Investigation of N-terminal glutamate cyclization of recombinant monoclonal antibody in formulation development.

The N-terminal glutamic acid (Glu) can be cyclized to form pyroglutamate (pGlu). Recent studies have suggested that N-terminal pGlu formation is an important posttranslational or co-translational event and is greatly facilitated by the enzyme glutaminyl cyclase, although the impact of the N-terminal cyclization on the potency and overall stability of mAbs is not been well known. Since most recombinant monoclonal antibodies (mAbs) contain glutamic acid and/or glutamine at their N-terminus, understanding the cyclization mechanisms may shed light on the factors that control the pGlu formation in therapeutic mAb development.

Efficacy of olanzapine and haloperidol in an animal model of mania.

Purpose: Intracerebroventricular (ICV) administration of ouabain, a potent sodium pump inhibitor, has been used to model mania. Antipsychotic agents have demonstrated efficacy in the management of acute mania. This study was undertaken to determine the prophylactic efficacy of olanzapine and haloperidol in the ouabain mania model.

Incidence of galactorrhea in young women using Depot-Medroxyprogesterone Acetate.

Galactorrhea is rarely mentioned as a possible side effect of the use of Depot-Medroxyprogesterone Acetate (DMPA). Over the last few years, we have noticed an increased number of patients complaining of galactorrhea. A review of clinical data showed that between 1999 and 2005, 360 adolescents in our clinic used DMPA for at least 6 months.

Gender difference in the activity but not expression of estrogen receptors alpha and beta in human lung adenocarcinoma cells.

The higher frequency of lung adenocarcinoma in women smokers than in men smokers suggests a role for gender-dependent factors in the etiology of lung cancer. We evaluated estrogen receptor (ER) alpha and beta expression and activity in human lung adenocarcinoma cell lines and normal lung fibroblasts. Full-length ERalpha and ERbeta proteins were expressed in all cell lines with higher ERbeta than ERalpha.

The ACAT inhibitor avasimibe increases the fractional clearance rate of postprandial triglyceride-rich lipoproteins in miniature pigs.

Previously, we have shown, in vivo, that the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe decreases hepatic apolipoprotein (apo) B secretion into plasma. To test the hypothesis that avasimibe modulates postprandial triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg) containing retinol was given to 9 control miniature pigs and to 9 animals after 28 days treatment with avasimibe (10 mg/kg/day, n=5; 25 mg/kg/day, n=4). The kinetic parameters for plasma retinyl palmitate (RP) metabolism were determined by multi-compartmental modeling using SAAM II.

Metalloendoprotease cleavage triggers gelsolin amyloidogenesis.

Amyloid diseases like Alzheimer's disease and familial amyloidosis of Finnish type (FAF) stem from endoproteolytic cleavage of a precursor protein to generate amyloidogenic peptides that accumulate as amyloid deposits in a tissue-specific manner. FAF patients deposit both 8 and 5 kDa peptides derived from mutant (D187Y/N) plasma gelsolin in the extracellular matrix (ECM). The first of two aberrant sequential proteolytic events is executed by furin to yield a 68 kDa (C68) secreted fragment.

A novel inhibitor of oxidosqualene:lanosterol cyclase inhibits very low-density lipoprotein apolipoprotein B100 (apoB100) production and enhances low-density lipoprotein apoB100 catabolism through marked reduction in hepatic cholesterol content.

Objective: Inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), an enzyme in the cholesterol synthesis pathway, has the unique ability to inhibit cholesterol synthesis while simultaneously enhancing oxysterol synthesis. Our objectives were to determine, in vivo, if a novel OSC inhibitor reduced low-density lipoprotein (LDL) cholesterol and to define the mechanism(s) involved.

Methods And Results: Miniature pigs received the OSC inhibitor RO0717625 or placebo and a diet containing fat (34% of energy) and 400 mg per day of cholesterol.

Lord of the rings--the mechanism for oxidosqualene:lanosterol cyclase becomes crystal clear.

The enzyme oxidosqualene:lanosterol cyclase (OSC) represents a novel target for the treatment of hypercholesterolemia. OSC catalyzes the cyclization of the linear 2,3-monoepoxysqualene to lanosterol, the initial four-ringed sterol intermediate in the cholesterol biosynthetic pathway. OSC also catalyzes the formation of 24(S),25-epoxycholesterol, a ligand activator of the liver X receptor.

Quality assessment of microarray experiments.

Objectives: We describe a quality assurance procedure to maximize the value of oligonucleotide microarray expression profiles.

Design, Methods, And Results: Background microarray noise was 82.2+/-54.

Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogen-activated protein kinase pathway in hepatocytes.

Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK(erk)) pathway.

AAV vector-mediated correction of brain pathology in a mouse model of Niemann-Pick A disease.

Niemann-Pick A disease (NPA) is a fatal lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. The lack of functional ASM results in cellular accumulation of sphingomyelin and cholesterol within distended lysosomes throughout the brain. In this study, we investigated the potential of AAV-mediated expression of ASM to correct the brain pathology in an ASM knockout (ASMKO) mouse model of NPA.

Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol efflux.

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation.

Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology.

Niemann-Pick disease is caused by a genetic deficiency in acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. In the present study, we evaluated the effects of direct intracerebral transplantation of neural progenitor cells (NPCs) on the brain storage pathology in the ASM knock-out (ASMKO) mouse model of Type A Niemann-Pick disease. NPCs derived from adult mouse brain were genetically modified to express human ASM (hASM) and were transplanted into multiple regions of the ASMKO mouse brain.

Ca2+ binding protects against gelsolin amyloidosis.

Amyloid diseases occur when native or mutant polypeptides misfold and aggregate to form deposits in the extracellular space. There are at least 20 proteins associated with amyloid diseases, including the well-known amyloid-beta peptide that is the causative agent for Alzheimer's disease (AD). This review describes familial amyloidosis of Finnish type (FAF), an amyloid disease caused by mutations in plasma gelsolin, a secreted protein that contains multiple Ca2+-binding domains.

Human smooth muscle cell subpopulations differentially accumulate cholesteryl ester when exposed to native and oxidized lipoproteins.

Background: Vascular smooth muscle cells (SMCs) manifest diverse phenotypes and emerging evidence suggests this is caused by inherently distinct SMC subtypes. Recently, Li et al (Circ Res 2001;89:517-525) successfully cloned 2 uniquely responsive SMC subpopulations from a single human artery and we used this unique resource to test the hypothesis that distinct SMC subtypes are differential precursors of foam cell formation.

Methods And Results: When challenged with human atherogenic native or oxidized hypertriglyceridemic very-low-density lipoprotein (HTG-VLDL), the larger, slower-growing, spindle-shaped HITB5 SMC clone accumulated significantly more cholesteryl ester (CE) and triglyceride (TG) than the smaller, faster-growing epithelioid-shaped HITA2 SMC clone (10 versus 2 microg CE/mg cell protein [PN] and 60 versus 7 microg TG/mg PN, P<0.

Metabolite-initiated protein misfolding may trigger Alzheimer's disease.

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease.

Pathological and functional amyloid formation orchestrated by the secretory pathway.

Amyloidogenesis has historically been associated with pathology in a class of neurodegenerative diseases known as amyloid diseases. Recent studies have shown that proteolysis by furin during secretion initiates both variant gelsolin amyloidogenesis, associated with the disease familial amyloidosis of Finnish type, and Pmel17 fiber formation, which is necessary for the functional biogenesis of melanosomes. Proteolysis combined with organelle-dependent environment changes orchestrate amyloidogenesis associated with both pathological processes and a functional pathway.

Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of finnish type.

Mutation of aspartic acid 187 to asparagine (D187N) or tyrosine (D187Y) in domain 2 of the actin-modulating protein gelsolin causes the neurodegenerative disease familial amyloidosis of Finnish type (FAF). These mutations render plasma gelsolin susceptible to aberrant proteolysis by furin in the trans-Golgi network, the initial proteolytic event in the formation of 71 and 53 residue fragments that assemble into amyloid fibrils. Ca(2+) binding stabilizes wild-type domain 2 gelsolin against denaturation and proteolysis, but the FAF variants are unable to bind and be stabilized by Ca(2+).

Dietary cholesterol, cholesterol absorption, postprandial lipemia and atherosclerosis.

The relationship among dietary cholesterol, cholesterol absorption, the metabolism of cholesterol-rich chylomicron remnants and atherosclerosis is complex; however, recent advances have provided insight into the mechanisms involved. Although dietary cholesterol is an independent risk factor for atherosclerosis, the attributable risk is low compared with dietary variables such as the amount and type of fat. Clinical studies have demonstrated that in humans consuming a typical Western-type diet, decreasing the amount of dietary cholesterol intake results in only small changes in low-density lipoprotein (LDL)-cholesterol and little or no change in the ratio of total cholesterol to high-density lipoprotein cholesterol.

Intracerebroventricular administration of ouabain as a model of mania in rats.

Background: Human bipolar illness is characterized by mood state- and diagnosis-associated abnormalities of cellular cation distribution and transport. These include reduced sodium pump activity and expression and increased intracellular sodium. If these alterations are related to the pathophysiology of the disease, rather than secondary or ancillary abnormalities, then one would expect that modeling of these changes in vivo would produce lithium-preventable behavioral abnormalities.

Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes.

Enhanced synthesis of the oxysterol 24(S),25-epoxycholesterol in macrophages by inhibitors of 2,3-oxidosqualene:lanosterol cyclase: a novel mechanism for the attenuation of foam cell formation.

Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux.

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization.

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects.