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High dietary fat intake is associated with metabolic dysregulation, but little is known regarding the effects of a high fat diet (HFD) on photoreceptor cell functioning. We explored the intersection of an HFD and the visual cycle adducts that form in photoreceptor cells by nonenzymatic reactions. In black C57BL/6J mice and albino C57BL/6J c2j mice raised on an HFD until age 3, 6, or 12 months, chromatographically quantified bisretinoids were increased relative to mice on a standard diet. In vivo measurement of fundus autofluorescence, the source of which is bisretinoid, also revealed a significant increase in the HFD mice. Additionally, mice provided with a diet high in fat presented with elevated retinol-binding protein 4, the protein responsible for transporting retinol in plasma. Vitamin A was elevated in plasma although not in ocular tissue. Bisretinoids form in photoreceptor cell outer segments by random reactions of retinaldehyde with phosphatidylethanolamine. We found that the latter phospholipid was significantly increased in mice fed an HFD versus mice on a control diet. In leptin-deficient ob/ob mice, a genetic model of obesity, plasma levels of retinol-binding protein 4 were higher but bisretinoids in retina were not elevated. Photoreceptor cell viability measured as outer nuclear layer thickness was reduced in the ob/ob mice relative to WT. The accelerated formation of bisretinoid we observed in diet-induced obese mice is related to the high fat intake and to increased delivery of vitamin A to the visual cycle., Competing Interests: Conflicts of interest The authors declare that there are no conflicts of interest related to the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Congenital hyperinsulinism (CHI), a rare disease of excessive and dysregulated insulin secretion, can lead to prolonged and severe hypoglycemia. Dextrose infusions are a mainstay of therapy to restore normal glycemia, but can be associated with volume overload, especially in infants. By releasing intrahepatic glucose stores, glucagon infusions can reduce dependency on dextrose infusions. Recent studies have reported positive outcomes with glucagon infusions in patients with CHI; however, to date, there are no reports describing the clinical utility of titrated doses of infused glucagon to achieve glycemic stability. Objective: To assess the potential clinical utility of dose-titrated glucagon infusions in stabilizing glycemic status in pediatric patients with CHI, who were managed by medical and/or surgical approaches. Methods: Patients with CHI ( N = 33), with or without mutations in the ATP-sensitive K + channel genes, ABCC8 , and KCNJ11 requiring glucagon by dose titration in addition to intravenous dextrose and medical therapy with diazoxide/octreotide to achieve glycemic stability were recruited. Following glucagon titration and a 24-h glucose stable period, glucose infusion rate (GIR) was reduced over a 24-h period. Achievement of glycemic stability and decrease in GIR were considered end points of the study. Results: All patients achieved glycemic stability with glucagon infusion, demonstrating clinical benefit. GIR reduced from 15.6 (4.5) to 13.4 (4.6) mg/kg/min mean (SD) ( p = 0.00019 for difference; n = 32; paired t -test) over 24 h. By univariate analysis, no individual baseline characteristic was associated with changes in the GIR. However, by baseline-adjusted modeling, mutational status of the patient ( p = 0.011) was inversely associated with a reduction in GIR. Adverse events were infrequent with diarrhea possibly attributed to glucagon treatment in 1 patient. With long-term treatment following GIR reduction, necrolytic migratory erythema was observed in another patient. Conclusion: These data suggest that dose-titrated glucagon infusion therapy aids hypoglycemia prevention and reduction in GIR in the clinical management of patients with CHI., (Copyright © 2020 Salomon-Estebanez, Yau, Dunne, Worth, Birch, Walewski and Banerjee.)

The processes causing increased hepatic triglycerides (TGs) in mouse models of hepatic steatosis (HS) due to high fat diet (HFD)-induced obesity (DIO), EtOH consumption, or obesity mutations ( ob/ob, db/db ) are uncertain. This report summarizes two studies. Study 1 focused on regulation by five transcription factors (TFs) (NfKb, Srebp-lc, AMPK, PPARα, PPARγ) of seven, much-studied hepatic long-chain fatty acid (LCFA) transporters (FABPpm, CD36, FATPl, FATP2, FATP4, FATP5, & Caveolin-1 [CAV-1]), and expression of genes for enzymes of LCFA synthesis (SCD-1, FASN) in mice with HS from various causes. Study 2 examined the effects of spexin, a novel adipokine, on obesity, type 2 diabetes mellitus (T2DM), and HS in these mice. Study 1 showed that: (1) processes underlying HS differed in mice with normal leptin signaling (DIO, EtoH-fed) versus those without it ( ob/ob, db/db ). Increased hepatocellular LCFA uptake was the principal cause of HS in the former, but increased hepatocellular LCFA synthesis predominated in the latter. (2) Expression of individual transporters was variable in the HS models studied, but strong correlations between TF expression and mean expression of four transporter genes across multiple HS models suggested regulatory interaction, and support the postulate that complexes of several different transporters mediate hepatic LCFA uptake. Study 2 indicated (1) that obese DIO mice often also have T2DM and/or nonalcoholic fatty liver disease (NAFLD); (2) confirmed that spexin treatment caused weight loss in DIO mice; (3) in DIO mice with T2DM, spexin also improved glucose tolerance, decreasing insulin resistance and HbAlc. Incubation with spexin directly inhibited LCFA uptake by hepatocytes isolated from DIO mice with HS/NAFLD by ≤70%. Spexin treatment in vivo for 4 weeks reduced hepatic lipids by 60%, and reduced serum alanine and aspartate aminotransferases. These studies in mice with DIO, T2DM, and HS/NAFLD suggest spexin may be an effective treatment for all three conditions., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Objective: This study examined whether changes in adipocyte long chain fatty acid (LCFA) uptake kinetics explain the weight regain increasingly observed following bariatric surgery., Methods: Three groups (10 patients each) were studied: patients without obesity (NO: BMI 24.2 ± 2.3 kg m(-2) ); patients with obesity (O: BMI 49.8 ± 11.9); and patients classified as super-obese (SO: BMI 62.6 ± 2.8). NO patients underwent omental and subcutaneous fat biopsies during clinically indicated abdominal surgeries; O were biopsied during bariatric surgery, and SO during both a sleeve gastrectomy and at another bariatric operation 16 ± 2 months later, after losing 113 ± 13 lbs. Adipocyte sizes and [(3) H]-LCFA uptake kinetics were determined in all biopsies., Results: Vmax for facilitated LCFA uptake by omental adipocytes increased exponentially from 5.1 ± 0.95 to 21.3 ± 3.20 to 68.7 ± 9.45 pmol/sec/50,000 cells in NO, O, and SO patients, respectively, correlating with BMI (r = 0.99, P < 0.001). Subcutaneous results were virtually identical. By the second operation, the mean BMI (SO patients) fell significantly (P < 0.01) to 44.4 ± 2.4 kg m(-2) , similar to the O group. However, Vmax (40.6 ± 11.5) in this weight-reduced group remained ~2X that predicted from the BMI:Vmax regression among NO, O, and SO patients., Conclusions: Facilitated adipocyte LCFA uptake remains significantly upregulated ≥1 year after bariatric surgery, possibly contributing to weight regain., (© 2015 The Obesity Society.)

Objective: Microarray studies identified Ch12:orf39 (Spexin) as the most down-regulated gene in obese human fat. Therefore, we examined its role in obesity pathogenesis., Methods: Spexin effects on food intake, meal patterns, body weight, respiratory exchange ratio (RER), and locomotor activity were monitored electronically in C57BL/6J mice or Wistar rats with diet-induced obesity (DIO). Its effects on adipocyte [(3)H]-oleate uptake were determined., Results: In humans, Spexin gene expression was down-regulated 14.9-fold in obese omental and subcutaneous fat. Circulating Spexin changed in parallel, correlating (r = -0.797) with Leptin. In rats, Spexin (35 µg/kg/day SC) reduced caloric intake ∼32% with corresponding weight loss. Meal patterns were unaffected. In mice, Spexin (25 µg/kg/day IP) significantly reduced the RER at night, and increased locomotion. Spexin incubation in vitro significantly inhibited facilitated fatty acid (FA) uptake into DIO mouse adipocytes. Conditioned taste aversion testing (70 µg/kg/day IP) demonstrated no aversive Spexin effects., Conclusions: Spexin gene expression is markedly down-regulated in obese human fat. The peptide produces weight loss in DIO rodents. Its effects on appetite and energy regulation are presumably central; those on adipocyte FA uptake appear direct and peripheral. Spexin is a novel hormone involved in weight regulation, with potential for obesity therapy., (Copyright © 2014 The Obesity Society.)

Alcohol, a major cause of human cardiomyopathy, decreases cardiac contractility in both animals and man. However, key features of alcohol-related human heart disease are not consistently reproduced in animal models. Accordingly, we studied cardiac histology, contractile function, cardiomyocyte long chain fatty acid (LCFA) uptake, and gene expression in male C57BL/6J mice consuming 0, 10, 14, or 18% ethanol in drinking water for 3months. At sacrifice, all EtOH groups had mildly decreased body and increased heart weights, dose-dependent increases in cardiac triglycerides and a marked increase in cardiac fatty acid ethyl esters. [(3)H]-oleic acid uptake kinetics demonstrated increased facilitated cardiomyocyte LCFA uptake, associated with increased expression of genes encoding the LCFA transporters CD36 and Slc27a1 (FATP1) in EtOH-fed animals. Although SCD-1 expression was increased, lipidomic analysis did not indicate significantly increased de novo LCFA synthesis. By echocardiography, ejection fraction (EF) and the related fractional shortening (FS) of left ventricular diameter during systole were reduced and negatively correlated with cardiac triglycerides. Expression of myocardial PGC-1α and multiple downstream target genes in the oxidative phosphorylation pathway, including several in the electron transport and ATP synthase complexes of the inner mitochondrial membrane, were down-regulated. Cardiac ATP was correspondingly reduced. The data suggest that decreased expression of PGC-1α and its target genes result in decreased cardiac ATP levels, which may explain the decrease in myocardial contractile function caused by chronic EtOH intake. This model recapitulates important features of human alcoholic cardiomyopathy and illustrates a potentially important pathophysiologic link between cardiac lipid metabolism and function., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hepatitis C virus infection is a major public health concern. Approximately 4 million people are reported to be infected with the virus in the United States, and the annual death rate due to HCV-associated decompensated liver failure or hepatocellular carcinoma is estimated to be approximately 18,000 within the next decade. Therapeutic success, as measured by a sustained virologic response, is approximately 50 % in G1 patients with pegylated-interferon/ribavirin-based therapies. Independent studies have reported significant variation in response rates depending on the ethnicity or race of the patient, though the underlying reasons are not well understood. Historically, ethnic populations have been underrepresented in most large clinical trials of HCV therapies, even though these populations have disproportionately high rates of HCV infection. Recent clinical trials have investigated genetic variations in key biological pathways that may underlie the mechanisms responsible for the different rates of HCV clearance and treatment outcomes in ethnic populations treated with pegylated-interferon/ribavirin. However, as novel direct-acting antiviral drugs are added to, and eventually replace, existing treatment regimens, the role of the innate immune response in determining treatment outcomes will diminish. Socioeconomic and biological factors can impact rates of HCV infection, disease progression, and treatment outcomes in minority populations. Improved access to health care, novel antiviral treatments, and a better understanding of the host factors that contribute to disparities in treatment outcomes are expected to result in optimized treatment paradigms that directly target the virus, leading to improved outcomes for all patients.

A nonarteriosclerotic cardiomyopathy is increasingly seen in obese patients. Seeking a rodent model, we studied cardiac histology, function, cardiomyocyte fatty acid uptake, and transporter gene expression in male C57BL/6J control mice and three obesity groups: similar mice fed a high-fat diet (HFD) and db/db and ob/ob mice. At sacrifice, all obesity groups had increased body and heart weights and fatty livers. By echocardiography, ejection fraction (EF) and fractional shortening (FS) of left ventricular diameter during systole were significantly reduced. The V(max) for saturable fatty acid uptake was increased and significantly correlated with cardiac triglycerides and insulin concentrations. V(max) also correlated with expression of genes for the cardiac fatty acid transporters Cd36 and Slc27a1. Genes for de novo fatty acid synthesis (Fasn, Scd1) were also upregulated. Ten oxidative phosphorylation pathway genes were downregulated, suggesting that a decrease in cardiomyocyte ATP synthesis might explain the decreased contractile function in obese hearts.

Unlabelled: Hepatic stellate cell (HSC) lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT) mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS) based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP) mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i) increased expression of typical markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1., Conclusion: Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.

Many anti-HCV antibodies are available, but more are needed for research and clinical applications. This study examines whether ascitic fluid from cirrhotic patients could be a source of reagent-grade antibodies. Ascitic fluid from 29 HCV patients was screened by ELISA for anti-HCV antibodies against three viral proteins: core, NS4B, and NS5A. Significant patient-to-patient variability in anti-HCV antibody titers was observed. Total ascitic fluid IgG purified by Protein-A chromatography reacted with HCV proteins in immunoblots, cell extracts, and replicon-expressing cells. Affinity-purification using synthetic peptides as bait allowed the preparation of cross-genotypic antibodies directed against pre-selected regions of HCV core, NS4B, and NS5A proteins. The performance of the polyclonal antibodies was comparable to that of monoclonal antibodies. Anti-NS4B antibody preparations reacted with genotype 1a, 1b, and 2a NS4B proteins in immunoblots and allowed NS4B to be localized in replicon-expressing cells. Ascitic fluid is an abundant source of human polyclonal cross-genotypic antibodies that can be used as an alternative to blood. This study shows the utility of selectively purifying human polyclonal antibodies from ascitic fluid. Affinity purification allows antibodies to be selected that are comparable to monoclonal antibodies in their ability to react with targeted regions of viral proteins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: The obesity epidemic causes significant morbidity and mortality. Knowledge of cellular function and gene expression in obese adipose tissue will yield insights into obesity pathogenesis and suggest therapeutic targets. The aim of this work is to study the processes determining fat accumulation in adipose tissue from obese patients., Methods: Omental fat was collected from two cohorts of obese bariatric surgery patients and sex-matched normal-weight donors. Isolated adipocytes were compared for cell size, volume, and long-chain fatty acid (LCFA) uptake. Omental fat RNAs were screened by 10K microarray (cohort 1: three obese, three normal) or Whole Genome microarray (cohort 2: seven obese, four normal). Statistical differences in gene and pathway expression were identified in cohort 1 using the GeneSifter Software (Geospiza) with key results confirmed in cohort 2 samples by microarray, quantitative real-time polymerase chain reaction, and pathway analysis., Results: Obese omental adipocytes had increased surface area, volume, and V (max) for saturable LCFA uptake. Dodecenoyl-coenzyme A delta isomerase, central to LCFA metabolism, was approximately 1.6-fold underexpressed in obese fat in cohorts 1 and 2. Additionally, the Kyoto Encyclopedia of Genes and Genomics pathway analysis identified oxidative phosphorylation and fatty acid metabolism pathways as having coordinate, nonrandom downregulation of gene expression in both cohorts., Conclusions: In obese omental fat, saturable adipocyte LCFA uptake was greater than in controls, and expression of key genes involved in lipolysis, beta-oxidation, and metabolism of fatty acids was reduced. Thus, both increased uptake and reduced metabolism of LCFAs contribute to the accumulation of LCFAs in obese adipocytes.

The hepatitis C virus (HCV) core gene is more conserved at the nucleic acid level than is necessary to preserve the sequence of the core protein, suggesting that it contains information for additional functions. We used a battery of anticore antibodies to test the hypothesis that the core gene directs the synthesis of core protein isoforms. Infectious viruses, replicons, and RNA transcripts expressed a p8 minicore containing the C-terminal portion of the p21 core protein and lacking the N-terminal portion. An interferon resistance mutation, U271A, which creates an AUG at codon 91, upregulated p8 expression in Con1 replicons, suggesting that p8 is produced by an internal initiation event and that 91-AUG is the preferred, but not the required, initiation codon. Synthesis of p8 was independent of p21, as shown by the abundant production of p8 from transcripts containing an UAG stop codon that blocked p21 production. Three infectious viruses, JFH-1 (2a core), J6/JFH (2a core), and H77/JFH (1a core), and a bicistronic construct, Bi-H77/JFH, all expressed both p8 and larger isoforms. The family of minicores ranges in size from 8 to 14 kDa. All lack the N-terminal portion of the p21 core. In conclusion, the core gene contains an internal signal that stimulates the initiation of protein synthesis at or near codon 91, leading to the production of p8. Infectious viruses of both genotype 1 and 2 HCV express a family of larger isoforms, in addition to p8. Minicores lack significant portions of the RNA binding domain of p21 core. Studies are under way to determine their functions.

Background: Neurocognitive deficits in patients with hepatitis C virus (HCV) infection prompted a search for HCV in brain., Results: HCV was present in the brains of 7 (54%) of 13 patients with viremia, as determined by 5' UTR and E1 (envelope 1) gene analysis. Brain HCV RNA consensus sequences differed from those in plasma and liver in 4 (57%) of 7 patients. The quality of HCV RNA from postmortem brain and liver was assessed and demonstrated to be suitable for sequence analysis. Quasispecies analysis revealed that several mutations present in clones from >1 brain region were absent in clones from liver and plasma. Brain-specific mutations defined several families of related sequences. The patterns of brain-specific mutations in these families were consistent with the evolution of HCV RNA from a common ancestor. Single-nucleotide-polymorphism analysis confirmed that a prominent brain-specific mutation constituted approximately 10% of HCV RNA in cerebellum and medulla but that this mutation was undetectable in the liver and plasma of the same patient., Conclusions: This study introduces novel methods for assessing RNA from postmortem samples. It increases the reported cases of HCV in the brain, provides the first E1 sequences from the brain, and contributes to the growing evidence that HCV replicates and evolves within the brain.

Hepatitis C virus (HCV) has been detected in the brain tissues of 10 individuals reported to date; it is unclear what clinical factors are associated with this, and with what frequency it occurs. Accordingly, a pilot analysis utilizing reverse transcriptase-polymerase chain reaction (RT- PCR) to detect and sequence HCV in premortem plasma and postmortem brain and liver from 20 human immunodeficiency virus (HIV)-infected and 10 HIV-naive individuals was undertaken. RNA encoding the first 126 amino acids of the HCV E1 envelope protein and the majority of the E1 signal sequence was analyzed in parallel with an 80-base-long segment of the 5' untranslated region (UTR). Liver HCV was detected only in subjects with premortem HCV viremia (10 HIV-infected and 3 HIV-naive). Brain HCV was detected in 6/10 HCV/HIV-coinfected and 1/3 HCV-monoinfected subjects. In the setting of HIV, the magnitude of plasma HCV load did not correlate with the presence of brain HCV. However, coinfected patients with brain HCV were more often off antiretroviral therapy and tended to have higher plasma HIV loads than those with HCV restricted to liver. Furthermore, premortem cerebrospinal fluid (CSF) analysis revealed that HCV/HIV-coinfected patients with brain HCV had detectable CSF HIV, whereas those without brain HCV had undetectable CSF HIV loads (P = .0205). Neuropsychologic tests showed a trend for hierarchical impairment of abstraction/executive functioning in HIV/HCV coinfection, with mean T scores for HIV monoinfected patients 43.2 (7.3), for liver-only HCV 39.5 (9.0), and for those with HCV in brain and liver 33.2 (5.1) (P = .0927). Predominant brain HCV sequences did not match those of the plasma or liver in 4 of the 6 coinfected patients analyzed. We conclude that in the setting of HIV/HCV coinfection, brain HCV is a common phenomenon unrelated to the magnitude of HCV viremia, but related to active HIV disease and detectable CSF HIV. Furthermore, there is sequence evidence of brain compartmentalization. Differences in abstraction/executive function of HCV/HIV coinfected patients compared to HIV monoinfected warrant further studies to determine if neuropsychiatric effects are predicated upon brain infection.

This study tested the hypothesis that hepatitis C virus (HCV) RNA and core antigen levels rise more rapidly after liver transplantation (LT) in recipients of grafts from living donors (LD) versus deceased donors (DD). Eleven consecutive LD and 15 DD recipients were followed prospectively. Before LT, median HCV RNA levels were similar: 5.42 (LDLT) and 5.07 (DDLT) log(10) IU/mL (P = NS). During the first 7 hours after LT a trend toward a greater HCV RNA decrease in LDLT patients was seen, although they received fewer blood replacement products during surgery. HCV RNA levels rose more rapidly in LDLT patients between days 1 and 3 (P = .0059) and were higher in this group on days 2, 3, 4, and 5. Core antigen levels were significantly higher in LDLT patients on days 3 and 5, although they were similar before LT (P = NS). Alanine aminotransferase (ALT) values were higher among LDLT patients from 8 to 14 days and from 4 to 24 months. Two-year graft and patient survival were 73% for LDLT patients and 80% for DDLT patients (P = NS). In conclusion, viral load rose more rapidly in LD recipients and reached higher levels shortly after surgery. Greater ALT elevations were evident in the LDLT group, but survival rates were similar. The trend toward a greater initial viral load decrease in patients with LD grafts and the significantly sharper increase suggest that the liver plays a predominant role in both HCV clearance and replication.

The BTNL2 gene is a member of the B7 receptor family that probably functions as a T-cell costimulatory molecule. It resides in the class II major histocompatibility complex (MHC) region of chromosome 6p and has recently been associated with sarcoidosis susceptibility in a white German population. We sought to replicate the BTNL2 association in an African American family-based study population (n=219 nuclear families) and two case-control populations--one African American (n=295 pairs) and one white (n=366 pairs). Ten SNPs were detected within a 490-bp region spanning exon/intron 5 of BTNL2. Haplotype variation within this region was significantly associated with sarcoidosis in all three study populations but more so in whites (P=.0006) than in the African American case-control (P=.02) or family-based (P=.03) samples. The previously reported BTNL2 SNP with the strongest sarcoidosis association, rs2076530, was also the SNP with the strongest association in our white population (P<.0001). The A allele of rs2076530 results in a premature exon-splice site and increases risk for sarcoidosis (odds ratio=2.03; 95% confidence interval 1.32-3.12). Although rs2076530 was not associated with sarcoidosis in either African American sample, a three-locus haplotype that included rs2076530 was associated with sarcoidosis across all three study samples. Multivariable logistic regression analyses showed that BTNL2 effects are independent of human leukocyte antigen class II genes in whites but may interact antagonistically in African Americans. Our results underscore the complexity of genetic risk for sarcoidosis emanating from the MHC region.

The hepatitis C virus (HCV) has an alternate reading frame (ARF) that overlaps the core protein gene. The overlapping reading frame distinguishes HCV from all of its known viral relatives, with the possible exception of GB virus B (GBV-B). The ARF is expressed during natural HCV infections and stimulates specific immune responses. Like several essential genes in other viruses (e.g., the human immunodeficiency virus polymerase) the ARF lacks an in-frame AUG start codon, suggesting that its expression involves unusual translation-level events. In vitro studies indicate that ribosomal frameshifting may be one of several processes that can lead to translation of the ARF. Frameshifting yields chimeric proteins that have segments encoded in the core gene covalently attached to amino acids encoded in the ARF. A consistent nomenclature for the ARF's protein products has yet to be established. We propose that all proteins that contain amino acids encoded in the + 1 ARF be called alternate reading frame proteins (ARFPs) and that specific ARFPs, such as the ARFP/F-protein, the double-frameshift protein, and the short form of core + 1, be designated as follows: ARFP/F (ARFP/F-protein), ARFP/DF (double-frameshift), and ARFP/S (short form of core + 1). The roles of ARFPs in the HCV life cycle are not yet known. There is a significant possibility that ARFPs may be responsible for some of the effects attributed to the core protein, given that most studies seeking to define the function of the core protein have employed materials likely to contain a combination of the core protein and ARFPs. The observed effects of the core protein include the induction of liver cancer, transformation of cells, and alterations of immune responses. This article reviews the discovery of ARF, describes the RNA structural elements involved in core/ARF gene expression, discusses possible functions of ARFPs, and considers the potential usefulness of ARFPs in vaccines. The HCV ARF is the focus of a new and rapidly expanding area of research, and the results of many ongoing studies are currently available in abstract form only. The preliminary nature of investigations that have not yet been reviewed by peers is noted in the text.

The RNA genome of the hepatitis C virus (HCV) undergoes rapid evolutionary change. Efforts to control this virus would benefit from the advent of facile methods to identify characteristic features of HCV RNA and proteins, and to condense the vast amount of mutational data into a readily interpretable form. Many HCV sequences are available in GenBank. To facilitate analysis, consensus sequences were constructed to eliminate the overrepresentation of certain genotypes, such as genotype 1, and a novel package of sequence analysis tools was developed. Mutation Master generates profiles of point mutations in a population of sequences and produces a set of visual displays and tables indicating the number, frequency, and character of substitutions. It can be used to analyze hundreds of sequences at a time. When applied to 255 HCV core protein sequences, Mutation Master identified variable domains and a series of mutations meriting further investigation. It flagged position 4, for example, where 90% or more of all sequences in genotypes 1, 2, 4, and 5, have N4, whereas those in genotypes 3, 6, 7, 8, 9, and 10 have L4. This pattern is noteworthy: L (hydrophobic) to N (polar) substitutions are generally rare, and genotypes 1, 2, 4, and 5 do not form a recognized super family of sequences. Thus, the L4N substitution probably arose independently several times. Moreover, not one member of genotypes 1, 2, 4, or 5 has L4 and not one member of genotypes 3, 6, 7, 8, 9, or 10 has N4. This nonoverlapping pattern suggests that coordinated changes at position 4 and a second site are required to yield a viable virus. The package generated a table of genotype-specific substitutions whose future analysis may help to identify interacting amino acids. Three substitutions were present in 100% of genotype 2 members and absent from all others: A68D, R74K, and R114H. Finally, this study revealed thatARFP, a novel protein encoded in an overlapping reading frame, is as conserved as conventional HCV proteins, a result supporting a role for ARFP in the viral life cycle. Whereas most conventional programs for phylogenetic analysis of sequences provide information about overall relatedness of genes or genomes, this program highlights and profiles point mutations. This is important because determinants of pathogenicity and drug susceptibility are likely to result from changes at only one or two key nucleotides or amino acid sites, and would not be detected by the type of pairwise comparisons that have usually been performed on HCV to date. This study is the first application of Mutation Master, which is now available upon request (http://tandem.biomath.mssm.edu/mutationmaster.html).

Gene expression profiling allows the level of activity of thousands of genes to be monitored simultaneously. Profiling is often carried out on specialized chips or slides, which have microarrays of gene targets at predetermined addresses. In the immediate future, microarrays promise to yield new insights into hepatitis C virus (HCV) pathogenesis and to produce 'signatures' that can be used in molecular diagnostics. In the longer-term, they may aid the development of serological tests by identifying genes encoding secretory proteins produced by HCV-infected livers, and they may suggest new avenues for disease intervention by detecting genes whose products are retained in the infected liver.

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p < 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

Lidocaine effects were studied at the single channel level on batrachotoxin-activated eel electroplax (muscle-derived) and on rat brain sodium channels in planar lipid bilayers to investigate whether these effects were the same on structurally different sodium channels. Lidocaine blocked the open state of brain channels with the same voltage dependence, but with 15-times as high a potency as muscle-derived channels. In brain channels, but not muscle-derived ones, the level of the open channel block showed periods of relief. Lidocaine at microM concentrations stabilized the highest conductance state in both channel types and at mM concentrations stabilized subconductance-like states in electroplax, but not in brain channels. In both channel types, lidocaine increased the lifetime and rate of entry to a long-nonconducting state. Since both channel types were studied under identical lipid and ionic conditions, the observed functional differences in the lidocaine action (effects, potency) must reflect channel structural differences.

The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine. The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incomplete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc.

Drug interactions between ethanol and pentobarbital and ethanol and chlordiazepoxide were investigated utilizing mice. At the peak of oral ethanol (0-4 g/kg), either sodium pentobarbital (1-120 mg/kg) or chlordiazepoxide hydrochloride (2-400 mg/kg) was given intraperitoneally. Blood concentrations of ethanol, pentobarbital, chlordiazepoxide, and its pharmacologically active major metabolites were monitored utilizing either gas chromatography or high performance liquid chromatography. Lethality and loss-of-righting reflex were measured as indexes of behavioral drug interactions. It was evident from the isobolographic plot that the interactions between ethanol and pentobarbital and ethanol and chlordiazepoxide were more than additive. Interaction between ethanol and pentobarbital was greater than that between ethanol and chlordiazepoxide. Furthermore, with increasing ethanol pretreatment the shift in dose-response curves for the loss-of-righting reflex was affected more than the shift in dose-response curves for lethality. Blood concentration monitoring of each drug indicated that the rate of biotransformation of pentobarbital was significantly decreased; sequential biotransformation of chlordiazepoxide was also altered, resulting in a large accumulation of demethylchlordiazepoxide in the blood.

Many studies have suggested the importance of the rate of drug elimination in the development of pharmacodynamic tolerance and physical dependence to sedative-hypnotic drugs. Our previous study demonstrated that the role of individual variation in elimination kinetics played an important part in producing pharmacodynamic tolerance and physical dependence when the drug was given at a fixed dose-frequency schedule. The present study investigated the effect of various dose-frequency schedules on the production of tolerance and physical dependence. Controlling the frequency of administration was thought to be the most rational clinical approach in avoiding the production of tolerance and physical dependence with repeated sedative-hypnotic medication. Groups of animals were treated with Na pentobarbital according to the "maximally tolerable" dosing schedule described previously, except that with different dose-frequency schedules, i.e., twice a day, once every day, once every 1.5 days and once every other day for 70 consecutive doses and then withdrawn abruptly. The relationship between the number of Na pentobarbital doses under each dose-frequency schedule and the pharmacokinetic and pharmacodynamic tolerance production, plus the intensity of withdrawal, were studied. The withdrawal intensity was further correlated to the time lag between each drug administration by estimating the maximal time allowed between doses that does not produce any spontaneous withdrawal convulsions and overt withdrawal signs. It was concluded that the time required for repeated Na pentobarbital administration that does not produce withdrawal convulsion and overt withdrawal signs was 4.9 and 5.7 times the average pentobarbital half-life, respectively.