richard Willstatter's synthesis and observation that 1,3,5,7-cyclooctatetraene is not aromatic (early 1900s). Sir Robert Robinson's proposal of the ‘aromatic sextet’ (1925). Erich Huckel's molecular orbital treatment of benzene and other unsaturated compounds that separated sigma and pi electrons (early 1930s). Tetsuo Nozoe and Michael Dewar's independent proposal of a new aromatic structure for a cycloheptatrieneone (mid-1940s). Ronald Breslow's demonstration that cyclopropenyl cation is aromatic (1958). Breslow's proposal of ‘antiaromaticity’ (1965). I offer several comments. First, in the field of aromaticity, Breslow is in mighty exceptional company. Second, his research has extended well beyond aromaticity. Yes, Breslow is applying the concepts of antiaromaticity toward the development of highly conductive organic materials. But his research has encompassed enzyme mimics, remote functionalization reactions, unnatural DNA analogues and cancer chemotherapy. Indeed, Breslow is the co-discoverer of a highly successful drug, Vorinostat, which is FDA-approved for the treatment of cutaneous T-cell lymphoma. We thank Professor Breslow for joining our project honoring his and our friend, Tetsuo Nozoe. Tetsuo Nozoe would have would have been deeply touched. —Jeffrey I. Seeman Guest Editor University of Richmond Richmond, Virginia 23173, USA E-mail: jseeman@richmond.edu Ronald Breslow with Koji Nakanishi (left) and Tetsuo Nozoe (center), Sendai, 1970. Abstract We contributed to the field of non-benzenoid aromatic compounds by creating the cyclopropenyl cation and various of its derivatives, including cyclopropenone; it was the first aromatic system with other than six pi electrons in a single ring, and the simplest aromatic system. The pioneering work of Tetsuo Nozoe in tropolone chemistry was celebrated with the founding of ISNA, the International Symposium on Non-Benzenoid Aromatic Compounds, where I described our work in the field. It fit the prediction that aromaticity would be found in systems with 4n + 2 pi electrons, where n is an integer. I was also concerned with the properties of monocyclic systems with 4n cyclically conjugated pi electrons. They were expected not to be aromatic, but the interesting question was whether they were actually antiaromatic, especially destabilized by the cyclic conjugation in such 4n species as the cyclopropenyl anion, cyclobutadiene, and cyclopentadienyl cation. The evidence supports antiaromaticity in these cases. We also examined compounds where 4n cyclic pi systems were fused with aromatic systems, and most interestingly systems in which two 4n pi systems were fused. In these cases the periphery of the molecules had 4n + 2 pi electrons, for aromaticity, but the components were antiaromatic. Recently we have studied electrical conductivities in aromatic molecules such as thiophene and saw that aromaticity added resistance to the systems, so non-aromatic compounds are better conductors and antiaromatic compounds are predicted to be the best of all.

A tetrahydroquinoline ring was used to mount the critical functional groups of pyridoxal, and also two examples of rigidly held chirally mounted basic groups. They were able to selectively catalyze decarboxylation, aldol reaction, and retro-aldol reaction of amino acids rather than transamination, and with stereoselectivity. In the aldol reaction of glycine with acetaldehyde to synthesize threonine and allo-threonine, one of the catalysts reversed its stereoselectivity when the basic group was protonated. The observed stereoselectivities were all consistent with prediction.

A novel catalyst has been synthesized in which a manganese–porphyrin unit is linked to four hydrophobic cyclophane binding groups. Relative to a previous catalyst in which the binding groups were cyclodextrins, the new catalyst shows somewhat higher catalytic turnovers before it is destroyed, but less selective product formation.