Showing papers by "Heidelberg University published in 1989"

Monoclonal antibody-mediated tumor regression by induction of apoptosis

Abstract: To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation of cells bearing APO-1 in vitro in a manner characteristic of a process called programmed cell death or apoptosis. Cell death was preceded by changes in cell morphology and fragmentation of DNA. This process was distinct from antibody- and complement-dependent cell lysis and was mediated by the antibody alone. A single intravenous injection of anti-APO-1 into nu/nu mice carrying a xenotransplant of a human B cell tumor induced regression of this tumor within a few days. Histological thin sections of the regressing tumor showed that anti-APO-1 was able to induce apoptosis in vivo. Thus, induction of apoptosis as a consequence of a signal mediated through cell surface molecules like APO-1 may be a useful therapeutic approach in treatment of malignancy.

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.

Abstract: NEUROTRANSMISSION effected by GABA (γ-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor1 exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates2. The primary structures of GABAA receptor α- and β-subunits have been deduced from cloned complementary DNAs3,4. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates3–5. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines4,5. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed γ2, which shares approximately 40% sequence identity with α-and β-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the γ2 subunit with α1 and β1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.

Type I and type II GABAA-benzodiazepine receptors produced in transfected cells

Abstract: GABAA (gamma-aminobutyric acid A)-benzodiazepine receptors expressed in mammalian cells and assembled from one of three different alpha subunit variants (alpha 1, alpha 2, or alpha 3) in combination with a beta 1 and a gamma 2 subunit display the pharmacological properties of either type I or type II receptor subtypes. These receptors contain high-affinity binding sites for benzodiazepines. However, CL 218 872, 2-oxoquazepam, and methyl beta-carboline-3-carboxylate (beta-CCM) show a temperature-modulated selectivity for alpha 1 subunit-containing receptors. There were no significant differences in the binding of clonazepam, diazepam, Ro 15-1788, or dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) to all three recombinant receptors. Receptors containing the alpha 3 subunit show greater GABA potentiation of benzodiazepine binding than receptors containing the alpha 1 or alpha 2 subunit, indicating that there are subtypes within the type II class. Thus, diversity in benzodiazepine pharmacology is generated by heterogeneity of the alpha subunit of the GABAA receptor.

A nuclear DNA attachment element mediates elevated and position-independent gene activity.

Abstract: The transcriptional activity of genes that have randomly integrated into the genomes of transfected cells and transgenic organisms is in general unpredictable, varying with the chromosomal site of the insertion. This effect of chromosomal position on gene expression may reflect the organization of chromosomes into topologically constrained loops and functional domains. To assess the biological significance of these loop domains, the anchorage of DNA to the nuclear scaffold has been studied at specific gene loci. We have previously defined cis-acting regions flanking the chicken lysozyme-gene domain that mediate the attachment of the chromatin to the nuclear scaffold. These 'A-elements' map to the 5' and 3' boundaries of the region of general DNase sensitivity in the active chromatin, which contains the lysozyme gene and its cis-regulatory elements. Here we report that when a reporter gene is flanked by 5' A-elements from the lysozyme gene, its expression in stably transfected cells is significantly elevated and is independent of chromosomal position.

Determination of atom positions at stacking-fault dislocations on Au(111) by scanning tunneling microscopy

Abstract: The determination of atom positions around Shockley-type partial dislocations [Burgers vector \( \frac{1}{6}(1,1, - 2)] \)at the (111) surface of gold has been achieved by operating a scanning tunneling microscope with atomic resolution on regions containing several unit cells of the\( (23 \times \sqrt {3} ) \) stacking-fault reconstruction of this surface. The data show directly the occupation of both hexagonal-closepacked and face-centered-cubic sites in the surface layer, verifying earlier reconstruction models.

GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs

Abstract: Cloned cDNAs encoding two new beta subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The beta 2 and beta 3 subunits share approximately 72% sequence identity with the previously characterized beta 1 polypeptide. Northern analysis showed that both beta 2 and beta 3 mRNAs are more abundant in the brain than beta 1 mRNA. All three beta subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each beta subunit, when co-expressed in Xenopus oocytes with an alpha subunit, forms functional GABAA receptors. These results, together with the known alpha subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations.

High-resolution imaging of copper-phthalocyanine by scanning-tunneling microscopy.

Abstract: Scanning-tunneling microscopy (STM) images of isolated Cu-phthalocyanine molecules on a Cu(100) substrate exhibit atomic scale features which agree well with Huckel molecular-orbital calculations. Images which show a molecule atop a measurably corrugated Cu(100) surface, a hopping molecule, packed molecular arrays, and a novel molecular binding site at a Cu step edge further illustrate the potential of STM for molecular observations.

Synaptophysin and chromogranins/secretogranins-widespread constituents of distinct types of neuroendocrine vesicles and new tools in tumor diagnosis

Abstract: Normal and neoplastic neuroendocrine (NE) cells have been identified for many years by morphological criteria only. With the advent of immunocytochemistry, antibodies against NE-specific polypeptides have been used to identify NE cells that had been missed by conventional techniques, thus improving the diagnosis of NE cells. In this review article we discuss (i) the biochemical, cell biological and molecular biological data obtained so far for two major types of NE markers, synaptophysin, which is characteristic of the small "transparent-looking" neurosecretory vesicles, and the chromogranins/secretogranins, which are widespread constituents of the larger "dense-cored" secretory granules; (ii) the immunohistochemical data obtained for these marker proteins in normal and neoplastic human NE cells and tissues; and (iii) future possible developments involving these as well as other proteins that are associated with these two distinct secretory organelles of NE cells and may serve as potential markers in NE cell diagnosis.

Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Abstract: Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

In vivo binding pattern of a trans-regulator of homoeotic genes in Drosophila melanogaster.

Abstract: The specification and maintenance of the metameric pattern in Drosophila melanogaster is regulated by complicated gene interactions. The differential expression of the homoeotic genes of the Antennapedia complex (ANT-C) and bithorax complex (BX-C), which determine segmental identities, is partly controlled by cross-regulatory interactions of loci within the two clusters and partly by trans-acting factors located outside the two complexes. One of the trans-regulatory genes, Polycomb (Pc), acts as a repressor of the ANT-C and BX-C1–4. Mutations of Polycomb result in a complete derepression of the homoeotic genes, leading to abdominal transformations of all body segments. Polycomb is part of a large class of trans-regulatory genes (Pc-group), estimated to comprise up to 40 loci5. We have raised antibodies against the Polycomb protein, and, using an improved immunostaining technique, showed that the Polycomb protein binds to 60 discrete sites along the polytene chromosomes of salivary glands. These sites comprise the ANT-C and the BX-C as well as several locations of Pc-group genes. This is the first clear evidence for a direct interaction of Polycomb with homoeotic loci and other Pc-group genes.

Aeromonas as a human pathogen.

Abstract: Although the first Aeromonas strain was described by Zimmermann as early as in 1890, it took 60 years until Caselitz established human pathogenicity of strains then called "Vibrio jamaicensis". Since then, and especially in the last 10 years, there have been increasing numbers of reports on different infections caused by members of the genus Aeromonas. These include sepsis; meningitis; cellulitis; necrotizing fasciitis; ecthyma gangrenosum; pneumonia; peritonitis; conjunctivitis; corneal ulcer; endophthalmitis; osteomyelitis; suppurative arthritis; myositis; subphrenic abscess; liver abscess; cholecystitis and/or ascending cholangitis; urinary tract infection; endocarditis; ear, nose, and throat infections; balanitis; etc. The role of Aeromonas in gastrointestinal disease is very controversial. Increasing epidemiological data suggest that these organisms play a major role in enteric infections, but so far enteropathogenicity has not been demonstrable in experiments where volunteers were given high numbers of Aeromonas possessing different virulence factors. Virulence factors include hemolysin(s), enterotoxin(s), hemagglutinins, invasivity, and others; but these are not found more frequently in strains isolated from patients with diarrhea than from healthy controls. Whether there is a correlation between species and disease remains to be elucidated and requires more information about the taxonomy of this genus.

Occupation of the c-fos serum response element in vivo by a multi-protein complex is unaltered by growth factor induction.

Abstract: Rapid, transient induction of the human c-fos proto-oncogene by extracellular signals requires the presence in cis of the serum response element (SRE). Two protein factors that bind to the SRE in vitro are the serum response factor (p67SRF) and polypeptide p62. These polypeptides must interact with one another and the SRE for efficient serum induction of the c-fos gene. Here we use dimethyl sulphate genomic footprinting to establish the in vivo protein contacts on the SRE and flanking sequences. In human A431 cells the patterns of protection and hyper-reactivity that we find are consistent with the presence of p67SRF, p62, and at least one other protein immediately 3' to p67SRF. The protein-DNA contacts we observe within the SRE are present before induction by epidermal growth factor and are unchanged during gene activation and subsequent repression. Our results indicate that a specific DNA-protein architecture may be maintained at the c-fos SRE, regardless of changes in the transcriptional state of the gene. Such established structures could be important generally in rapid transcriptional responses to extracellular signals.

Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus.

Abstract: The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. The single conformational determinant responsible for HBc antigenicity in the assembled core (HBc) and a linear HBe-related determinant (HBe1) were both mapped to an overlapping hydrophilic sequence around amino acid 80; a second HBe determinant (HBe2) was assigned to a location in the vicinity of amino acid 138 but found to require for its antigenicity the intramolecular participation of the extended sequence between amino acids 10 and 140. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis B virus nucleocapsid.

J1-160 and J1-180 are oligodendrocyte-secreted nonpermissive substrates for cell adhesion

Abstract: The glia-derived J1 extracellular matrix glycoproteins have been referred to as J1-160/J1-180 (the developmentally late appearing lower molecular weight group) and J1-200/J1-220 (the developmentally early appearing higher molecular group immunochemically related to tenascin). Members of the two groups show distinct cross-reactivities. To characterize the structural and functional differences between these J1 glycoproteins, two monoclonal antibodies were generated which recognize only the members of the lower molecular weight group. The two antibodies detect immunochemical similarities among the members of the lower molecular weight group, but do not react with J1/tenascin. J1-160 and J1-180 are specifically expressed by differentiated oligodendrocytes in culture and by myelin of the central nervous system and have not been found in the peripheral nervous system nor in any other organ of the adult mice tested. Electron microscopic examination of rotary-shadowed J1-160 and J1-180 reveals, respectively, dimeric and trimeric (tribrachion) kink-armed rodlike structures, which are linked by disulfide bridges. J1-160/J1-180 are nonpermissive substrates for the attachment and spreading of early postnatal small cerebellar neurons, astrocytes, and fibroblasts. In a mixture with laminin, J1-160/J1-180 are nonpermissive substrates for neurons, but not for astrocytes or fibroblasts. The repulsive effect toward neurons can be neutralized by one of the monoclonal antibodies, but not by the other. These observations are discussed in the context of cell interactions during regeneration in the mammalian nervous system.

The promoter of Alzheimer's disease amyloid A4 precursor gene.

Abstract: The promoter of the gene for the precursor of Alzheimer's Disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. A typical TATA box is missing, and transcription initiates at multiple sites. It shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in vivo. Upstream of the RNA start sites we found sequences homologous to the consensus binding sites of transcription factor AP-1 and the heat shock control element binding protein. Six copies of a 9bp long GC-rich element are located between positions -100 and -200 of the sequence. A protein-DNA interaction could be mapped to this element. The ratio of the dinucleotide CpG, the target for DNA methylation, versus GpC is about 1:1 around the RNA start site, in contrast to the normal ratio of 1:5 in eucaryotic DNA. These findings suggest that four mechanisms may participate in the regulation of the PAD gene: the stress-related heat shock; the AP-1/Fos binding; the GC-rich element, and the possible methylation of the CpG region. PAD gene regulation could be of relevance for the progression of amyloid deposition in Alzheimer's Disease.

Similar Risks of Nephropathy in Patients with Type I or Type II Diabetes Mellitus

Abstract: It is commonly assumed that in patients the risks of developing nephropathy and uraemia are high in type I and low in type II diabetes mellitus. Since type II occurs mostly in elderly individuals with limited life expectancy and high cardiovascular mortality, the true risk may have been underestimated, as many patients do not survive to experience renal complications. To assess renal risk further, we evaluated all patients with type II and type I diabetes mellitus without severe secondary disease who were followed in the outpatient clinic between 1970 and 1985. The cumulative risk of proteinuria after 20 years of diabetes mellitus was 27% in type II and 28% in type I, the findings after 25 years were 57% and 46% respectively. The cumulative risk of renal failure, i.e. serum creatinine greater than 1.4 mg/dl, after 3 years of persisting proteinuria was 41% in both type II and type I, and after 5 years of proteinuria were 63% and 59% respectively. We conclude that the renal risk is similar in patients with type II and type I diabetes mellitus.

Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.

Abstract: The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.

A4 protein in Alzheimer's disease: primary and secondary cellular events in extracellular amyloid deposition.

Abstract: This study was designed to investigate the role of serum proteins, microglia, glial fibrillary acidic protein (GFAP) positive cells and dystrophic neurites in the genesis of cerebral amyloid Using A4 protein antisera, we found an amorphous non-congophilic, form of plaque, which was not seen in Bielschowsky silver staining or Bodian impregnations GFAP-positive glial cells, cells immunolabelled for some macrophage markers and dystrophic neurites were detected in congophilic plaques with crystalline amyloid, but not in the amorphous, non-congophilic plaques The presence of alpha l-antichymotrypsin, complement factors and P component, but not of common serum proteins in both the amorphous and congophilic plaques, indicates that these three proteins may have a pathogenetic role in amyloid formation Amorphous plaques may be the earlier forms of plaque and consequently, the presence of reactive cells and dystrophic neurites may be secondary phenomena

Immunohistological localization of the adhesion molecules L1, N-CAM, and MAG in the developing and adult optic nerve of mice.

Abstract: The localization of the cell adhesion molecules L1, neural cell adhesion molecule (N-CAM), and myelin-associated glycoprotein (MAG) was studied immunohistologically at the light and electron microscopic levels and immunochemically in the developing and adult mouse optic nerve and retina. The neural adhesion molecule L1 is strongly expressed on the shafts of fasciculating unmyelinated axons at all ages studied from embryonic day 15 through adulthood. Growth cones of retinal ganglion cell axons were weakly L1-positive or L1-negative when contacting glial cells. Unmyelinated axons were not only L1-positive when contacting each other but also when contacting glia, whereas contacts between glial cells were L1-negative at all developmental stages. The same axons that expressed L1 in their fasciculating state in the unmyelinated retinal nerve fiber layer or in the unmyelinated optic nerve head became L1-negative when enwrapped by myelin in the optic nerve proper. At all stages of development N-CAM showed profuse labeling on fasciculating axons, growth cones, and their contact sites with glial cells as well as contacts between glial cells. In contrast to L1, axons remained N-CAM-positive when becoming myelinated. Sometimes, N-CAM was found in compact myelin. However, N-CAM was absent from glial surfaces contacting basement membranes at the interface to meninges, blood vessels, and the vitreous body of the eye. MAG was first detectable intracellularly in oligodendrocytes associated with the endoplasmic reticulum and Golgi apparatus before it became apparent at the cell surface. There it was present on oligodendrocytes prior and during the first stages of ensheathment of axons, both on cell body and processes. After formation of compact myelin MAG remained strongly expressed periaxonally and was only weakly detectable in noncompacted myelin including inner mesaxon and paranodal loops. None of the adhesion molecules was detectable on extracellular matrix, in the meninges, or on endothelial cells. Immunochemical analysis of antigen expression at different developmental stages was in agreement with the immunohistological data. We infer from these observations that L1 is involved in stabilization not only of axon-axon, but also axon-glia contacts, while the more dynamic structure of the growth cone generally expresses less L1. A differential expression of L1 along the course of an axon—being present on its unmyelinated, but absent on its myelinated part—further supports the notion that L1 may be involved in the stabilization of axonal fascicles but not of axon-myelin contacts. Since axon-astrocyte appositions are L1-negative at the node of Ranvier and L1-positive in nonmyelinated areas, L1 appears to play a regionally differential functional role in neuron-astrocyte interactions. MAG seems to be involved in the initiation of axon-oligodendrocyte interactions before the onset of my-elination and in the stabilization of neuron-oligodendrocyte and oligodendro- cyte-oligodendrocyte contacts in the mature myelin. Because of its general occurrence at contacts between all cell types studied except for the astroglial- basement membrane apposition, N-CAM appears to be responsible for the general stabilization of tissue integrity at all developmental stages studied and in the adult.

A detailed chemical kinetic reaction mechanism for the oxidation of iso-octane and n-heptane over an extended temperature range and its application to analysis of engine knock

Abstract: A detailed chemical kinetic reaction mechanism is developed to describe the oxidation of n -heptane, iso-octane, and their mixtures over a wide range of operating conditions. In addition to a high temperature submechanism, reaction paths are included to describe the lower temperature regimes in which the rate and intermediate products of oxidation are controlled by addition of molecular oxygen to alkyl and isomerized alkylperoxy radicals, internal H atom abstractions, and reactions involving O-heterocyclic species. This overall reaction mechanism is validated through comparisons between computed results and experimental data from shock tubes, turbulent flow reactor, and low temperature static and stirred reactors. The mechanism is then used to study the influence of fuel composition on knocking in internal combustion engines. Autoignition of mixtures of iso-octane and n -heptane is examined, in which experimentally measured variations of engine pressure with time were used to simulate the conditions encountered by the end-gases responsible for knocking operation. The computations reproduce the variations of autoignition delay time with octane number and these variations are interpreted in terms of detailed differences in the structure of the two primary reference fuels. Sensitivity analyses of the computations are presented, indicating those portions of the reaction mechanisms which have the greatest influence on the model results.