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Chronic alcohol consumption is a major reason for several human diseases, and alcoholism has been associated with a variety of societal problems. Changes in fatty acid metabolism in alcoholics and its effects leading to membrane damage are largely unknown. Therefore, we aimed to investigate the fatty acid composition of erythrocyte membrane phospholipids in relation with plasma lipid profile and other plasma metabolites in chronic alcoholics in comparison with controls. We systematically measured the levels of glucose, lactate and pyruvate in the blood and free amino acids, free fatty acids, mucoproteins and glycolipids, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein (VLDL) cholesterol and triglycerides (TG) in plasma of chronic alcoholics and controls. Furthermore, we measured fatty acid composition by gas chromatographic analysis. The fatty acid composition clearly revealed certain changes in chronic alcoholic erythrocyte membrane, chiefly increments in C16:0 and a decrease in C22:4 and C22:6 fatty acids besides the presence of unidentified fatty acids, probably C-24 or C-26 fatty acids. In addition, a significant increase in blood lactate, decrease in blood pyruvate and increased levels of free amino acids and free fatty acids, mucoproteins, VLDL cholesterol, TG and HDL-C in chronic alcoholics were observed with no significant change in plasma TC, LDL-C and glycolipids when compared with controls. Alcohol-induced alterations in plasma and erythrocyte membranes of chronic alcoholics in the present study might be an adaptive response to counteract the deleterious effects of alcohol. The implications of our findings warrant further investigation and needs further in-depth study to explore the mechanisms of alcohol-induced membrane changes.

Tissue microarray (TMA) is a new high-throughput method that enables simultaneous analysis of the profiles of protein expression in multiple tissue samples. TMA technology has not previously been adapted for physiological and pathophysiological studies of rodent kidneys. We have evaluated the validity and reliability of using TMA to assess protein expression in mouse and rat kidneys. A representative TMA block that we have produced included: (1) mouse and rat kidney cortex, outer medulla, and inner medulla fixed with different fixatives; (2) rat kidneys at different stages of development fixed with different fixatives; (3) mouse and rat kidneys with different physiological or pathophysiological treatments; and (4) built-in controls. As examples of the utility, immunostaining for cyclooxygenase-2, renin, Tamm Horsfall protein, aquaporin-2, connective tissue growth factor, and synaptopodin was carried out with kidney TMA slides. Quantitative analysis of cyclooxygense-2 expression in kidneys confirms that individual cores provide meaningful representations comparable to whole-kidney sections. These studies show that kidney TMA technique is a promising and useful tool for investigating the expression profiles of proteins of interest in rodent kidneys under different physiological and pathophysiological conditions. (J Histochem Cytochem 58:413–420, 2010)

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine. (J Histochem Cytochem 56:495–509, 2008)

Recombinant human interleukin-18 (rHuIL-18) is currently in clinical trials for treatment of cancer. This report presents results of preclinical toxicity studies with rHuIL-18 in cynomolgus monkeys and recombinant murine IL-18 (rMuIL-18) in mice. The rHuIL-18 was administered intravenously in 1 or 2 different 5-day cycles at doses 0.3 to 75 mg/kg/day in monkeys. Decreases in red cell mass, neutrophil, and platelet counts, increases in monocyte and large unstained cell counts, and lymphoid hyperplasia in spleen and lymph nodes were mild, reversible, and likely related to the pharmacologic activity of IL-18. The only toxic effect was protein cast nephropathy, secondary to coprecipitation of administered IL-18 and Tamm-Horsfall protein in the distal nephron, that only occurred at 75 mg/kg/day. Other adverse effects of rHuIL-18 were related to strong immunogenicity in monkeys and were manifest only during a second dosing cycle. The rMuIL-18, at similar dosing levels and cycles in mice, resulted in reduced red cell mass, increased white blood cell counts, spleen and lymph node hyperplasia, and mild, reversible changes in intestine, liver, and lungs. Protein cast nephropathy occurred in mice at doses ≥30 mg/kg/day. In conclusion, preclinical safety studies showed that rIL-18 was well tolerated at pharmacologically active doses in both monkeys and mice.

Secondary cultures from murine spiral ligament (SL) fibrocytes were stimulated with proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), and expression of various adhesion molecules was investigated. Cultures without cytokine stimulation did not show positive immunostaining for vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although staining was also negative after stimulation with IL-1beta, VCAM-1 and ICAM-1 staining was observed after the cells were stimulated with TNF-alpha. Reverse transcription-polymerase chain reaction analysis showed messenger RNAs for both VCAM-1 and ICAM-1 expression to be present after fibrocytes were stimulated with TNF-alpha. These data suggest that activated fibrocytes may cause inflammatory cells to persist in the SL. Given that SL fibrocytes may play a role in homeostasis of cochlear fluid and ion concentrations, prolongation of the inflammatory response could lead to fibrocyte damage that might ultimately result in cochlear malfunction.

The putative anion channel mCLCA3 (alias gob-5) is the third murine member of the recently discovered family of calcium-activated chloride channels (CLCA family). Preliminary data suggest that mCLCA3 may play a significant role in diseases with secretory dysfunctions, including asthma and cystic fibrosis. In this study, the mCLCA3 protein was characterized biochemically and its cellular and subcellular distribution pattern was established in normal murine tissues. Polyclonal rabbit antibodies were generated and affinity-immunopurified using synthetic oligopeptides corresponding to the extracellular amino terminus of the mCLCA3 polypeptide. After in vitro translation and glycosylation, protein-ase K protection assay, and heterologous expression in COS-7 or HEK 293 cells, SDS-PAGE and immunoblotting revealed a protein structure similar to that of previously characterized CLCA proteins. A systematic light, confocal laser scanning, and transmission electron microscopic immunolocalization study, including virtually all murine tissues, identified the mCLCA3 protein exclusively associated with mucin granule membranes of gastrointestinal, respiratory, and uterine goblet cells and other mucin-producing cells. The results suggest that mCLCA3 may be involved in the synthesis, condensation, or secretion of mucins.

The transmigration of lymphocytes across vascular endothelium is a critical step for the localization of lymphocytes to lymph nodes in both naïve and immune reactive states. Mice deficient in lymphotoxin-alpha (LT- α)lack peripheral and gut associated lymph nodes. Lymphocyte function and homing ability are reported to be normal in these mice yet information regarding cell adhesion molecules and counterpart vascular addressins is lacking. The phenotype of peripheral lymphocytes from LT- α deficient mice was investigated by the use of fluorescent activated cell sorting and immunohistochemistry. No difference was detected in the splenocyte and tissue expression of L-selectin, α4 β7 or its individual integrin components, mucosal addressin cell adhesion molecule (MAdCAM-1), intracellular adhesion molecule (ICAM-1), peripheral node addressin (PNAd), or platelet/endothelial cell adhesion molecule (PECAM-1) between wild-type and LT- α deficient mice. Therefore, impaired expression of these lymphocyte homing and vascular addressin molecules is apparently not included in the phenotype of the LT- α deficient mouse.

CA 549 is one of several carcinoma associated mucin antigens proposed as a breast cancer tumor marker. In this study, the performance characteristics of the CA 549 assay were validated and the clinical utility of the test was compared with that of other breast cancer markers including CA 15-3, CA M26, CA M29 and carcinoembryonic antigen. The upper limit of normal was established as 15.5 U/ml based on data for 250 control subjects apparently free of disease. Overall, CA 549 had a low negative predictive value (0.51) due to a low sensitivity in the detection of early breast cancer. However, the test had a high positive predictive value (0.93) reflecting a high specificity for the disease. In 56 patients with advanced breast cancer, the sensitivity was 0.71 for CA 549 alone and 0.79-0.84 for CA 549 combined with any of the other markers studied.

AimTo investigate possible age-related changes in glycosaminoglycans (GAGs) in the human cornea. The substances today called GAGs were previously referred to as mucopolysaccharides.MethodsSamples of human cornea were taken from 12 younger (age 21 ± 1.2) and 12 older (age 72 ± 1.6) male subjects. Samples were weighed, homogenized, and used for biochemical and molecular analyses. All the quantitative results were statistically analyzed.ResultsThe human cornea appears to undergo age-related changes, as evidenced by our biochemical and molecular results. The total GAG and hyaluronic acid counts were significantly higher in the younger subjects than in the older subjects. The sulfated heavy GAGs, such as chondroitin, dermatan, keratan, and heparan sulfate, were lower in the younger subjects than in the older subjects.DiscussionGAGs of the human cornea undergo numerous age-related changes. Their quantity is significantly altered in the elderly in comparison with younger subjects. GAGs play an important role in age-related diseases of the human cornea.

SummaryThis study has demonstrated the presence of a proteolytic enzyme, chymopapain C, in the extract of Carica papaya. This enzyme differs significantly from papain in enzymatic activity, thermal half-life and action of human IgG. However, this enzyme is similar to commercial chymopapain in its activity and in its digestion of human IgG, but differs in molecular weight, thermal half-life and relative electrophoretic mobility.

Various treatments carried out prior to the concanavalin A-horseradish perioxidase (HRP) method have been found to affect the staining and have permitted differentiation of three main classes of complex carbohydrates in the rat alimentary tract. Class I mucosubstances lose and class II and III paradoxically gain concanavalin A-horseradish peroxidase reactivity after periodate oxidation. Class II mucosubstances lose whereas class III retain or increase their reactivity with a reduction step interposed between oxidation and concanavalin A-horseradish peroxidase staining. Mucous neck cells, pyloric glands, Brunner's glands and mast cells exhibit strong class III staining, whereas other sites such as intestinal goblet and salivary gland acini differ widely in their type of staining. Liver glycogen stains like mucosubstances in an unstable subgroup of class III. The paradoxical increase in concanavalin A binding during oxidation correlates with the appearance of Schiff reactivity implicating oxidation of vicinal hydroxyls as the basis for the effect. The periodate-induced staining is therefore, thought to result from an oxidative disruption of linkages between vicinal hydroxyls of neighboring sugars and hydroxyls of mannose required for concanavalin A binding. Staining with the described concanavalin A-horseradish peroxidase variants appears to afford information concerning cytochemical distribution of mannose-rich glycoproteins as well as differences among these substances in the relation of mannose to neighboring sugars.

The behaviour of the perchlorsoluble mucoproteic fraction from plasma, from neoplastic tissue and from peritumoral connective neoplastic tissue has been studied in rats hearing experimental cancers (Galliera sarcoma, 3,4-benzpyrene tumour, Warker adenocarcinoma). The mucoproteic content of the neoplastic tissue was observed to be constant during the tumour growth; on the contrary the level of the same fraction from peritumoral connective tissue gradually increases till a rather advanced stage of the tumour growth, after which the increase is no more evident. The hypothesis is advanced that such local process may be responsible (at least partially) for the increase of the circulating perclor-soluble protein which is also observed to be gradually and markedly increased during the growth of the grafted malignant tissue.

Variations in the perchlorsoluble mucoprotein contents (expressed as tyrosine and hydrocarbon equivalent) have been studied in plasma and subcutaneous connective tissue of rats of both sexes treated with synthetic estrogen (dioxy-diethylstilbene). An increase in the contents was observed both in blood and connective tissue after administration of 9, 24, 50 mg in males and 24, 50 mg in females. No significant modifications were observed after administration of 100 mg in males and 9, 100 mg in females. Some possible interpretations are advanced.

SummaryThe electrophoretic behavior of mucoproteins isolated from perchloric acid filtrates of human plasma has been studied over the pH range of 2 to 8.4. There are at least 3 electrophoretic components in such preparations, the isoelectric points being approximately 2.3, 3.4 and 4.3. The major component has the lowest isoelectric point and travels with a mobility characteristic of α1-globulin at pH 8.4. The increase in the mucoprotein level isolated from the plasma of a patient with gastric cancer was largely in this acid mucoprotein fraction.

Sera from various mammalian species, solutions of various fractions of serum proteins, and solutions of certain other substances were tested by ultrafiltration in the presence of Ca⁴⁵ and Sr⁶⁵ for their binding of Ca, and Sr. For 6 species, human, dog, horse, cow, sheep, and rat, the ratio, Ca bound/Sr bound, was 1.29, 1.14, 1.19, 1.15, 1.17, and 1.13, respectively. The absolute degree of binding shown by these species falls into 2 groups. The human, dog, and horse sera bound the 2 ions to a lesser extent, 57% Ca and 47% Sr; cow, sheep, and rat sera bound the ions to a greater extent, 69% Ca and 60% Sr. Ultrafiltration of various serum protein fractions of the human and bovine revealed that fraction V (albumin) showed no difference between the binding of Ca and Sr but differences were noted with the globulin fractions (IV, III, II) that could account for the discrimination observed in whole serum. Results of the ultrafiltration of a variety of other proteins and polyelectrolytes led to the speculation that mucoproteins in the serum globulins could account for the large fiuctuations in binding of both ions by globulin fractions of various species; however, the results did notmore » show any indication of the type of substance or chemical grouping which could account for the slight but persistent discrimination between Ca and Sr that has been observed in whole serum. (auth)« less

The form of iron which binds specifically to mucins in the Mayer (10) and Hale (2) reactions and their variants appears to be the complex ion FeOH++. The reagent is produced in two ways, the better by carefully computed H2O2 oxidation of ferrous chloride (or acetate in the Mayer reaction), the second by addition of NaOH to FeCl3 solutions, best at a 1:1 M ratio. The first reagent is relatively stable, an 0.1 M solution remaining useful for 4 months; 10 mM solutions of both are usable repeatedly for 4 and 3 weeks, respectively, for the 1st and 2nd variants. The binding of this form of iron is prevented by methylation, impaired variably by acetylation and unaffected by a deamination adequate to prevent anionic dye staining. These characters distinguish FeOH++ sharply from Fe++ and Fe+++ which bind to lysyl and arginyl residues and whose binding is prevented in lysyl areas by both methylation (amine alkylation) and deamination. The action on a number of specific mucosubstances in man and laboratory animals is described. Gastric epithelial mucoprotein is reactive (rat and guinea pig), in distinction from its nonreactivity to most metachromatic basic dyes (7). Reactivity was recorded for guinea pig tracheal cartilage and glands, duodenal goblet cells, Brunner gland mucin, crypt and goblet cell mucins of ileum and colon, for rat mast cells, sublingual and submaxillary but not parotid glands, for human umbilical cord matrix and nucleus pulposus.

SummaryQualitative and quantitative profiles were established for 14 proteins and mucoproteins in meconium from meconium ileus and control subjects. Three of these substances were found in both meconiums, at different concentrations. The abnormal meconium characteristically contained 6 and lacked 5 proteins and mucoproteins.

Summary1. Both rabbit and human seromucoproteins isolated by Winzler's method exhibited the same patterns of digestibility to enzymes. 2. They were digested completely by the common proteolytic enz...

SummaryTo determine the time of appearance of the haptoglobin serum proteins in the developing human being, starch gel zone electrophoresis was performed on sera from 5 fetuses, 63 umbilical cord bloods, and 86 infants. Haptoglobins were absent in sera from the fetuses, but present in 10% of the umbilical cord bloods at birth, in 75% of the sera from infants up to 2 weeks old, and in 100% of the sera from the infants over 2 weeks of age.For cooperation and assistance in securing the cord and newborn bloods necessary for this study, we wish to thank Dr. Marion Erlandson and Dr. Margaret Hilgartner, Dept. of Pediatric Hematology, New York Hospital, Dr. Roy Bonsnes and Miss Dorothy Wendel, Central Laboratories of New York Hospital, and Dr. Wha Shim Han, Bellevue Hospital. We also wish to thank Dr. Bartholomeus Hoogstraten, Mt. Sinai Hospital, for obtaining the fetal bloods. Dr. German Castillo, Univ. of the Philippines, participated in the early phases of the study.

SummaryPartial hepatectomy resulted in an increase in the relative synthetic rates (RSR) of albumin, fibrinogen, and the seromucoid fraction which extended over a period of at least 14 days following the operation. The response of the seromucoid fraction showed a biphasic pattern; an early marked increase closely related to the initial stress and a less marked secondary response. The response of albumin was less dramatic than that of the other fractions and showed no evidence of a biphasic character.Laparotomy caused an increase in the RSR of the seromucoid and fibrinogen fractions but had no effect on albumin synthesis. The patterns of response for fibrinogen and the seromucoid fraction following laparotomy were different from those following partial hepatectomy; RSR values were back to normal by 8 days and the secondary wave of seromucoid synthesis was absent.

Variations in the perchlorsoluble mucoprotein contents have been studied in plasma and in normal as well as peritumoral subcutaneous connective tissue of rats bearing sarcoma Galliera and treated with dioxydiethylstilbene. In the groups treated with 9, 24, 50 mg (males) and 24, 50 mg (females) these variations (generally similar to those observed in non-bearing tumour animals likewise treated) consist in an increase of the mucoprotein contents which, on the contrary, is unmodified in animals of both sexes treated with 100 mg. Quantitative variations in this protein fraction are observed also within the neoplasm (the maximum increase is observed in males treated with 100 mg and in females treated with 24 mg).

The blood lymphocyte culture «in vitro» has been employed in order to investigate (a) the presence of neoplastic antigens in reticulum cell sarcoma exudates, and (b) the ability of patients with reticulum cell proliferative diseases (reticulum cell sarcoma, reticulum cell reticulosis) to give immune reactions to such antigens. Lymphocytes from a patient with reticulum cell reticulosis showed a 7.3% transformation to blast cells in the presence of the pleural exudate from a reticulum cell sarcoma patient. However, this same exudate did not induce transformation of lymphocytes from two other patients with reticulum cell sarcoma nor of normal lymphocytes. Lymphocytes from the exudate donor patient did show a small percentage transformation in the presence as well as in the absence of the exudate (2.3, and 2.1%, respectively). With the purpose of identification of the transformating factor, fractionation studies were performed yielding a carbohydrate-rich, partially dialyzable mucoprotein. The conclusion is drawn that lymphocytes from the patient with reticulum cell reticulosis were immunologically reactive to a neoplastic antigen contained in the reticulum cell sarcoma exudate, and possibly produced by the proliferating reticulum cells.

SummarySerum haptoglobin was studied in guinea pigs and rabbits in normal animals and after experimental production of connective tissue damage such as in acute scurvy, carrageenan granuloma, turpentine-induced amyloidosis, and intravenous injection of different enzymes (papain, elastase, hyaluronidase, trypsin, chymotrypsin); controls received intravenous ovalbumin and saline. Significant increase of serum haptoglobin was found in acute scurvy (average 8 times normal), carrageenan granuloma (4 times normal) or both combined (5 times normal), and after intravenous injection of papain. elastase and of hyaluronidase (average of 2 to 2.5 times normal). Particularly striking results were obtained with papain and elastase which in some cases produced an increase in haptoglobin up to 7 times the starting value. These methods may be used to obtain reproducible experimental hyperhaptoglobinemia.

Sialidase digestion of histological sections releases sialic acid from some sialic acid rich nonsulfated mucins. Such treatment eliminates the metachromatic staining and alcian blue affinity of these mucopolysaccharides. The procedure furnishes a method for histochemical localization of certain sialomucins.

SummarySerum total proteins, glycopro-teins, mucoproteins, albumin and different fractions of globulins were determined in normal monkeys fed ad lib, in scorbutic monkeys, in normal monkeys pair-fed with the scorbutic, and in monkeys which recovered from scurvy after treatment with ascorbic acid. Following changes from pair-fed normal were observed in scorbutic monkeys: Serum total proteins, a2-globulin and γ-globulin did not change. Serum albumin diminished. Glycoproteins, mucoproteins, a1-globulin and β-globulin expressed as percent of total protein of serum increased. The changes in serum proteins were reversed to normal when the scorbutic monkeys recovered after supplementation with ascorbic acid with the exception of serum a2-globulin which diminished significantly. The changes in serum proteins of scorbutic monkeys seems to be a specific effect of ascorbic acid deficiency.

Summary1. Liver cell contains lipids, potassium, free aldehyde groups, sulfuric acid esters of mucopolysaccharide origin, protein bound sulfhydryl groups, mucoproteins, calcium, non-specific esterase, ribosenucleic acid and vit. C. 2. The lipid of the liver cell showing fatty metamorphosis is histochemically different than normal liver cell lipid. 3. The non-specific esterase activity is increased and clumped in the fatty liver. 4. Cytoplasmic liver cell RNA appears to be decreased in patients with morphologically active cirrhosis. 5. In the presence of clinical jaundice, alkaline phosphatase activity is increased in the Kupffer cells, in the liver cells adjacent to the central veins in the bile plugging the ducts. 6. Fatty liver appears to contain more vit. C than the normal liver.

SummaryMucoproteins obtained from hog gastric mucosa exert a protective effect in experimental poliomyelitis (Type II) infection of mice, while they have no action on the virus in tissue cultures. The protective action is attributed to some mechanism increasing nonspecific resistance of the host.

SummarySerum glycoproteins, mucoproteins, phospholipid, total lipid, total cholesterol and lipoproteins were determined in patients suffering from cholera and in normal subjects. In cholera serum glycoproteins, mucoproteins, phospholipids, total lipids and β-lipoproteins were significantly increased and α-lipoproteins and total cholesterol were diminished.

SummarySerum albumin, α2- and β2- globulins which diminished and a1-globulin which increased in scurvy became approximately normal after insulin treatment. The changes in protein pattern of serum might be due to insulin deficiency of scorbutic guinea pigs. Serum glycoproteins, mucoproteins and β-lipoproteins increased and α-lipoprotein and “o” fraction diminished in scurvy.