Effect of α-methylnorepinephrine,an α2-adrenergic agonist,on jejunal absorption in neurally intact conscious dog

Although ₂-adrenergic agonists stimulate absorption in the mammalian small and large intestinein vitro, the possibility of central neural effects have confounded interpretation ofin vivo studies. Our aim was to assess the effects of intravenous administration of -methylnorepinephrine (MNE), and ₂-adrenergic agonist that does not cross the blood-brain barrier, on net jejunal absorption of water and electrolytes in the neurally intact, conscious dog. Absorption from a 30-cm proximal jejunal segment was studied using a triple-lumen perfusion technique in seven dogs. A warmed, isosmolar, balanced electrolyte solution containing [¹⁴C]polyethylene glycol was infused at 5 ml/min. Net jejunal fluxes of water and electrolytes were determined before, during, and after a 1.5-hr infusion of MNE (900 nmol/kg/hr). MNE increased net jejunal water absorption (from 12.9±1.8 to 22.5±1.5 l/cm/min,P<0.05). Peripheral ₂-adrenergic receptors mediate a net proabsorptive response in the neurally intact canine jejunumin vivo independent of direct central neural effects.Parts of this work were published as an abstract inGastroenterology (100:A689, 1991). Supported in part by the United States Surgical Corporation, a grant provided by Harry S. Tan, and USPHS NIH grant DK39337 (M.G.S.).     

Size-dependent forced PEG partitioning into channels: VDAC,OmpC, and α-hemolysin

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.Partitioning of polymers into nanosize cavities has broad relevance (1), generally in biology, where the consequences of molecular crowding are well appreciated but not completely understood (2, 3), and in biotechnology for single-molecule sensing and characterization based on the variation of current through ion-conducting aqueous pores (4–7). The partitioning of nonionic polymers such as PEG into α-hemolysin (aHL) from Staphylococcus aureus has been previously studied and shown to be size-dependent at relatively low salt concentrations (8–13). In a different way, namely, as the amplitude of channel blockage, polymer size dependency has also been observed in single-molecule studies at high salt concentrations (4 M KCl) with aHL (14) and recently with aerolysin from Aeromonas hydrophila (15), and was shown to exhibit pronounced size sensitivity with resolution in the submonomer range.We studied passive size-dependent partitioning and size discrimination that can be manipulated to force polymers into nanosize pores under strong nonideality, when polymer partitioning is qualitatively modified by polymer–polymer repulsion that allows polymers, which are excluded in dilute solutions, to enter the channel pore (13). This concentration-dependent partitioning was rationalized by an argument that the overlap concentration of the polymer in the pore is higher than that in the bath (16, 17). For polymer mixtures, where one component is used to preferentially push another into a cavity, this phenomenon of forced polymer partitioning was referred to as “polymers pushing polymers” (PPP) (18). Using the osmotic pressure of a polymer solution composed of various sizes of the same type of polymers, these theoretical advances quantified the forced preferential entry of polymers into a nanopore depending on their size and the pore penetration energy penalty. The theoretical analysis (18) was formulated specifically for a binary polymer mixture, where only one component is allowed to penetrate the pore, whereas the other polymer is excluded. An equation of state (EOS) of a polymer mixture (osmotic pressure as a function of composition) is first validated with osmotic measurements for a binary polymer mixture. Then this EOS, together with the observed polymer selectivity of the pore, is used to interpret the polymer partitioning coefficient.We apply this approach to different β-barrel pores to assess to what extent it can be useful in understanding concrete examples of size-dependent forced polymer partitioning. We performed partitioning measurements of differently sized PEGs into three pores: mitochondrial voltage-dependent anion channel (VDAC), bacterial porin OmpC (outer membrane protein C), and bacterial toxin aHL. In what follows we limit ourselves exclusively to the case of PEG mixtures, composed of “short” (PEG200) and “long” (PEG3400) polymer components.     

Efficacy of thymosin α-1 plus peginterferon α-2a combination therapy compared with peginterferon α-2a monotherapy in HBeAg-positive chronic hepatitis B: A prospective, multicenter, randomized, open-label study

Abstract Objective. Thymosin α-1 plus interferon α-2a offers superior efficacy over interferon α-2a alone in patients with chronic hepatitis B. The aim was to compare the antiviral efficacy of thymosin α-1 plus peginterferon α-2a and peginterferon α-2a alone in HBeAg-positive chronic hepatitis B patients. Materials and methods. HBeAg-positive CHB patients were enrolled in this prospective, randomized, open-label study. Fifty-one patients were assigned to either combination (26 patients; 180 μg of peginterferon α-2a weekly for 48 weeks and 1.6 mg of thymosin α-1 twice a week for the first 12 weeks) or monotherapy (25 patients; 180 μg of peginterferon α-2a weekly for 48 weeks) groups. Results. The rates of the combined response, defined as HBeAg seroconversion, HBV DNA suppression, and normalization of serum ALT, were 4/26 (15.4%) and 3/25 (12.0%) for the combination group and the monotherapy group at the end of treatment (p = 0.725), and 6/26 (23.1%) and 5/25 (20.0%) at the end of follow-up (p = 0.789), respectively. Based on multiple logistic regression analysis, a >2 log(10) IU/mL reduction of HBV DNA at week 12 was identified as an independent predictor for combined response (OR, 9.72; 95% CI, 1.33-71.06; p = 0.025) at the end of follow-up. A lower pretreatment HBV DNA level (≤7 log(10) IU/mL) was another predictor for combined response (OR, 9.64; 95% CI, 1.23-75.32; p = 0.031). No significant differences in adverse events were observed. Conclusions. The short-term addition of thymosin α-1 was not superior to peginterferon α-2a alone in HBeAg-positive CHB patients on the basis of antiviral efficacy.     

Efficacy of thymosin α-1 plus peginterferon α-2a combination therapy compared with peginterferon α-2a monotherapy in HBeAg-positive chronic hepatitis B: A prospective,multicenter, randomized,open-label study

Abstract

Objective. Thymosin α-1 plus interferon α-2a offers superior efficacy over interferon α-2a alone in patients with chronic hepatitis B. The aim was to compare the antiviral efficacy of thymosin α-1 plus peginterferon α-2a and peginterferon α-2a alone in HBeAg-positive chronic hepatitis B patients. Materials and methods. HBeAg-positive CHB patients were enrolled in this prospective, randomized, open-label study. Fifty-one patients were assigned to either combination (26 patients; 180 μg of peginterferon α-2a weekly for 48 weeks and 1.6 mg of thymosin α-1 twice a week for the first 12 weeks) or monotherapy (25 patients; 180 μg of peginterferon α-2a weekly for 48 weeks) groups. Results. The rates of the combined response, defined as HBeAg seroconversion, HBV DNA suppression, and normalization of serum ALT, were 4/26 (15.4%) and 3/25 (12.0%) for the combination group and the monotherapy group at the end of treatment (p = 0.725), and 6/26 (23.1%) and 5/25 (20.0%) at the end of follow-up (p = 0.789), respectively. Based on multiple logistic regression analysis, a >2 log₁₀ IU/mL reduction of HBV DNA at week 12 was identified as an independent predictor for combined response (OR, 9.72; 95% CI, 1.33–71.06; p = 0.025) at the end of follow-up. A lower pretreatment HBV DNA level (≤7 log₁₀ IU/mL) was another predictor for combined response (OR, 9.64; 95% CI, 1.23–75.32; p = 0.031). No significant differences in adverse events were observed. Conclusions. The short-term addition of thymosin α-1 was not superior to peginterferon α-2a alone in HBeAg-positive CHB patients on the basis of antiviral efficacy.     

Randomized,double-blind,placebo-controlled trial of eight-week course of recombinant α-interferon for chronic non-A,non-B hepatitis

Forty-nine Japanese patients were enrolled in a randomized, placebo-controlled, doubleblind trial of -interferon for chronic non-A, non-B hepatitis: 24 patients received 3 million units of recombinant human alpha -interferon (-2a) thrice weekly for eight weeks, and 25 patients received placebo in a similar schedule. The mean serum alanine aminotransferase (ALT) dropped from 155±91 (sd) to 69±72 during interferon treatment, but remained unchanged (158±140 to 147±130) during placebo treatment (P<0.001). Serum ALT level fell to the normal range in 29% of interferon-treated patients, but in only 4% of placebo-treated patients. Pre- and posttreatment liver biopsies were obtained in all but one case. Average histological activity indices (HAI) were markedly improved in the interferon-treated group (9.5±3.7 to 7.0±4.3), but were unchanged in the placebo group (8.5±4.3 to 8.5±4.9). In addition, we compared the efficacy of interferon treatment between anti-hepatitis C virus (HCV) antibody positive and negative groups. Biochemical and histological improvements were similar and statistically significant in patients with and without antibody to hepatitis C virus. These data indicate that a eight-week course of -interferon induces biochemical and histological improvement in more than half the patients with chronic non-A, non-B hepatitis.This study was supported by a grant from Japanese Ministry of Health and Welfare.     

Molecular screening of the Hbs Constant Spring (codon 142, TAA>CAA, α2) and Paksé (codon 142, TAA>TAT, α2) mutations in Thailand

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies.     

Salivary chromogranin A, but not α-amylase, correlates with cardiovascular parameters during high-intensity exercise

Introduction Several studies have shown that activation of the sympathetic nervous system results in the increased secretion of α‐amylase (sAA), an enzyme produced by salivary glands. Recently, chromogranin A (CgA), a soluble protein costored and coreleased with catecholamines from the adrenal medulla and sympathetic nerve endings, has been proposed as a marker of sympathoadrenal medullary system (SAM) activity. The aim of this study was to investigate the behaviour of salivary chromogranin A (sCgA) and sAA during high‐intensity exercise and to analyse their possible correlation with cardiovascular and psychological parameters. Methods Before and during a standardized treadmill stress test, and at 5, 15 and 30 min during the recovery phase, sCgA and sAA were monitored in 21 healthy men. The double product (DP) of blood pressure and heart rate responses, and the product of the subjective ratings of perceived exertion recorded at the final step (RPE) and the exercise duration were used as indices of cardiovascular and exercise intensity, respectively. Results With respect to baseline, significant (P < 0·001) increases in peak sCgA (median 64%) and sAA (median 86%) were observed at the end of exercise. During the recovery phase, sAA levels fell abruptly, whereas sCgA remained elevated (P < 0·001). Significant correlations emerged only for sCgA with respect to %DP (r = 0·84; P < 0·001) and last step‐RPE (r = 0·82; P = 0·024). Conclusions These data suggest sCgA as a reliable marker of SAM activation. Furthermore, the relationship between sCgA and exercise intensity highlights the potential use of this noninvasive parameter in monitoring the adrenergic response during intense physical stress.     

Effect of α1-adrenergic blockade on canine ileal water,electrolyte, and glucose absorption

Meal ingestion stimulates an increase in small intestinal water and electrolyte absorption. Endogenous norepinephrine may at least partially mediate this meal-stimulated proabsorptive response. Luminally administered ₁-adrenergic agonists such as norepinephrine and phenylephrine cause significant small bowel absorption, which can be prevented by the selective ₁-adrenergic antagonist terazosin. This study tested two hypotheses: (1) a meal stimulates ileal water, electrolyte, and glucose absorption; and (2) meal-stimulated ileal absorption is mediated via ₁-adrenergic receptor activation. Absorption studies (N=27) were performed on dogs with 25-cm ileal Thiry-Vella fistulas (TVF). Perfusion with [¹⁴C]PEG was used to calculate absorption of water, electrolytes, and glucose from the TVF. Three groups were randomly studied over 4 hr: (1) terazosin alone, (2) meal alone, and (3) terazosin plus meal. Terazosin (10^–4 M) was administered to the TVF in groups 1 and 3 following the first hour. A 480-kcal mixed canine meal was ingested at the end of the second hour in groups 2 and 3. Ileal water, electrolyte, and glucose absorption increased significantly in response to meal ingestion (P<0.05). Luminal terazosin did not significantly alter basal or meal-stimulated ileal absorption. In conclusion, meal ingestion stimulates ileal absorption of water, electrolytes, and glucose. Neither basal nor meal-stimulated ileal absorption is altered by ₁-adrenergic receptor blockade. These data suggest that nonadrenergic neural pathways or humoral factors are the likely mediators of meal-induced intestinal absorption.Presented in part at a poster session of the Annual Meeting of The American Gastroenterological Association, Boston, Massachusetts, May 1993. Published in abstract form inGastroenterology 104:A608, 1993.Supported in part by National Institutes of Health grant R29-DK41178 (C.J.Y.)     

The antiviral protein,viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

Lipid droplets are intracellular lipid-storage organelles that are thought to be derived from the endoplasmic reticulum (ER). Several pathogens, notably hepatitis C virus, use lipid droplets for replication. Numerous questions remain about how lipid droplets are generated and used by viruses. Here we show that the IFN-induced antiviral protein viperin, which localizes to the cytosolic face of the ER and inhibits HCV, localizes to lipid droplets. We show that the N-terminal amphipathic α-helix of viperin that is responsible for ER localization is also necessary and sufficient to localize both viperin and the fluorescent protein dsRed to lipid droplets. Point mutations in the α-helix that prevent ER association also disrupt lipid droplet association, and sequential deletion mutants indicate that the same number of helical turns are necessary for ER and lipid droplet association. Finally, we show that the N-terminal amphipathic α-helix of the hepatitis C viral protein NS5A can localize dsRed and viperin to lipid droplets. These findings indicate that the amphipathic α-helices of viperin and NS5A are lipid droplet-targeting domains and suggest that viperin inhibits HCV by localizing to lipid droplets using a domain and mechanism similar to that used by HCV itself.     

A combination of α-fetoprotein,midkine, thioredoxin and a metabolite for predicting hepatocellular carcinoma

Introduction and objectivesThe heterogenous nature of hepatocellular carcinoma (HCC) motivated this attempt at developing and validating a model based on combined biomarkers for improving early HCC detection.Patients/materials and methodsThis study examined 196 patients for an estimation study (104 patients with HCC, 52 with liver cirrhosis and 40 with liver fibrosis) and 122 patients for the validation study (80 patients with HCC, 42 with liver cirrhosis). All patients were positive for hepatitis C virus. Four markers were measured: Midkine and thioredoxin using ELISA, 1-methyladenosine and 1-methylguanosine using a gas chromatography–mass spectrometry (GC–MS). The results were compared with alpha-fetoprotein (AFP). The performance of the model was estimated in BCLC, CLIP and Okuda staging systems of HCC.ResultsThe model yielded high performance with an area under ROC (AUC) of 0.94 for predicting HCC in patients with liver cirrhosis, compared with AUC of 0.69 for AFP. This model had AUCs of 0.93, 0.94 and 0.94 in patients who had only one single nodule, absent macrovascular invasion and tumor size <2 cm, respectively, compared with AUCs of 0.71, 0.6 and 0.59 for AFP. The model produced AUCs of 0.91 for BCLC (0-A), 0.92 for CLIP (0–1) and 0.94 for Okuda (stage I) compared with AUCs of 0.56, 0.58 and 0.64 for AFP. No significant difference was found between AUC in the estimation and the validation groups.ConclusionThis model may enhance early-stage HCC detection and help to overcome insufficient sensitivity of AFP.     

Leukocyte integrin αLβ2 headpiece structures: The αI domain,the pocket for the internal ligand,and concerted movements of its loops

High-resolution crystal structures of the headpiece of lymphocyte function-associated antigen-1 (integrin α_Lβ₂) reveal how the αI domain interacts with its platform formed by the α-subunit β-propeller and β-subunit βI domains. The α_Lβ₂ structures compared with α_Xβ₂ structures show that the αI domain, tethered through its N-linker and a disulfide to a stable β-ribbon pillar near the center of the platform, can undergo remarkable pivoting and tilting motions that appear buffered by N-glycan decorations that differ between α_L and α_X subunits. Rerefined β₂ integrin structures reveal details including pyroglutamic acid at the β₂ N terminus and bending within the EGF1 domain. Allostery is relayed to the αI domain by an internal ligand that binds to a pocket at the interface between the β-propeller and βI domains. Marked differences between the α_L and α_X subunit β-propeller domains concentrate near the binding pocket and αI domain interfaces. Remarkably, movement in allostery in the βI domain of specificity determining loop 1 (SDL1) causes concerted movement of SDL2 and thereby tightens the binding pocket for the internal ligand.Integrins are α/β heterodimeric metallo-receptors that bidirectionally transduce chemical cues together with mechanical forces across cell membranes (1). The β₂ integrin subfamily contains lymphocyte function-associated antigen-1 (LFA-1, integrin α_Lβ₂), complement receptor 3 (CR3, α_Mβ₂, Mac-1), CR4 (α_Xβ₂, p150,95), and α_Dβ₂ and is exclusively expressed on leukocyte lineages (2). LFA-1 on the surface of lymphocytes, natural killer cells, neutrophils, and monocytes binds to intercellular adhesion molecules (ICAMs) on the surface of other cells to mediate adaptive and innate immune responses, trafficking across endothelium, and migration within tissues (3). Leukocyte adhesion deficiency (4) and clinical approval of an antibody to LFA-1 to treat autoimmunity (5) illustrate the immunological significance of LFA-1 interaction with ICAMs.β₂ integrin α subunits contain an inserted (αI) domain that binds to external ligands such as ICAMs. Crystal and NMR structures have revealed open/high-affinity and closed/low-affinity conformations of the LFA-1 αI domain, how it binds to ICAMs, and its dynamics, but only in isolation from other integrin domains (6–12). Here, we characterize crystal structures of the LFA-1 headpiece and rerefine previous α_Xβ₂ ectodomain (13) and β₂ leg fragment (14, 15) crystal structures. The LFA-1 αI domain adopts a markedly different orientation relative to the remainder of the integrin head than seen with α_Xβ₂ (13, 16) and suggests that a surprising range of αI domain orientations are compatible with relay of allostery. We also find that the βI domain SDL2 loop in β₂ integrins moves in allostery and describe the responsible SDL1–SDL2 interactions, which are present in only a subset of integrin β subunits.     

The Spectrum of α-Thalassemia Mutations in Kermanshah Province,West Iran

Abstract

Thalassemia is a hereditary blood disorder that results from genetic defects causing deficient synthesis of hemoglobin (Hb) polypeptide chains. Although thalassemia mostly affects developing countries, there is limited knowledge of its accurate frequency and distribution in these regions. Knowing the prevalence of thalassemia and the frequency of responsible mutations is therefore an important step in the prevention and control program as well as treatment strategies. α-Thalassemia (α-thal) is prevalent in Middle East Asian populations, including Iran. In this study, 678 unrelated α-thal carriers, attending the Kermanshah Medical Genetics Laboratory, Kermanshah, Iran, were investigated for α-globin gene mutations by multiplex polymerase chain reaction (PCR) and direct sequencing. The most common mutation among our patients was ?α^3.7 (rightward) (60.9%) deletion, which is also known to occur in high frequencies in other parts of Iran, in Southeast Asia and Mediterranean countries. Other prevalent α-thal mutations were α^{?5 nt} (10.6%), α^polyA4 (9.9%), α^polyA6 (3.7%), –?–^MED (3.2%), ?α^4.2 (leftward) (3.1%) deletion and codon 59 (Hb Adana; HBA1: c.179?G?>?A) (2.5%). These comprehensive new data are useful for establishing a screening strategy for the effective control of α-thal in Kermanshah Province.     

Deletion of Serpina1a, a murine α1-antitrypsin ortholog, results in embryonic lethality

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States Approximately 1% to 2% of COPD patients suffer from α(1)-antitrypsin (A1AT) deficiency, the major inheritable predisposition to COPD/emphysema. To further study the role of A1AT deficiency in the pathogenesis of COPD/emphysema, the authors attempted to generate null-mutant mice for Serpina1a, 1 of 2 A1AT orthologs in mice. Here the authors show that targeted deletion of Serpina1a results in embryonic lethality prior to 8.5 days post conception (dpc). The results are surprising given that A1AT-null humans exist and therefore do not require this gene product for normal development. The Serpina1 gene cluster is substantially different between mouse and man. Through gene duplication, mice have 3 to 5 (depending on the strain) highly homologous proteinase inhibiting (Pi) genes, 2 of which inhibit neutrophil elastase. Despite the abundance of Pi genes in mice, Serpina1a serves a critical, nonredundant function during early mouse development. A1AT-deficient mice have been highly sought after to study emphysema, cancer, and liver disease, and as a model to perfect gene replacement therapy. These results highlight important differences between human and murine serpins and point to the difficulty inherent to using gene-targeted mice to study this common human genetic disease.     

Solid Serous Cystadenoma of the Pancreas: A Case Report of 2 Patients Revealing Vimentin, β-Catenin, α-1 Antitrypsin,and α-1 Antichymotrypsin as New Immunohistochemistry Staining Markers

Solid serous cystadenoma (SCA) of the pancreas is a rare type of pancreatic solid tumors. Postoperative pathological evaluation is of particular importance for distinguishing solid SCA of the pancreas from other pancreatic solid tumors.Here we present 2 cases of solid SCA of the pancreas, both preoperatively diagnosed with pancreatic neuroendocrine tumors. One case had positive OctreoScan test.Surgical resections were done for both cases. Postoperative immunohistochemistry assays were conducted with marker panels for SCA and 2 types of pancreatic solid tumors, which were neuroendocrine tumor (pNET) and solid pseudopapillary tumor (SPT).Two cases showed typical staining patterns for SCA markers. Notably, both cases showed positivity for 4 SPT markers (vimentin, β-catenin, α-1 antitrypsin, and α-1 antichymotrypsin).Emphasis should be paid to those 4 new markers for future pathological diagnosis of solid SCA of the pancreas.     

Peroxisome proliferator-activated receptor α, a potential therapeutic target for alcoholic liver disease

Alcoholic liver injury represents a progressive process with a range of consequences including hepatic steatosis, steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Targeting key molecular regulators involved in the development of alcoholic liver injury may be of great value in the prevention of liver injury. Peroxisome proliferator-activated receptor α (PPARα) plays a pivotal role in modulation of hepatic lipid metabolism, oxidative stress, inflammatory response and fibrogenesis. As such, PPARα may be a potential therapeutic target for the treatment of alcoholic liver disease.