Determination of carbohydrate-deficient transferrin separated by lectin affinity chromatography for detecting chronic alcohol abuse.

Carbohydrate-deficient transferrin (CDT) has been established as a valuable biological marker for detecting chronic alcohol abuse. To improve the diagnostic efficiency, we studied new CDT determination procedures involving the use of lectin affinity chromatography with Allomyrina dichotoma agglutinin (allo A) and Trichosanthes japonica agglutinin I (TJA-I) to isolate the CDT isoforms CDT-allo A and CDT-TJA, respectively. These procedures, based on detection of the CDT-allo A and CDT-TJA isoforms in sera, showed high sensitivity (100% and 98%, respectively) and high specificity (93% and 85%, respectively). These results demonstrate that the new procedures involving the use of lectin affinity chromatography are more useful for isolating markers in the CDT test than the conventional charge-based separation method.     

Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry

Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.     

Carbohydrate-deficient transferrin (CDT) as a marker of alcohol abuse: a critical review of the literature 2001-2005

The diagnosis of alcohol abuse based on objective data is a necessary requirement in both clinical and forensic environments. Among the different biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is world wide recognized as the most reliable indicator. However, several problems about the real meaning of CDT and the reliability of its use for the diagnosis of alcohol abuses are still open, as reported by numerous research articles and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2001 to 2005 (included). The article is organized in the following sections: (1) introduction, (2) definition and structure of human serum CDT, (3) pathomechanisms of the ethanol-induced CDT increase, (4) preanalysis, (5) analysis, (6) data interpretation, (7) review papers, (8) conclusions. As many as 127 papers appeared in the international literature and retrieved by the search engines PubMed and Scopus are quoted.     

Different Binding Affinities of Pb2+ and Cu2+ to Glycosylation Variants of Human Serum Transferrin Interfere with the Detection of Carbohydrate-Deficient Transferrin

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse, and for diagnosis of chronic alcohol, abuse is often determined using isoelectric focusing (IEF) and chromatographic techniques. To allow this method to be used for the diagnosis of alcohol abuse, inferences of various physical and chemical factors with the detection of CDT have been investigated. However, few reports have focused thus far on whether different metal ions have different binding affinities to CDT and HTf variants or further interfere in the detection of CDT. Here, in order to figure out whether and how metal ions such as Pb(2+) and Cu(2+) bind to holo-human serum transferrin (holo-HTf) and further interfere in CDT detection, the binding characteristics and the binding parameters of holo-HTf with metal ions such as Pb(2+) and Cu(2+) were investigated using UV-visible spectroscopy, Fluorescence spectroscopy, and ICP-MS. Moreover, whether the metal ions such as Pb(2+) and Cu(2+) will reduce the diagnostic accuracy of CDT in clinic was investigated using IEF. The present study demonstrates that Pb(2+) and Cu(2+) have different binding affinities to holo-HTf variants and produce different changes in the relative amounts of each glycosylation isoforms of HTf. Accordingly, the glycosylation chains of HTf will affect the binding affinities of glycosylation isoforms with Pb(2+) and Cu(2+), causing further interferences in CDT detection.     

High adiponectin and TNF-alpha levels in moderate drinkers suffering from liver steatosis: comparison with non drinkers suffering from similar hepatopathy

Moderate alcohol consumption is associated with increased insulin sensitivity and a reduced risk for type 2 diabetes. An important endogenous mediator of insulin sensitivity is adiponectin (AN), an adipokine that displays numerous antiatherogenic, antidiabetogenic and antiinflammatory effects. Recently, acute increase in alcohol consumption has been shown to be associated with increase in plasma adiponectin and, concomitantly, insulin sensitivity. Whether chronic alcohol consumption predicts an increase in plasma AN and whether this is independent of adiposity, markers of liver dysfunction, and plasma adipokines such as tumor necrosis factor (TNF)-alpha is not known. We, therefore, investigated these relationships in 75 men who were diagnosed with liver steatosis using ultrasound/liver biopsy. We examined 75 men, who were diagnosed for having liver steatosis (ultrasound/liver biopsy). Each filled in a questionnaire on alcohol intake. Subjects were divided into two subgroups according to alcohol history and CDT concentrations--drinkers and non-drinkers. All individuals were examined for serum concentrations of AN, glucose, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate transferase (GMT) activity; carbohydrate-deficient transferrin (CDT%) a marker of chronic alcohol consumption, insulin and TNF-alpha. The Quicki insulin sensitivity index was calculated. Forty-eight individuals were found to be moderate drinkers and 27 subjects non-drinkers. Moderate drinkers had significantly higher concentrations of AN (13.8 +/- 3,7 versus 9.1 +/- 5.4 mg/l, means +/- SD, p = 0.012) compared with non-drinkers, independent of adiposity. Plasma AN concentrations in the whole group were positively correlated with TNF-alpha concentrations (r = 0.6; p = 0.0001), CDT (r = 0.26; p = 0.0084), AST/ALT index (r = 0.3, p = 0.009), AST (r = 0.29; p = 0.011) and GMT (r = 0.29; p = 0.011) and negatively with BMI (r = -0.48; p = 0.0002) and glycemia (r = -0.22; p = 0.049). The positive associations of AN with TNF-alpha (0.8; p = 0.001), CDT (0.55; p = 0.017), AST/ALT index (0.55; p = 0.019) and the negative correlation with glycemia (-0.35; p = 0.0158) were independent of BMI. Stratified according to alcohol intake, in moderate drinkers, a positive correlation was found between AN and TNF-alpha concentrations (r = 0.6, p = 0.0001, AST/ALT index (r = 0.34, p = 0.0295) whereas in non-drinkers no such correlations were found. The concentration of AN and BMI displayed a negative correlation in both drinker and nondrinker patients (r = -0.42, p = 0.01 and -0.61; p = 0.012, respectively). We concluded that plasma AN is higher in moderate drinkers compared to non-drinkers, even after correction for BMI. Drinkers suffering from liver steatosis were found to have a positive correlation between AN concentrations, laboratory markers of liver disease and TNF-alpha. Such correlation was absent in non-drinkers suffering from liver steatosis. This suggests that alcohol may modulate the inhibitory effect of TNF-alpha on AN production, and thus, increase its plasma concentrations.     

Glycoconjugates in the detection of alcohol abuse

Up to 30% of all hospital admissions and health-care costs may be attributable to alcohol abuse. Ethanol, its oxidative metabolites, acetaldehyde and ROS (reactive oxygen species), non-oxidative metabolites of alcohol [e.g. FAEEs (fatty acid ethyl esters)] and the ethanol-water competition mechanism are all involved in the deregulation of glycoconjugate (glycoprotein, glycolipid and proteoglycan) metabolic processes including biosynthesis, modification, transport, secretion, elimination and catabolism. An increasing number of new alcohol biomarkers that are the result of alcohol-induced glycoconjugate metabolic errors have appeared in the literature. Glycoconjugate-related alcohol markers are involved in, or are a product of, altered glycoconjugate metabolism, e.g. CDT (carbohydrate-deficient transferrin), SA (sialic acid), plasma SIJ (SA index of apolipoprotein J), CETP (cholesteryl ester transfer protein), β-HEX (β-hexosaminidase), dolichol, EtG (ethyl glucuronide) etc. Laboratory tests based on changes in glycoconjugate metabolism are useful in settings where the co-operativeness of the patient is impaired (e.g. driving while intoxicated) or when a history of alcohol use is not available (e.g. after trauma). In clinical practice, glycoconjugate markers of alcohol use/abuse let us distinguish alcoholic from non-alcoholic tissue damage, having important implications for the treatment and management of diseases.     

Chromatographic methods for the determination of markers of chronic and acute alcohol consumption

The development in chromatographic methods for the determination of markers of alcohol consumption is summarized in this review. The markers included in this article are ethanol in body fluids, ethanol congeners, fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), cocaethylene (CE), carbohydrate-deficient transferrin (CDT), phosphatidylethanol (PEth), 5-hydroxytryptophol (5-HTOL), dolichol, ketone bodies, acetaldehyde-protein adducts, and salsolinol (SAL). Some of these markers for alcohol consumption do not only indicate previous ethanol ingestion, but also approximate the amount of intake and the time when ethanol ingestion last occurred. Basic information about the procedures, work-up, and chromatographic conditions are summarized in tables. Also the main metabolic pathways and reaction schemes are demonstrated in figures. Some examples of typical applications are presented. The author points out that in many of the reviewed papers validation data of the procedures as well as specificities and sensitivities were not clearly presented and consequently were not comparable.     

ReviewA Review of the Role of Reactive Oxygen and Nitrogen Species in Alcohol-induced Mitochondrial Dysfunction

Our understanding of the mechanisms involved in the development of alcohol-induced liver disease has increased substantially in recent years. Specifically, reactive oxygen and nitrogen species have been identified as key components in initiating and possibly sustaining the pathogenic pathways responsible for the progression from alcohol-induced fatty liver to alcoholic hepatitis and cirrhosis. Ethanol has been demonstrated to increase the production of reactive oxygen and nitrogen species and decrease several antioxidant mechanisms in liver. However, the relative contribution of the proposed sites of ethanol-induced reactive species production within the liver is still not clear. It has been proposed that chronic ethanol-elicited alterations in mitochondria structure and function might result in increased production of reactive species at the level of the mitochondrion in liver from ethanol consumers. This in turn might result in oxidative modification and inactivation of mitochondrial macromolecules, thereby contributing further to mitochondrial dysfunction and a loss in hepatic energy conservation. Moreover, ethanol-related increases in reactive species may shift the balance between pro- and anti-apoptotic factors such that there is activation of the mitochondrial permeability transition, which would lead to increased cell death in the liver after chronic alcohol consumption. This article will examine the critical role of these reactive species in ethanol-induced liver injury with specific emphasis on how chronic ethanol-associated alterations to mitochondria influence the production of reactive oxygen and nitrogen species and how their production may disrupt hepatic energy conservation in the chronic alcohol abuser.     

A review of the role of reactive oxygen and nitrogen species in alcohol-induced mitochondrial dysfunction

Our understanding of the mechanisms involved in the development of alcohol-induced liver disease has increased substantially in recent years. Specifically, reactive oxygen and nitrogen species have been identified as key components in initiating and possibly sustaining the pathogenic pathways responsible for the progression from alcohol-induced fatty liver to alcoholic hepatitis and cirrhosis. Ethanol has been demonstrated to increase the production of reactive oxygen and nitrogen species and decrease several antioxidant mechanisms in liver. However, the relative contribution of the proposed sites of ethanol-induced reactive species production within the liver is still not clear. It has been proposed that chronic ethanol-elicited alterations in mitochondria structure and function might result in increased production of reactive species at the level of the mitochondrion in liver from ethanol consumers. This in turn might result in oxidative modification and inactivation of mitochondrial macromolecules, thereby contributing further to mitochondrial dysfunction and a loss in hepatic energy conservation. Moreover, ethanol-related increases in reactive species may shift the balance between pro- and anti-apoptotic factors such that there is activation of the mitochondrial permeability transition, which would lead to increased cell death in the liver after chronic alcohol consumption. This article will examine the critical role of these reactive species in ethanol-induced liver injury with specific emphasis on how chronic ethanol-associated alterations to mitochondria influence the production of reactive oxygen and nitrogen species and how their production may disrupt hepatic energy conservation in the chronic alcohol abuser.     

Effect of Separation Conditions on Automated Isoelectric Focusing of Carbohydrate-Deficient Transferrin and Other Human Isotransferrins Using the PhastSystem

To investigate the effect of automated isoelectric focusing conditions in the PhastSystem, e.g., the point of sample application, prerun and separation times, and minimized gels on isotransferrin band pattern, human sera were analyzed with native transferrin iron load, after iron saturation or iron depletion in vitro. Varying the focusing conditions we found (i) Point of sample application (anode, middle of the gel, cathode) strongly affected transferrin iron loss. It was greatest at the anode and least at the cathode. (ii) Without prerun, distinct transferrin iron loss also occurred. A short prerun time prevented iron loss, but increasing it did not improve transferrin iron load stability as stated by others. (iii) An inappropriately long separation time inevitably yielded iron loss. In conclusion, inappropriate isoelectric focusing conditions strongly affect iron load stability of isotransferrins (obviously via low pH within the gel), resulting in transferrin iron release and cofocusing of isotransferrins with different sialic acid or iron contents. For determination of carbohydrate-deficient transferrin, such conditions resulted in overestimation of the marker of chronic alcohol abuse. Our findings may be of guiding importance for isoelectric focusing of protein-ligand complexes. We recommend the procedure described for development of isoelectric focusing of protein-ligand complexes.     

Sex-dependent differences in the regulation of myocardial protein synthesis following long-term ethanol consumption

Chronic heavy alcohol consumption alters cardiac structure and function. Controversies remain as to whether hearts from females respond to the chronic ethanol intake in a manner analogous to males. In particular, sex differences in the myocardial response to chronic alcohol consumption remain unresolved at the molecular level. The purpose of the present set of experiments was to determine whether alterations in cardiac structure and protein metabolism show sexual dimorphism following chronic alcohol consumption for 26 wk. In control animals, hearts from female rats showed lowered heart weights and had thinner ventricular walls compared with males. The smaller heart size was associated with a lower protein content that occurred in part from a reduced rate of protein synthesis. Chronic alcohol consumption in males, but not in females, caused a thinning of the ventricular wall and intraventricular septum, as assessed by echocardiography, correlating with the loss of heart mass. The alterations in cardiac size occurred, in part, through a lowering of the protein content secondary to a diminished rate of protein synthesis. The decreased rate of protein synthesis appeared related to a reduced assembly of active eukaryotic initiation factor (eIF)4G.eIF4E complex secondary to both a diminished phosphorylation of eIF4G and increased formation of inactive 4Ebinding protein (4EBP1).eIF4E complex. The latter effects occurred as a result of decreased phosphorylation of 4EBP1. None of these ethanol-induced alterations in hearts from males were observed in hearts from females. These data suggest that chronic alcohol-induced impairments in myocardial protein synthesis results, in part, from marked decreases in eIF4E.eIF4G complex formation in males. The failure of female rats consuming ethanol to show structural changes appears related to the inability of ethanol to affect the regulation protein synthesis to the same extent as their male counterparts.     

Carbohydrate deficient transferrin: a marker for alcohol abuse.

OBJECTIVE--To assess the value of serum carbohydrate deficient transferrin as detected by isoelectric focusing on agarose as an indicator of alcohol abuse. DESIGN--Coded analysis of serum samples taken from patients with carefully defined alcohol intake both with and without liver disease. Comparison of carbohydrate deficient transferrin with standard laboratory tests for alcohol abuse. SETTING--A teaching hospital unit with an interest in general medicine and liver disease. PATIENTS--22 "Self confessed" alcoholics admitting to a daily alcohol intake of at least 80 g for a minimum of three weeks; 15 of the 22 self confessed alcoholics admitted to hospital for alcohol withdrawal; 68 patients with alcoholic liver disease confirmed by biopsy attending outpatient clinics and claiming to be drinking less than 50 g alcohol daily; 47 patients with non-alcoholic liver disorders confirmed by biopsy; and 38 patients with disorders other than of the liver and no evidence of excessive alcohol consumption. INTERVENTION--Serial studies performed on the 15 patients undergoing alcohol withdrawal in hospital. MAIN OUTCOME measure--Determination of relative value of techniques for detecting alcohol abuse. RESULTS--Carbohydrate deficient transferrin was detected in 19 of the 22 (86%) self confessed alcohol abusers, none of the 47 patients with non-alcoholic liver disease, and one of the 38 (3%) controls. Withdrawal of alcohol led to the disappearance of carbohydrate deficient transferrin at a variable rate, though in some subjects it remained detectable for up to 15 days. Carbohydrate deficient transferrin was considerably superior to the currently available conventional markers for alcohol abuse. CONCLUSION--As the technique is fairly simple, sensitive, and inexpensive we suggest that it may be valuable in detecting alcohol abuse.     

One ring to rule them all? Another cellular responsibility for PCNA

To prevent duplication or loss of genomic regions during DNA replication, it is essential that the entire genome is copied precisely once every S phase. Cells achieve this by mutually exclusive regulation of origin firing and licensing. A crucial protein that is involved in origin licensing is chromatin licensing and DNA replication factor 1 (CDT1) and, therefore, activity of this protein must be strictly controlled. Four recent articles have demonstrated that proliferating cell nuclear antigen (PCNA), an essential sliding clamp used in replication and DNA repair, has a crucial role in this process by mediating the proteasomal degradation of CDT1.     

Acute Binge Drinking Increases Serum Endotoxin and Bacterial DNA Levels in Healthy Individuals

Binge drinking, the most common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its biological consequences are poorly defined. Previous studies demonstrated that chronic alcohol use results in increased gut permeability and increased serum endotoxin levels that contribute to many of the biological effects of chronic alcohol, including alcoholic liver disease. In this study, we evaluated the effects of acute binge drinking in healthy adults on serum endotoxin levels. We found that acute alcohol binge resulted in a rapid increase in serum endotoxin and 16S rDNA, a marker of bacterial translocation from the gut. Compared to men, women had higher blood alcohol and circulating endotoxin levels. In addition, alcohol binge caused a prolonged increase in acute phase protein levels in the systemic circulation. The biological significance of the in vivo endotoxin elevation was underscored by increased levels of inflammatory cytokines, TNFα and IL-6, and chemokine, MCP-1, measured in total blood after in vitro lipopolysaccharide stimulation. Our findings indicate that even a single alcohol binge results in increased serum endotoxin levels likely due to translocation of gut bacterial products and disturbs innate immune responses that can contribute to the deleterious effects of binge drinking.     

Alcohol myopathy: impairment of protein synthesis and translation initiation

Alcohol consumption leads to numerous morphological, biochemical and functional changes in skeletal and cardiac muscle. One such change observed in both tissues after either acute alcohol intoxication or chronic alcohol consumption is a characteristic decrease in the rate of protein synthesis. A decrease in translation efficiency appears to be responsible for at least part of the reduction. This review highlights advances in determining the molecular mechanisms by which alcohol impairs protein synthesis and places these observations in context of earlier studies on alcoholic myopathy. Both acute and chronic alcohol administration impairs translational control by modulating various aspects of peptide-chain initiation. Moreover, this alcohol-induced impairment in initiation is associated with a decreased availability of eukaryotic initiation factor (eIF) 4E in striated muscle, as evidenced by an increase in the amount of the inactive eIF4E.4E-BP1 complex and decrease in the active eIF4E.eIF4G complex. In contrast, alcohol does not produce consistent alterations in the control of translation initiation by the eIF2 system. The etiology of these changes remain unresolved. However, defects in the availability or effectiveness of various anabolic hormones, particularly insulin-like growth factor-I, are consistent with the alcohol-induced decrease in protein synthesis and translation initiation.     

Identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry

Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. The currently available markers, however, are limited in sensitivity and specificity for screening of excessive alcohol drinkers. Therefore, searches for novel markers are warranted. Recently, surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) has been successfully used to detect disease-associated proteins in complex biological specimens. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence from a total of 16 chronic alcoholic patients hospitalized for a rehabilitation program. We recognized two peaks (5.9 and 7.8 kDa), both of which had been downregulated on admission, the expression level of which significantly increased after a one-week abstinence. These changes were also seen in nonresponders of gamma-glutamyltransferase. These two proteins were partially purified and subjected to amino acid sequencing. The 5.9 kDa protein was identified as a fragment of fibrinogen alphaE chain and the 7.8 kDa was a fragment of apoprotein A-II. These novel protein fragments may be promising biomarkers for excessive alcohol drinking.     

Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.     

Mapping a locus for alcohol physical dependence and associated withdrawal to a 1.1 Mb interval of mouse chromosome 1 syntenic with human chromosome 1q23.2-23.3

Physiological dependence and associated withdrawal episodes are thought to constitute a motivational force perpetuating continued alcohol use/abuse. Although no animal model duplicates alcoholism, models for specific factors, like the withdrawal syndrome, are useful to identify potential determinants of liability in humans. We previously detected quantitative trait loci (QTLs) with large effects on predisposition to physical dependence and associated withdrawal following chronic or acute alcohol exposure to a large region of chromosome 1 in mice ( Alcdp1 and Alcw1 , respectively). Here, we provide the first confirmation of Alcw1 in a congenic strain, and, using interval-specific congenic strains, narrow its position to a minimal 1.1   Mb (maximal 1.7   Mb) interval syntenic with human chromosome 1q23.2-23.3. We also report the development of a small donor segment congenic that confirms capture of a gene(s) affecting physical dependence after chronic alcohol exposure within this small interval. This congenic will be invaluable for determining whether this interval harbors a gene(s) involved in additional alcohol responses for which QTLs have been detected on distal chromosome 1, including alcohol consumption, alcohol-conditioned aversion and -induced ataxia. The possibility that this QTL plays an important role in such diverse responses to alcohol makes it an important target. Moreover, human studies have identified markers on chromosome 1q associated with alcoholism, although this association is still suggestive and mapped to a large region. Thus, the fine mapping of this QTL and analyses of the genes within the QTL interval can inform developing models for genetic determinants of alcohol dependence in humans.     

Both calmodulin and the unconventional myosin Myr4 regulate membrane trafficking along the recycling pathway of MDCK cells

In epithelial cells, endocytosed transferrin and its receptor, which cycle basolaterally, have been shown to transit through recycling endosomes which can also be accessed by markers internalized from the apical surface. In this work, we have used an in vitro assay to follow transfer of an endocytosed marker from apical or basolateral early endosomes to recycling endosomes labeled with transferrin. We show that calmodulin (CaM) function is necessary for transfer and identified myr4, a member of the unconventional myosin superfamily known to use CaM as a light chain, as a possible target protein for CaM. Since myr4 is believed to act as an actin-based mechanoenzyme, we tested the role of polymerized actin in the assay. Our data show that conditions which either prevent actin polymerization or induce the breakdown of existing filaments strongly inhibit interactions between recycling endosomes and either set of early endosomes. Altogether, our data indicate that trafficking at early steps of the endocytic pathway in Madin–Darby Canine Kidney cells depends on the actin-based mechanoenzyme myr4, its light chain CaM, and polymerized actin.     

Cyclosporin A inhibits an initial step in folding of transferrin within the endoplasmic reticulum.

As resolved by electrophoresis in non-reducing SDS gels, transferrin newly made in Hep G2 cells migrates as a very diffuse set of species. During a subsequent 1-h chase all transferrin polypeptides are converted to a single, rapidly migrating species. These changes in gel mobility are due to alterations in the pattern of disulfide bonding, are not caused by carbohydrate processing, and occur while the protein is in the rough endoplasmic reticulum. Cyclosporin A causes an approximately 10-min lag in transferrin folding, after which folding resumes at the normal rate. Cyclosporin A also retards transferrin maturation from the endoplasmic reticulum and its secretion, at concentrations that do not affect secretion of other hepatoma proteins. Neither FK506 nor rapamycin affect transferrin folding. We conclude that an initial stage in transferrin folding is accelerated by an endoplasmic reticulum peptidyl-proline isomerase that is inhibited by cyclosporin A.