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Biomedical subjects

W Hunziker

Publications and source records attributed to W Hunziker.

At least 73 records · Page 4Linked to original sources

Allelic deletions in the long arm of chromosome 12 identify sites of candidate tumor suppressor genes in male germ cell tumors.

Human male germ cell tumors (GCTs) result from malignant transformation of premeiotic or early meiotic germ cells and exhibit embryonal-like differentiation of the three germinal layers. The genetic basis of origin and expression of differentiated phenotypes by GCTs are poorly understood. Our recent cytogenetic analysis of a large series of GCTs has shown that two chromosome 12 abnormalities, an isochromosome for the short arm [i(12p)] and deletions in the long arm [del(12q)], characterize these tumors, which led us to suggest that the deletions represent loss of one or more candidate tumor suppressor genes whose products regulate the normal proliferation of the spermatogonial stem cells. We undertook a molecular mapping of the deletions by comparing germ-line and tumor genotypes of eight polymorphic loci in paired normal/tumor DNA samples from 45 GCT patients. Analysis of loss of constitutional heterozygosity at these loci revealed two regions of frequent loss (> 40%), one at 12q13 and the other at 12q22, identifying the sites of the postulated tumor suppressor genes. One tumor (no. 143A) exhibited a homozygous deletion of a region of 12q22, which included the MGF gene. The KIT and MGF genes have been shown to play key roles in embryonal and postnatal development of germ cells; therefore, we evaluated their expression by Northern blot analysis in a panel of three GCT cell lines and 24 fresh GCT biopsies. Deregulated expression of MGF and KIT, which was discordant between seminomatous and nonseminomatous lesions, was observed.

Alleles↗

The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.

Animals↗

Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2. Differences in colipase dependence and in lipase activity.

We have isolated cDNAs coding for two novel human pancreatic lipase (hPL)-related human proteins, referred to as hPL-related proteins 1 and 2 (hPLRP1 and hPLRP2) and for hPL. The two novel proteins show an amino acid sequence identity to hPL of 68 and 65% for hPLRP1 and 2, respectively. All three proteins are secreted into the medium after transfection of COS cells with the corresponding cDNAs. The size of the three expressed proteins is similar and ranges between 45 and 50 kDa. The expressed hPLRP2 shows a lipolytic activity that is, however, in contrast to that of hPL only marginally dependent on the presence of colipase, whereas hPLRP1 shows no activity in this assay. A Northern analysis of normal human pancreas mRNA shows that the expression levels of hPLRP1 and hPLRP2 are about 4-fold and 24-fold lower, respectively, than that of hPL. hPLRP2 is, additionally, most closely related to a lipase reported to be expressed in mouse T-cells. A comparison of the sequences of the three proteins with sequences described as pancreatic lipases of other animal species shows three subfamilies of closer kinship. This suggests that the two novel proteins also exist in other species and that some of the sequences reported to be pancreatic lipase might more likely be the orthologues of hPLRP1 or hPLRP2 in those species.

Amino Acid Sequence↗

Brefeldin A and the endocytic pathway. Possible implications for membrane traffic and sorting.

A number of recent observations have suggested that the endocytic and biosynthetic pathways may share fundamentally similar transport mechanisms at the molecular level. Some of the more striking of these suggestions have come from a comparison of the effects of the macrocyclic lactone brefeldin A (BFA) on endosomes and the Golgi complex. BFA is thought to affect Golgi-specific coat proteins that may be involved in maintaining the structural integrity of the organelle and in regulating membrane transport in the secretory pathway. Many of the effects of BFA on the endocytic system, such as the guanine nucleotide and aluminum fluoride (AlF4-)-regulated induction of microtubule-dependent endosomal tubules, are strikingly reminiscent of the action of the drug on the Golgi complex. Therefore, the similar mechanisms of action of the drug on endosomes suggest that organelles of the endocytic pathway may be associated with similar cytoplasmic coats that could regulate endosome function and integrity.

Brefeldin A↗

Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.

B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton.

Amino Acid Sequence↗

Absence of calbindin-D28 expression in nonclassical 1,25-dihydroxyvitamin D targets: analysis by polymerase chain reaction.

CaBP-D28 mRNA expression in rat heart, testis, and lung was assessed by polymerase chain reaction (PCR). The animal model used was the hyperinduced vitamin D-treated rat (100 ng 1,25-dihydroxyvitamin D subcutaneously, daily for 7 days). For the PCR studies, two pairs of 20 mer oligonucleotide primers (designated 1-4 according to their position on the coding strand, but with primers 3 and 4 in reverse orientation) derived from the rat CaBP-D28 cDNA sequence were tested in various combinations. Optimal conditions were established using a 1:100 dilution of cDNA from normal rat kidney. Bands of the predicted sizes of 869 (1, 3), 994 (1, 4), 725 (2, 3), and 850 (2, 4) nucleotide base pairs resulted, but with varying intensities: 2,4 approximately 1,3 > 1,4 > 2,3. Repeat PCR (recycling after 1:100 dilution and readdition of reagents and primers with at least one different primer) provided strong additional amplification, particularly with the 1,4/2,4 combination. Under these conditions, mixing experiments showed that CaBP-D28 transcripts were detectable at 10(-7)- to 10(-9)-fold lower levels of expression than in D+ kidney. When RNA was isolated and cDNA generated from test tissues from 4 individual vitamin D-stimulated (D+) and vitamin D-deficient (D-) rats, repeat PCR (1,4/2,4 primer combination) provided no evidence of significant CaBP-D28 mRNA expression in the nonclassic target tissues, in contrast to strong bands in both the D- kidney (undiluted) and D+ kidney (1:100 dilution) preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis.

In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects of olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) - 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) - 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) - 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70-80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) - 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) - 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.

Animals↗

Subregional mapping of 13 single-copy genes on the long arm of chromosome 12 by fluorescence in situ hybridization.

Subregional localization of 13 single-copy DNA sequences previously assigned to the long arm of chromosome 12 has been performed using the fluorescence in situ hybridization (FISH) technique. The following order is suggested for the 13 mapped genes: cen-->COL2A1-->(VDR-D12S15)-->(D12S17-D12S4++ +-D12S14-D12S6)-->D12S8-->(IAPP-MGF- D12S7-D12S12)-->IGF1-->qter. Eight of the mapped genes clustered at two regions, one at 12q13 (D12S17-D12S4-D12S14-D12S6) and the other at 12q22 (IAPP-MGF-D12S7-D12S12). Our results show that single-copy DNA sequences as small as 500 bp can be successfully mapped by FISH.

Chromosome Mapping↗

Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization.

Macrophages and B-lymphocytes express two major isoforms of Fc receptor (FcRII-B2 and FcRII-B1) that exhibit distinct capacities for endocytosis. This difference in function reflects the presence of an in-frame insertion of 47 amino acids in the cytoplasmic domain of the lymphocyte isoform (FcRII-B1) due to alternative mRNA splicing. By expressing wild type and mutant FcRII cDNAs in fibroblasts, we have now examined the mechanism by which the insertion acts to prevent coated pit localization and endocytosis. We first identified the region of the FcRII-B2 cytoplasmic domain that is required for rapid internalization. Using a biochemical assay for endocytosis and an immuno-EM assay to determine coated pit localization directly, we found that the distal half of the cytoplasmic domain, particularly a region including residues 18-31, as needed for coated pit-mediated endocytosis. Elimination of the tyrosine residues at position 26 and 43, separately or together, had little effect on coated pit localization and a partial effect on endocytosis of ligand. Since the FcRII-B1 insertion occurs in the membrane-proximal region of the cytoplasmic domain (residue 6) not required for internalization, it is unlikely to act by physically disrupting the coated pit localization determinant. In fact, the insertion was found to prevent endocytosis irrespective of its position in the cytoplasmic tail and appeared to selectively exclude the receptor from coated regions. Moreover, receptors bearing the insertion exhibited a temperature- and ligand-dependent association with a detergent-insoluble fraction and with actin filaments, perhaps in part explaining the inability of FcRII-B1 to enter coated pits.

Actins↗

Selective inhibition of transcytosis by brefeldin A in MDCK cells.

Treatment of most cells with brefeldin A (BFA) leads to the retrieval of the Golgi complex to the endoplasmic reticulum, presumably reflecting an inhibition of cytoplasmic coat protein binding to Golgi membranes. Although BFA has been thought to act only on biosynthetic organelles, we now show that this drug also reversibly blocks polymeric immunoglobulin receptor-mediated transcytosis in MDCK cells. The action of BFA on transcytosis was selective, since internalization, recycling, and intracellular degradation were unaffected. The block occurred early on the transcytotic pathway, probably before the translocation of IgA-containing vesicles from the basal to the apical cytoplasm. Although BFA caused MDCK cell endosomes to become more tubular, the organization of the Golgi and binding of the 110 kd Golgi coat protein beta-COP was surprisingly unaffected. These results suggest that in MDCK cells, endocytic organelles contain a BFA-sensitive coat that regulates their organization and function even though the Golgi coat is BFA resistant.

Animals↗

Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant.

In MDCK cells, Golgi to basolateral transport of several membrane proteins has been found to involve a cytoplasmic domain determinant. In some cases (Fc receptor, lysosomal glycoprotein Igp120), the determinant appears similar to that required for endocytosis via clathrin-coated pits; for Igp120, elimination of a single cytoplasmic domain tyrosine both blocks internalization and results in apical transport. In other cases (LDL receptor), the determinant does not involve the cytoplasmic domain tyrosine required for endocytosis. Thus, contrary to current models, basolateral transport in MCDK cells occurs not by default but depends on one or more cytoplasmic domain determinants, the precise nature of which is unknown. For some proteins, it is closely related to coated pit determinants. The fact that many membrane proteins can reach the apical surface in the absence of this determinant suggests that signals for apical transport are widely distributed.

Amino Acid Sequence↗

Efficient purification of recombinant human tumor necrosis factor beta from Escherichia coli yields biologically active protein with a trimeric structure that binds to both tumor necrosis factor receptors.

A fast and efficient method for medium scale purification of recombinant human tumor necrosis factor beta (rTNF-beta) from Escherichia coli cells is described. The purified rTNF-beta displayed biological activity similar to rTNF-alpha in a WEHI 164 cell cytotoxicity assay. The titration curve of rTNF-beta and elution profiles of rTNF-beta in gel filtration experiments were different from those of rTNF-alpha. However, light scattering and ultra-centrifugation studies showed that both cytokines have trimeric structures in solution at 0.5 mg/ml, with minor differences in the distribution of nontrimeric species. rTNF-beta bound to purified 55- and 75-kDa TNF receptors with high affinity. The binding of rTNF-beta to either receptor was analyzed on Scatchard plots and compared with that of rTNF-alpha.

Binding, Competitive↗

Parvalbumin and calbindin-D28K immunoreactivity as developmental markers of auditory and vocal motor nuclei of the zebra finch.

The posthatch developmental profiles of parvalbumin and calbindin-D28K immunoreactivity were compared for the auditory nucleus mesencephalicus lateralis pars caudalis in the midbrain, n. ovoidalis in the thalamus, and telencephalic field L, as well as for the telencephalic vocal motor nuclei hyperstriatum ventrale pars caudalis and n. robustus archistriatalis. The two calcium-binding proteins showed specific temporal patterns of expression in each nucleus, without following an ascending or descending sequence. Calbindin-D28K immunoreactivity usually preceded parvalbumin immunoreactivity. Onset of expression, especially of parvalbumin-immunostaining, was earlier in auditory nuclei than in vocal motor nuclei. The developmental order of appearance of immunoreactivity in somata, dendrites and axons was different in various brain regions. In some structures parvalbumin or calbindin-D28K immunoreactivity occurred only transiently. The two antibodies bound to separate but spatially complementary groups of cells in the nucleus mesencephalicus lateralis pars dorsalis and n. ovoidalis, as has previously been described in visual nuclei. This pattern was maintained into adulthood. These hitherto unknown subcompartments may reflect internal functional organization in these nuclei. A transitory neostriatal zone containing parvalbumin-positive neurons and fibres was observed between the immature field L and the emerging hyperstriatum ventrale pars caudalis. Some comparative aspects are discussed as to the way in which neurons distinguished by the two Ca-binding proteins may differ in energy metabolism, activity pattern and other functional mechanisms.

Animals↗

Assignment of human pancreatic lipase gene (PNLIP) to chromosome 10q24-q26.

Human pancreatic lipase (EC 3.1.1.3) is a 56-kDa protein secreted by the acinar pancreas and is essential for the hydrolysis and absorption of long-chain triglyceride fatty acids in the intestine. In vivo, the 12-kDa protein cofactor, colipase, is required to anchor lipase to the surface of lipid micelles, counteracting the destabilizing influence of bile salts. Southern blot analysis, using a pancreatic lipase cDNA to probe DNA from mouse-human somatic cell hybrids, indicated that the pancreatic lipase gene (PNLIP) resides on human chromosome 10. In situ hybridization to human metaphase chromosomes confirmed the cell hybrid results and further localized the gene to the 10q24-qter region with the strongest peak at q26.1.

Blotting, Southern↗

Intracellular calcium-binding proteins: more sites than insights.

Calcium ions as biological regulators exert their effects in part via interaction with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has about 170 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.

Binding Sites↗

Relationships between sorting in the exocytic and endocytic pathways of MDCK cells.

Epithelial cells use both the exocytic and endocytic pathways to generate and maintain the polarized distribution of membrane proteins. This review summarizes current information concerning the general features and functions of the exocytic and endocytic pathways in MDCK cells. We analyse the possible implications of similarities between signals for endocytosis and determinants for basolateral sorting in the TGN. Furthermore, we discuss the fundamental relationships that might also exist in the biochemical basis of membrane traffic in the two pathways.

Brefeldin A↗

Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.

Animals↗

Fc receptor phosphorylation during receptor-mediated control of B-cell activation.

It is well known that Fc receptors for IgG (FcRII) on macrophages mediate the endocytosis of antibody-antigen complexes and signal the release of inflammatory and cytotoxic agents. FcRII are also expressed at high levels on B cells where they are less involved in endocytosis than in modulating B-cell activation by membrane immunoglobulins. Although crosslinking of membrane immunoglobulins can result in B-cell differentiation and proliferation through stimulation of phospholipase C, mobilization of intracellular Ca2+, and activation of protein kinase C, crosslinking FcR with membrane immunoglobulins confers a dominant inhibitory signal that prevents or aborts activation. This form of regulation may have a role in the induction of tolerance by IgG and in controlling the B-cell repertoire by anti-idiotypes. The different functions of FcR on B cells and macrophages may reflect the fact that these cell types express closely related but distinct FcR isoforms. We have recently found that the main lymphocyte FcR isoform, FcRII-B1, is unable to mediate endocytosis by way of coated pits and coated vesicles owing to an in-frame insertion of 47 amino acids in its cytoplasmic tail. Here we show that this insert, absent from the FcRII-B2 macrophage isoform, also contains serine phosphorylation sites that may have a role in the ability of FcR to regulate B-cell activation through membrane immunoglobulins.

Alkaloids↗