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Biomedical subjects

J Roth

Publications and source records attributed to J Roth.

At least 433 records · Page 24Linked to original sources
PubMed1988

Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carrying polylactosaminoglycan.

Two major lysosomal membrane glycoproteins with apparent Mr approximately 120,000 were purified from chronic myelogenous leukemia cells. These glycoproteins are major sialoglycoproteins containing polylactosaminoglycan and represent approximately 0.1-0.2% of total cell proteins. A monoclonal antibody specific to one of the glycoproteins and polyclonal antibodies specific to the other glycoprotein were obtained. Immunoelectron microscopic examination of HeLa cells revealed that these two glycoproteins mainly reside in lysosomes and multivesicular bodies. Immunoprecipitation experiments showed that a number of different cell lines express these glycoproteins. However, the apparent molecular weights differed between cell lines; this probably represents differences in the amount of polylactosaminoglycan expressed by each cell line. As shown in the following paper (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928) one of the glycoproteins is very homologous to that of a mouse counterpart, m-lamp-1. The human form of this glycoprotein is therefore named human lamp-1 (h-lamp-1), while the other glycoprotein, to which the monoclonal antibody was made, is called human lamp-2 (h-lamp-2). Pulse-chase labeling experiments detected that h-lamp-1 and h-lamp-2 are produced first as precursor forms of 87.5 and 84 kDa, and treatment with endo-beta-N-acetylglucosaminidase H (endo-H) or endo-beta-N-acetylglucosaminidase F (endo-F) reduced their molecular masses to 39.5 and 41.5 kDa, respectively. It was estimated that h-lamp-1 has 18 N-linked saccharides and h-lamp-2 16, based on the results of partial digestions with endo-F. These results indicate that the two lysosomal membrane glycoproteins are extensively modified by N-glycans, and some of these were found to have polylactosaminyl repeats and sialic acid. Human lamp-1 and lamp-2, therefore, serve as good models for understanding polylactosaminoglycan formation and the biosynthesis and processing of polylactosaminoglycan-containing glycoprotein.

Amino Acids↗
PubMed1988

Characterization of GP120 binding to CD4 and an assay that measures ability of sera to inhibit this binding.

There is evidence that the initial interaction between HIV-1 and the host that is essential for infection is the specific binding of the viral envelope glycoprotein, gp120, to the CD4 molecule found on certain T cells and monocytes. Most individuals infected with HIV develop antibodies against the gp120 protein. Although in vitro treatment of CD4+ T cells with mAb to a specific epitope of the CD4 molecule (T4a) blocks virus binding, syncytia formation, and infectivity, it is unclear if antibodies to gp120 from an infected individual that can inhibit the binding of gp120 to CD4 is in any way related to the clinical course of disease. Our present study characterizes the binding of 125I-labeled rgp120 to CD4+ cells, and describes an assay system that measures a potentially relevant form of immunity to HIV infection, i.e., the blocking of HIV binding to CD4+ cells. Optimal binding conditions included a 2-h incubation at 22 degrees C, 4 x 10(6) CD4+ cells, and 1 nM gp120. The dissociation constant (KD) for gp120 binding to cell surface CD4 was 5 nM, and was inhibited by soluble CD4 and by mAb to T4a but not to T3 or T4. For the binding inhibition assay, negative controls included healthy seronegatives, seronegatives with connective tissue diseases, patients with HTLV-1 disease, and patients infected with HIV-2. In studying over 100 sera, the assay was highly sensitive (98%) and specific (100%). The majority of HIV+ sera could inhibit binding at dilutions of 1/100 to 1/1000. No correlation was noted between binding inhibition (BI) titer in this assay and clinical stage of HIV infection. In addition, there was no correlation between BI titer and HIV neutralizing activity. The BI titer was correlated with the titer of anti-gp160 (r = 0.63) and the titer of anti-gp120 (r = 0.52) antibodies determined by Western blot dilution. As with neutralizing antibodies and other forms of immune response to HIV, it is unclear what role antibody blocking of HIV binding to CD4+ cells may play in active immunity to HIV in infected individuals. This activity may prove to have some value in protection against initial HIV infection and, thus, the assay may be of use in monitoring vaccine trials.

Animals↗
PubMed1988

Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody.

During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.

Animals↗
PubMed1988

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands.

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.

Acetylgalactosamine↗
PubMed1988

Changes in water balance and in release of arginine vasopressin during thermal adaptation in guinea-pigs.

The following experiments were made to investigate whether any changes in water balance and in the release of arginine vasopressin (AVP) accompany the development of thermal adaptation. Twelve guinea-pigs (300-400 g initial weight) were kept in individual metabolic cages at 22 degrees C during weeks 1 and 5. During weeks 2-4, six of them were exposed to 5 degrees C, and six to 28 degrees C. Before the start of the experiment, eight animals were implanted with chronic arterial catheters for removal of blood samples. Food and water intake, body weight, and colon temperature, as well as the amounts of urine and feces, were recorded in each animal every morning. In urine and blood plasma samples (taken daily, resp. weekly), the osmolality was estimated by vapor pressure osmometry, and concentrations of AVP by a radioimmunoassay. It is apparent that the daily turnover of water increased from 94 ml in guinea-pigs adapted to 22 degrees C (N), to 111 ml in cold adapted (CA), and to 154 ml in warm adapted (WA) animals. In CA the amounts of AVP excreted in urine increased dramatically (being 10 times higher than in WA). This high release of AVP cannot be explained by changes in osmotic pressure and by alterations in volume of extracellular fluid. It is concluded that AVP is released in CA guinea-pigs mainly as a stressor hormone, in amounts which highly exceed the antidiuretic needs. The WA animals, having free access to water, did not use the AVP system to conserve water. They doubled their water intake, producing more urine of lower osmolality, corresponding to the reduced release of AVP.

Adaptation, Physiological↗
PubMed1988

Changes in body temperature and vasopressin content of brain neurons, in pregnant and non-pregnant guinea pigs, during fevers produced by Poly I:Poly C.

The synthetic polyribonucleotide pyrogen Poly I:Poly C (800 micrograms.kg-1) was injected intramuscularly on alternate days into pregnant and non-pregnant female guinea pigs. Pregnant animals, close to term, had smaller fevers in response to the pyrogen than did non-pregnant animals. Repeated injections of the pyrogen caused sequentially smaller fevers for the first 3-4 injections, particularly in non-pregnant animals, and this appeared to be like the tolerance usually developed to repeated injections of endotoxin. Continued pyrogen injections then caused, in non-pregnant animals, fevers of increasing magnitude until the original fever levels were reached, whereas in pregnant guinea pigs the fever responses remained reduced until parturition. The development of tolerance was associated with an increase in immunoreactivity for arginine vasopressin (AVP) in some neurons in the medial part of the paraventricular nucleus, and in terminals in the lateral septum and amygdala similar to changes found in these areas at term of pregnancy. These observations raise the possibility that AVP in these regions may have a role in the development of tolerance to pyrogens, and further quantitative studies of the AVP content of, and release from, nerve terminals projecting to the limbic system seem warranted.

Animals↗
PubMed1988

Influence of increased catecholamine levels in blood plasma during cold-adaptation and intramuscular infusion on thresholds of thermoregulatory reactions in guinea-pigs.

Catecholamines and some of their metabolites were determined in urine and blood plasma of guinea-pigs before, during and after acclimation to a cold or warm environment. During adaptation to 5 degrees C the amounts of noradrenaline in plasma and 24-h urine samples continuously increased up to 600% compared with values obtained at an ambient temperature of 22 degrees C. Higher levels of dihydroxyphenylglycol and 3-methoxy-4-hydroxyphenylglycol further indicated an increased turnover of noradrenaline during cold adaptation. Acclimation to an ambient temperature of 28 degrees C reduced the peripheral release of noradrenaline in comparison to the release observed at 22 degrees C. Cold-induced increases in metabolic rate and electrical muscle activity both occur at a considerably lower mean body temperature in cold-than in warm-adapted guinea-pigs. The shift of thermoregulatory cold defence reactions to a lower mean body temperature could also be observed in warm-adapted animals after intramuscular infusion of noradrenaline in amounts comparable to those released during cold adaptation. It is concluded that high peripheral sympathetic activity directly or indirectly inhibits noradrenergic neurons in the lower brain stem that modulate the thermoregulatory control system by means of their afferents to the hypothalamus. As a consequence of this peripheral influence the thermoregulatory set point is shifted to a lower mean body temperature.

Acclimatization↗
PubMed1988

Elderberry bark lectin--gold techniques for the detection of Neu5Ac (alpha 2,6) Gal/GalNAc sequences: applications and limitations.

The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (alpha 2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites of Sambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (alpha 2,6) galactose/N-acetylgalactosamine.

Acetylgalactosamine↗
PubMed1988

Long term inotropic effects of flecainide and propafenone.

This study was undertaken to evaluate the effect of flecainie (Fle) and propafenone (Pro) on rest and exercise myocardial contractility after prolonged oral administration. Standardized, fully automated, gated radionuclide angiograms at rest and during maximum exercise were performed single blinded and prospectively in 19 patients before and after oral treatment with either 150 mg b.i.d. of Fle or 150 mg t.i.d. of Pro. The left ventricular ejection fraction (EF%) as a hemodynamic indicator presented as follows: (Table: see text) In general, there was no significant effect of either drug on resting ventricular function. In only one patient of the Fle group with decreased baseline function did the left ventricular EF fall from 40% to 29%. During exercise, ventricular performance showed deterioration in the Pro group, but, interestingly, improvement in the Fle group. As a result of these opposite changes, the effect of the two drugs on the exercise response of ventricular function was significantly different (P less than 0.05). This study thus gives first evidence that no significant cardiodepressive effects are to be expected after prolonged application of Fle or Pro in normal hearts. There might even be a mild beneficial effect on exercise performance after Fle.

Administration, Oral↗
PubMed1988

Evaluation of polysialic acid in the diagnosis of Wilms' tumor. A comparative study on urinary tract tumors and non-neuroendocrine tumors.

The polysialic acid moiety of the neural cell adhesion molecule has been shown to represent an onco-developmental antigen which can be detected in both embryonic human kidney and Wilms' tumor but not in normal adult human kidney. In the present comparative study, Wilms' tumors, clear cell (bone-metastasizing) sarcomas of kidney, cystic nephromas, renal cell carcinomas, transitional cell carcinomas and papillomas of the renal pelvis, ureter and urinary bladder (as well normal transitional epithelium from these regions). Ewing sarcomas, hepatoblastomas, rhabdomyosarcomas, and carcinomas of the stomach, colon, exocrine pancreas, lung, and esophagus, were investigated immunohistochemically for the presence of polysialic acid. In addition, immunoblot analysis was performed in selected tumors. With the exception of Wilms' tumor, none of the tumors investigated was positive for polysialic acid. In Wilms' tumor, blastemal cells and all epithelial components were positive but no immunostaining was observed in the stroma. These observations emphasize the potential value of a monoclonal anti-polysialic acid antibody in identifying blastemal metanephric cells and their epithelial differentiatives in Wilms' tumor.

Animals↗
PubMed1988

Insulin-related materials in the nervous system of vertebrates and non-vertebrates: possible extrapancreatic production.

Studies from multiple laboratories with a range of methods raised the possibility that insulin production occurs naturally at extrapancreatic sites. Part A covers the presence of insulin-related materials in organisms that do not have an endocrine pancreas, including unicellular prokaryotes and eukaryotes as well as multicellular non-vertebrate animals (insects et al.) and plants. Part B covers possible production of insulin by extrapancreatic tissues of vertebrates that are remote from a source of pancreatic insulin e.g. early chick embryos and mammalian cells in culture. Part C covers possible extrapancreatic insulin production in mammals in vivo. Each section ends with an outline summary with evidence in favor of and against the hypothesis.

Amino Acid Sequence↗
PubMed1988

[Biliary and arterial obstructive processes in regional chemotherapy with FUDR].

By far the majority of 54 patients suffering from hepatic metastases due to colorectal primaries developed obliterations of the arterial vascular bed when being submitted to regional chemotherapy with FUDR. In addition, about 25% exhibited obstructive jaundice in the course of severe sclerosing cholangitis. Either effect, which can be detected by radiological procedures, has to be referred to irritating activities of the chemotherapeutic drug used.

Adult↗
PubMed1988

Reexpression of poly(sialic acid) units of the neural cell adhesion molecule in Wilms tumor.

A unique structural feature of the neural cell adhesion molecule N-CAM is the presence of homopolymers of alpha (2----8)-linked sialic acid units. We have used two specific probes for the detection of poly(sialic acid) in normal human kidney and Wilms tumor: a monoclonal antibody against meningococci group B capsular polysaccharide (homopolymers of alpha (2----8)-linked sialic acid units), which shows no crossreactivity with polynucleotides and denaturated DNA, and bacteriophage-induced endosialidases specifically hydrolyzing alpha (2----8)-linked poly(sialic acid) units. Additionally, for the detection of N-CAM, antibodies recognizing the polypeptide portion of the molecule and biotinylated antisense RNA transcribed from a cDNA clone for N-CAM were applied. Poly(sialic acid) was regionally detectable in human embryonic kidney but undetectable in normal adult kidney, as already reported for rat kidney. The malignant Wilms tumor, which is characterized by the presence of structural components resembling those found in embryonic kidney, reexpressed poly(sialic acid) units and showed positive immunostaining for the polypeptide portion of N-CAM. Immunoblot analysis of Wilms tumor as well as human embryonic kidney and brain with the monoclonal anti-poly(sialic acid) antibody revealed in each case the same high molecular mass broad band. In situ hybridization demonstrated the presence of mRNA for N-CAM in Wilms tumor. We conclude that poly(sialic acid), most probably present on N-CAM, is an oncodevelopmental antigen in human kidney.

Antigens, Surface↗
PubMed1988

Sucrase-isomaltase deficiency in humans. Different mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme.

Eight cases of congenital sucrase-isomaltase deficiency were studied at the subcellular and protein level with monoclonal antibodies against sucrase-isomaltase. At least three phenotypes were revealed: one in which sucrase-isomaltase protein accumulated intracellularly probably in the endoplasmic reticulum, as a membrane-associated high-mannose precursor, one in which the intracellular transport of the enzyme was apparently blocked in the Golgi apparatus, and one in which catalytically altered enzyme was transported to the cell surface. All patients expressed electrophoretically normal or near normal high-mannose sucrase-isomaltase. The results suggest that different, probably small, mutations in the sucrase-isomaltase gene lead to the synthesis of transport-incompetent or functionally altered enzyme which results in congenital sucrose intolerance.

Adolescent↗