Detection of thyrotropin-releasing hormone (TRH) mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary.

To investigate the presence of TRH mRNA in the human anterior pituitary tissue, total RNA from human normal and tumoral anterior pituitary, hypothalamus (positive control) and muscle tissues (negative control) was reverse transcribed (RT) to the first strand of cDNA. RT products were then amplified by polymerase chain reaction (PCR) using a set of three exon-specific primers (two external 5' and 3' primers and one internal 3' primer) for a target sequence of the TRH gene including an intronic sequence of about 650 base pairs (bp). Southern analysis of the RT-PCR products specifically hybridizing with a 45-mer TRH probe showed two bands of the predicted sizes (399 and 351 bp) far more intense in hypothalamus than in normal and tumoral anterior pituitary tissue. The 399 and 351 bp RT-PCR products contained the BglII enzyme restriction site included in the TRH cDNA sequences spanned by the primers and the two respective digested fragments which were, as predicted, 337 and 289 bp long, hybridized with the TRH probe. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target TRH sequences in the human normal and tumoral anterior pituitary tissue as well as in the hypothalamus. Our data imply TRH gene expression in the human anterior pituitary.


Missense mutations and a deletion of the p53 gene in human gastric cancer.

To investigate the molecular pathogenesis of human gastric cancers the p53 gene, a suppressor oncogene, was analyzed in 12 human gastric cell lines. Southern blot and Northern blot analysis revealed a total deletion of p53 gene in KATO-III cells but no major abnormality of p53 gene in other cell lines. By the use of the reverse-transcriptase polymerase chain reaction and direct sequencing 7 cell lines showed point mutations of p53 gene resulting in amino-acid substitutions. Most of them were rare mutations which had not been observed in other types of cancers. One of these mutations was also detected through the use of PCR and oligomer-specific hybridization. Six out of 7 cell lines with mutations of p53 gene also lost one allele of chromosome 17p. Immunoblotting of cell lysates with an antibody specific to p53 demonstrated the absence of p53 protein in KATO-III cell. By contrast, the high levels of the p53 protein were observed in 5 cell lines all of which contained mutations of p53 gene. These results further suggest that the inactivation of p53 gene may play an important role in the transformation of gastric cells to the malignant phenotype. KATO-III cells might be a good model for studying the significance of the loss of p53 gene in cellular transformation.


Detection of point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 in Alzheimer's brains.

Point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 (ND2) were detected in 10 of 19 Alzheimer's brains but not in 11 normal brains. The same mutations were also detected in 2 of 6 patients with amyotrophic lateral sclerosis (ALS). However, neurofibrillary tangles and neuritic plaques characteristic of Alzheimer's disease were found histologically in the brain of one ALS patient who was positive of the mutation. The finding suggests that a point mutation in ND2 is a potential risk factor for Alzheimer's disease.


Divergent effects of phorbol esters and insulin on insulin-like growth factor binding protein-1 (IGFBP-1) production and mRNA in rat H4IIE hepatoma cells.

125I-IGF-I binding assay, western ligand and immunoblotting, and northern analysis of total RNA reveal that phorbol ester agonists of protein kinase C rapidly enhance IGFBP-1 production and increase the abundance of IGFBP-1 mRNA in rat H4IIE hepatoma cells. In combination with insulin, a potent inhibitor of IGFBP-1 gene transcription, this early effect of phorbol esters is dominant. These results demonstrate divergent regulation of IGFBP-1 by phorbol esters and insulin and indicate that protein kinase C may play a critical role in the regulation of IGFBP-1 and modulation IGF bioactivity in metabolic disease.


Increased expression of retroviral sequences in progressionally advanced rat prostatic tumors.

Differential hybridization analysis revealed two cDNA clones, pBUS19 and pBUS30, to be overexpressed in progressionally advanced rat prostatic tumors. Northern blot analysis suggested the clones to be related although no homology in nucleotide sequence could be shown. Isolation and characterization of a pBUS19-related clone, pJG116, and computer-assisted database comparison showed that all three clones could be mapped within a rat-specific endogenous retrovirus. The importance of overexpression of retroviral sequences in advanced prostatic cancer remains unclear.


Activation of K+ current in macrophages by platelet activating factor.

Puff application of platelet activating factor (10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced an outward current at a holding potential of -63 mV. The current was suppressed by an antagonist Y-24180 but not by CV-3988. Charybdotoxin (10(-6) M) suppressed the current. Reversal potentials were dependent on external K+ concentrations. The current was not suppressed in Ca(2+)-free EGTA-containing solution but was completely abolished in BAPTA-AM containing solution. These results suggest that platelet activating factor activates a Ca(2+)-dependent K+ channel.


Attachment of Trypanosoma cruzi to host cells: a monoclonal antibody recognizes a trypomastigote stage-specific epitope on the gp 83 required for parasite attachment.

A set of monoclonal antibodies against the purified surface gp 83 of T. cruzi trypomastigotes was produced and the ability of these monoclonals to inhibit the attachment of trypomastigotes to heart myoblasts was investigated. Western blots of solubilized trypomastigotes, epimastigotes or amastigotes probed with this set of monoclonal antibodies show that the gp 83 is present in invasive trypomastigotes, but not in non-invasive epimastigotes or amastigotes. One monoclonal antibody (Mab 4A4) from this set inhibits the attachment of trypomastigotes to heart myoblasts, whereas the others (MAbs 2H6, 4B9, 2D11) do not. These results show that the Mab 4A4 recognizes an epitope on the gp 83 of invasive trypomastigotes required for parasite binding to host cells.


Decreased accessory cell function and costimulatory activity by 1,25-dihydroxyvitamin D3-treated monocytes.

To characterize the mechanism(s) by which 1,25-dihydroxyvitamin D3 (calcitriol) modulates the costimulatory capacity of monocytes, we examined the effect of calcitriol pretreatment of monocytes on their capacity to promote T cell proliferation (accessory cell function). Correlation of calcitriol-dependent changes in monocyte accessory cell function and alterations in phenotype and cytokine production, and the dependence of these changes on cell viability, were studied. Calcitriol pretreatment induced a defect in accessory cell function that was evident with fixed monocytes, suggesting a cell-surface-associated mechanism. Altered accessory cell function did not correlate with changes in HLA-DR antigen expression and was unaffected by concurrent treatment with interferon-gamma. Calcitriol treatment did not alter either the expression of adhesion molecules or monocytic production of interleukin-1 beta (IL-1 beta) or IL-6. Exogenous IL-1 or IL-6 did not overcome the impaired costimulatory activity of calcitriol-treated monocytes. Thus, calcitriol treatment reduces the capacity of monocytes to promote lectin-induced T cell activation at the level of the plasma membrane, perhaps through altered expression of an uncharacterized molecule important in monocyte-T cell interactions. At chronically inflamed sites, elaboration of calcitriol by activated macrophages may regulate the ability of monocytes to induce both antigen-dependent and antigen-independent T cell proliferation.


Detection of stromelysin and collagenase in synovial fluid from patients with rheumatoid arthritis and posttraumatic knee injury.

To quantify stromelysin and collagenase in synovial fluid (SF) from patients with rheumatoid arthritis (RA) or traumatic knee injury. Stromelysin and collagenase were measured in the SF of 33 patients with RA or posttraumatic knee injury, using specific double-antibody sandwich enzyme-linked immunosorbent assays. Stromelysin was fractionated from representative SF, and the molecular form was identified by immunoblot analysis. The stromelysin concentration was approximately 20-fold higher than the collagenase concentration in the fluids from patients with RA and approximately 8-fold higher in the fluids from patients with traumatic injury. For both metalloproteinases, there was a higher enzyme concentration in RA SF than in the SF from patients with trauma (stromelysin 40.1 +/- 26 micrograms/ml [mean +/- SD] in RA SF, 8.5 +/- 15 micrograms/ml in trauma SF; collagenase 2.2 +/- 3.3 micrograms/ml in RA SF, 1.1 +/- 2.3 micrograms/ml in trauma SF). The majority of the stromelysin within the SF bound to reactive red-agarose and was identified as prostromelysin based on electrophoretic mobility and immunoblotting with monospecific antibodies. The finding of high levels of stromelysin in SF from patients with RA supports the proposal that this enzyme may play a role in the connective tissue degradation observed in this disease.


Expression of cell-adhesion molecules in the salivary gland microenvironment of Sjögren's syndrome.

The potential role of cell adhesion molecules in the pathogenesis of Sjögren's syndrome (SS) was assessed by examining their expression in salivary gland (SGL) tissue. Intercellular adhesion molecule type 1 (ICAM-1), lymphocyte function-associated antigen type 1 (LFA-1), LFA-3, CD2, and CD44 expression were determined using indirect immunofluorescence techniques. In inflamed labial SGL tissue, ICAM-1 expression was evident on infiltrating LFA-1+/CD2+/LFA-3+ mononuclear cells, and to a limited extent on SGL acinar epithelial cells adjacent to sites of intense inflammation. In SS, the SGL microenvironment is characterized by only a modest up-regulation of ICAM-1 expression on epithelial cells, despite the presence of T cells bearing an activated phenotype.


Molecular genetic analysis of HLA-DR and HLA-DQ genes among anti-U1-70-kd autoantibody positive connective tissue disease patients.

We have recently found that the presence of autoantibodies against the 70-kd polypeptide of U1 RNP (U1-70-kd) is associated with HLA-DR4 and DR2. To further characterize this association, we performed a molecular genetic analysis of HLA-DR and DQ genes among patients with autoantibodies against U1-70-kd. The polymerase chain reaction (PCR), sequence-specific oligonucleotide hybridization, and solid-phase direct DNA sequencing of PCR-amplified DNA were utilized to analyze HLA-DRB1, DRB5, DQA1, and DQB1 genes. A comprehensive analysis of HLA-DRB1, DRB5, DQA1, and DQB1 from 27 patients and controls identified shared amino acids FDYFYQA (Phe, Asp, Tyr, Phe, Tyr, Gln, Ala) at positions 26, 28, 30-32, 70, and 73 of HLA-DRB1 on disease-associated haplotypes. A common cluster of shared amino acids, or a shared epitope, identified within HLA-DRB1 among anti-U1-70-kd autoantibody positive connective tissue disease patients may be important in regulating an autoimmune response to the U1-70-kd antigen.


Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato plants.

Cyclodextrins (CDs) are cyclic oligosaccharides containing six (alpha), seven (beta), or eight (gamma) glucose molecules, respectively. The cyclodextrin glycosyltransferases (CGT), which produce CDs from starch, are found only in bacteria and are used in batch fermentors with hydrolyzed starch to produce CDs commercially. Using a CGT gene from Klebsiella, we attempted to engineer the tubers of developing potatoes to produce these novel, high-value carbohydrates. A chimeric gene, consisting of (1) the patatin promoter for tuber-specific expression, (2) the small subunit of ribulose bisphosphate carboxylase (SSU) transit peptide for plastid targeting, (3) the CGT structural gene from Klebsiella and (4) the nopaline synthase 3' region, was introduced into potatoes. Both alpha and beta CDs were produced in tubers of transgenic potatoes at levels corresponding to 0.001-0.01% of the starch being converted to CDs.


Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer.

To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex. To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers. Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment. The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance. Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A. The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A. These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy. In the following paper in this issue [Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C., & von Hippel, P. H. (1992)], these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.


Physical properties of the Escherichia coli transcription termination factor rho. 2. Quaternary structure of the rho hexamer.

Under approximately physiological conditions, the transcription termination factor rho from Escherichia coli is a hexamer of planar hexagonal geometry [Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P., & von Hippel, P. H. (1992) Biochemistry (preceding paper in this issue)]. Here we describe studies that further define the quaternary structure of this hexamer. We use a combination of chemical cross-linking and treatment with mild denaturants to show that the fundamental unit within the rho hexamer is a dimer stabilized by an isologous (or pseudoisologous) bonding interface. Three identical dimers of rho interact via a second type of isologous bonding interface to yield a hexamer with C3 or D3 symmetry. Cross-linking and denaturation experiments definitely rule out C6 and C2 symmetry for the rho hexamer. Data from fluorescence quenching, lifetime, and energy transfer experiments also argue against C2 symmetry. The simplest symmetry assignment that is not contradicted by any experimental data is D3; thus we conclude that the rho hexamer has D3 symmetry. We also consider the positioning of the binding sites for RNA and ATP relative to the coordinate reference frame of the D3 hexamer. Fluorescence energy transfer data are presented and integrated with data from the literature to arrive at a self-consistent model for the quaternary structure of the rho hexamer.


Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.


Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine.

A method providing more sensitive detection of transglutaminase substrates was developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when [14C]putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r2 = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.


Contributions of phylogenetically variable structural elements to the function of the ribozyme ribonuclease P.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which participates in processing precursor tRNAs. The RNA subunit contains the catalytic site and is capable of catalysis in the absence of the protein subunit. RNase P RNAs from various eubacteria consist of a core of conserved sequence and secondary structure which is evolutionarily modified in different organisms by the presence of discrete helical elements at various sites in the RNAs. The variable occurrence of these helical elements suggests that they have no important functional role in the enzyme. The Escherichia coli RNase P RNA contains four such elements. It has been shown that simultaneous deletion of all four of them produces an RNA that is functional but has several significant defects which could arise from general disruption of the RNA or from the loss of element-specific functions. This paper describes a more detailed analysis of the role of the variable elements in E. coli RNase P RNA. Removal of one of the elements had no apparent effect on RNase P activity in vitro. Two other elements are required for correct folding of the RNA: their absence confers a requirement for extremely high monovalent salt concentrations, apparently to reduce intramolecular electrostatic repulsion. The fourth element that was tested participates in a long-range structural interaction (pseudoknot) which contributes to the structural stability of the enzyme and affects substrate binding affinity. In the absence of this helix, the RNA becomes temperature-sensitive, and the KM increases 100-fold.(ABSTRACT TRUNCATED AT 250 WORDS)


Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.

We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)


Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.

Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 to 3800 microseconds were observed, suggesting that band 3 exists in different aggregate states within the plane of the membrane. The oxidizing agent phenylhydrazine was used to induce hemichrome formation within intact erythrocytes. The immobilization of band 3 in membranes prepared from these erythrocytes suggests that the binding of hemichromes induces clustering of band 3. The addition of purified hemichromes to erythrocyte ghosts leads to a similar effect. We have also examined the mobility of the cytoplasmic domain of band 3. This region was labeled indirectly using a phosphorescently labeled antibody which binds to an epitope within the cytoplasmic domain. We observed very rapid motion of the cytoplasmic region of band 3, which was only partially restricted upon hemichrome binding. This suggests that the integral and cytoplasmic domains of band 3 may be independently mobile.


Detergent solubilization of the endocytic Ca(2+)-independent hyaluronan receptor from rat liver endothelial cells and separation from a Ca(2+)-dependent hyaluronan-binding activity.

Rat liver sinusoidal endothelial cells (LECs) mediate the removal of hyaluronan (HA) from the circulation via a specific Ca(2+)-independent endocytic receptor. To characterize the receptor biochemically, detergent-soluble extracts were prepared from crude LEC membranes. Using a dot blot assay to quantitate 125I-HA binding activity in CHAPS-solubilized membranes, we detected not only specific Ca(2+)-independent but also specific Ca(2+)-dependent HA-binding activity. Both HA-binding activities behave as integral membrane-associated proteins; they are not released from LEC membranes by treatment at pH 11, and they require detergent for extraction. The Ca(2+)-independent HA receptor was inactivated by treatment at 56 degrees C for 30 min or with 200 mM DTT at 4 degrees C for 30 min, whereas the Ca(2+)-dependent activity actually increased by 75% after treatment at 56 degrees C and only 20% of the Ca(2+)-dependent activity was lost after DTT treatment. A two-cycle membrane extraction protocol using CHAPS partially separated the two HA-binding activities. Eight millimolar KCl and 0.5% CHAPS extracted approximately 50% of the Ca(2+)-independent HA receptor, but only 4-11% of the Ca(2+)-dependent activity. When the KCl and CHAPS concentrations were increased to 2.0 M and 1.5%, respectively, the remaining HA receptor, as well as 89-96% of the Ca(2+)-dependent activity, was then extracted. The Ca(2+)-independent and Ca(2+)-dependent activities could also be further separated using Sephacryl S-400 gel filtration chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)


Role of glucose carrier in human erythrocyte water permeability.

Although the transport properties of human erythrocyte water channels have been well characterized, the identity of the protein(s) mediating water flow remains unclear. Recent evidence that glucose carriers can conduct water raised the possibility that the glucose carrier, which is abundant in human erythrocytes, is the water channel. To test this possibility, water permeabilities and glucose fluxes were measured in large unilamellar vesicles (LUV) containing human erythrocyte lipid alone (lipid LUV), reconstituted purified human erythrocyte glucose carrier (Glut1 LUV), or reconstituted glucose carrier in the presence of other human erythrocyte ghost proteins (ghost LUV). In glucose and ghost LUV, glucose carriers were present at 25% of the density of native erythrocytes, were oriented randomly in the bilayer, and exhibited characteristic inhibition of glucose flux when exposed to cytochalasin B. Osmotic water permeability (Pf, in centimeters per second; n = 4) averaged 0.0012 +/- 0.00033 in lipid LUV, 0.0032 +/- 0.0015 in Glut1 LUV, and 0.006 +/- 0.0014 in ghost LUV. Activation energies of water flow for the three preparations ranged between 10 and 13 kcal/mol; p-(chloromercuri)benzenesulfonate (pCMBS), an organic mercurial inhibitor of erythrocyte water channels, and cytochalasin B did not alter Pf. These results indicate that reconstitution of glucose carriers at high density increases water permeability but does not result in water channel activity. However, because the turnover number of reconstituted carriers is reduced from that of native carriers, experiments were also performed on erythrocyte ghosts with intact water channel function. In ghosts, Pf averaged 0.038 +/- 0.013 (n = 9), while the activation energy for water flow averaged 3.0 +/- 0.3 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase.

We have labeled the primer binding domain of HIV1-RT with 5'-32P-labeled (dT)15 primer using ultraviolet light energy. The specificity of the primer cross-linking to HIV1-RT was demonstrated by competition experiments. Both synthetic and natural primers, e.g., p(dA)15, p(dC)15, and tRNA(Lys), inhibit p(dT)15 binding and cross-linking to the enzyme. The observed binding and cross-linking of the primer to the enzyme were further shown to be functionally significant by the observation that tRNA(Lys) inhibits the polymerase activity on poly(rA).(dT)15 template-primer as well as the cross-linking of p(dT)15 to the enzyme to a similar extent. At an enzyme to p(dT)15 ratio of 1:3, about 15% of the enzyme can be cross-linked to the primer. To identify the domain cross-linked to (dT)15, tryptic peptides were generated and purified by a combination of HPLC on a C-18 reverse-phase column and DEAE-Sephadex chromatography. A single peptide cross-linked to p(dT)15 was identified. This peptide corresponded to amino acid residues 288-307 in the primary sequence of HIV1-RT as judged by amino acid composition and sequence analyses. Further, Leu(289)-Thr(290) and Leu(295)-Thr(296) of HIV1-RT appear to be the probable sites of cross-linking to the primer p(dT)15.


Tissue-specific and hormonal regulation of human prostate-specific glandular kallikrein.

Kallikreins are involved in the posttranslational processing of a number of specific polypeptide precursors. Previously, human glandular kallikrein (hGK-1) mRNA has been identified in the prostate; however, the hGK-1 protein has not been identified and characterized. Therefore, its physiologic function in the prostate is not known. In this study, we have isolated a full-length hGK-1 cDNA from a human adenocarcinoma cell line, LNCaP. In vitro translation experiments demonstrated that the molecular size of the hGK-1 protein generated from this cDNA is similar to that of prostate-specific antigen (PSA), a protein which is produced exclusively in the prostate. In situ hybridization with a hGK-1-specific oligonucleotide probe (77 bases), which can differentiate hGK-1 mRNA from PSA mRNA, demonstrated the hGK-1 mRNA to be located in the prostate epithelium. The hGK-1 mRNA was colocalized with PSA mRNA in prostatic epithelia. Moreover, in situ hybridization studies revealed that the level of hGK-1 mRNA in human benign prostatic hyperplasia tissues is approximately half that of PSA mRNA. Furthermore, we have demonstrated that hGK-1 mRNA is under androgenic regulation in LNCaP cells. Time course analysis revealed that hGK-1 mRNA levels increased significantly at 5 h of mibolerone treatment and reached maximal levels by 9 h. In addition, hGK-1 mRNA levels were increased by dihydrotestosterone, but not by dexamethasone or diethylstilbestrol treatments. Flutamide, a nonmetabolized anti-androgen, repressed the androgenic effects. These studies suggest that expression of hGK-1 mRNA is regulated by androgen via the androgen receptor.


Coculture of newborn rat skin epidermal cells with Swiss 3T3 cells: effect of the phases of 3T3 cells on attachment, growth and keratin synthesis of epidermal cells.

Swiss albino mouse 3T3 cells in various states were inoculated onto one side of Millipore filters. The other side of the filter was then coated with type I collagen and inoculated with newborn rat skin epidermal cells. On coculture of these cells, the attachment, growth and keratin synthesis of epidermal cells were found to depend on the state of the 3T3 cells: 3T3 cells in the stationary phase of growth were the most effective, followed by those in the logarithmic growth phase, those in the lag phase and plasmolyzed fibroblasts being only slightly effective. The effects of 3T3 cells in different states correlated well with their abilities to synthesize type IV collagen, but not type I collagen: with an increase in type IV collagen synthesis by the 3T3 cells, attachment of epidermal cells to the cell support, and their growth and synthesis of keratins increased. This culture system is concluded to mimic conditions in skin in vivo, and therefore to be suitable for studies on the effects of fibroblasts on the growth of epidermal cells.


Localization of a PlA1 epitope to the amino terminal 66 residues of platelet glycoprotein IIIa.

A platelet glycoprotein (GP) IIIa epitope library was constructed by insertion of randomly cleaved GPIIIa cDNA fragments in the prokaryotic expression vector lambda gt22 and screened with purified anti-PlA1 antibodies for clones expressing a PlA1 epitope. Five independent clones were isolated and characterized by nucleotide sequencing. The smallest anti-PlA1 reactive clone obtained encoded the amino terminal 66 residues of mature GPIIIa. Substitution of leucine33 (PlA1) with a proline33 (PlA2) by in vitro mutagenesis resulted in the loss of anti-PlA1 reactivity; however, this clone still reacted with anti-GPIIIa polyclonal antibodies. These data indicate that a PlA1 alloantigenic epitope is located within a small, unglycosylated fragment of GPIIIa containing the polymorphism responsible for the PIA phenotype. Furthermore, these results prove that small recombinant mimics of a PlA1 epitope may be synthesized and used for detection of these alloantibodies.


Serotherapy of B-cell neoplasms with anti-B4-blocked ricin: a phase I trial of daily bolus infusion.

Anti-B4-blocked Ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody (MoAb) and the protein toxin "blocked ricin." The anti-B4 MoAb is directed against the B-lineage-restricted CD19 antigen expressed on more than 95% of normal and neoplastic B cells. Blocked ricin is an altered ricin derivative that has its nonspecific binding eliminated by chemically blocking the galactose binding domains of the B chain. In vitro cytotoxicity studies demonstrate that the IC37 of Anti-B4-bR is 2 x 10(-11) mol/L compared with 4 x 10(-12) mol/L for native ricin. A phase I dose escalation clinical trial was conducted in 25 patients with refractory B-cell malignancies. Anti-B4-bR was administered by daily 1-hour bolus infusion for 5 consecutive days at doses ranging from 1 microgram/kg/d to 60 micrograms/kg/d. Serum levels above 1 nmol/L were achieved transiently in the majority of patients treated at the maximum tolerated dose of 50 micrograms/kg/d for 5 days for a total dose of 250 micrograms/kg. The dose-limiting toxicity was defined by transient, reversible grade 3 elevations in hepatic transaminases, without impaired hepatic synthetic function. Minor toxicities included transient hypoalbuminemia, thrombocytopenia, and fevers. Human antimouse antibody and human anti-ricin antibody were detected in nine patients. One complete response, two partial responses, and eight mixed or transient responses were observed. These results show the in vitro and in vivo cytotoxicity of Anti-B4-bR and indicate that this immunotoxin can be administered as a daily bolus infusion for 5 days with tolerable, reversible toxicity.


Role of c-kit ligand in the expansion of human hematopoietic progenitor cells.

To test the hypothesis that the c-kit ligand plays an important role in the regulation of early events occurring during human hematopoiesis, we determined the effect of a recombinant form of c-kit ligand, termed mast cell growth factor (MGF), on the high-proliferative potential colony-forming cell (HPP-CFC) and the cell responsible for initiating long-term hematopoiesis in vitro (LTBMIC). MGF alone did not promote HPP-CFC colony formation by CD34+ DR- CD15- marrow cells, but synergistically augmented the ability of a combination of granulocyte-monocyte colony-stimulating factor (GM-CSF) interleukin (IL)-3 and a recombinant GM-CSF/IL-3 fusion protein (FP) to promote the formation of HPP-CFC-derived colonies. MGF had a similarly profound effect on in vitro long-term hematopoiesis. Repeated additions of IL-3, GM-CSF, or FP alone to CD34+ DR- CD15- marrow cells in a stromal cell-free culture system increased cell numbers 10(3)-fold by day 56 of long-term bone marrow culture (LTBMC), while combinations of MGF with IL-3 or FP yielded 10(4)- and 10(5)-fold expansion of cell numbers. Expansion of the number of assayable colony-forming unit-granulocyte-monocyte (CFU-GM) generated during LTBMC was also markedly enhanced when MGF was added in combination with IL-3 or FP. In addition, MGF, IL-3, and FP individually led to a twofold to threefold increase in HPP-CFC numbers after 14 to 21 days of LTBMC. Furthermore, the effects of these cytokines on HPP-CFC expansion during LTBMC were additive. Throughout the LTBMC, cells receiving MGF possessed a higher cloning efficiency than those receiving IL-3, GM-CSF, or FP alone. These data indicate that the c-kit ligand synergistically interacts with a number of cytokines to directly augment the proliferative capacity of primitive human hematopoietic progenitor cells.


Increased erythropoietin-receptor expression on CD34-positive bone marrow cells from patients with chronic myeloid leukemia.

Erythropoietin-receptor (EpR) expression on bone marrow cells from normal individuals and from patients with chronic myeloid leukemia (CML) was examined by multiparameter flow cytometry after stepwise amplified immunostaining with biotin-labeled Ep, streptavidin-conjugated R-phycoerythrin, and biotinylated monoclonal anti-R-phycoerythrin. This approach allowed the detection of EpR-positive cells in all bone marrow samples studied. Most of the EpR-positive cells in normal bone marrow were found to be CD45-dull, CD34-negative, transferrin-receptor-positive and glycophorin-A-intermediate to -positive. This phenotype is characteristic of relatively mature erythroid precursors, ie, colony-forming units-erythroid and erythroblasts recognizable by classic staining procedures. Approximately 5% of normal EpR-positive cells displayed an intermediate expression of CD45, suggesting that these represented precursors of the CD45-dull EpR-positive cells. Some EpR-positive cells in chronic myeloid leukemia (CML) bone marrow had a phenotype similar to the major EpR-positive phenotype in normal bone marrow, ie, CD34-negative and CD45-dull. However, there was a disproportionate increase in the relative number of EpR-positive/CD45-intermediate cells in CML bone marrow. Even more striking differences between normal individuals and CML patients were observed when EpR-expression on CD34-positive marrow cells was analyzed. Very few EpR-positive cells were found in the CD34-positive fraction of normal bone marrow, whereas a significant fraction of the CD34-positive marrow cells from five of five CML patients expressed readily detectable EpR. These findings suggest that control of EpR expression is perturbed in the neoplastic clone of cells present in patients with CML. This may be related to the inadequate output of mature red blood cells typical of CML patients and may also be part of a more generalized perturbation in expression and/or functional integrity of other growth factor receptors on CML cells.


Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts.

The receptor encoded by the W (c-kit) locus (W receptor) is expressed on the surface of cultured mast cells (CMC) derived from normal (+/+) mice, whereas its ligand encoded by the Sl locus (Sl ligand) is expressed on the surface of fibroblast cell lines derived from murine embryos. Involvement of W receptors and Sl ligands in attachment of CMC to fibroblasts was investigated. CMC were cocultured with fibroblasts; nonattaching CMC were removed and the remaining CMC were counted. CMC derived from mice of the W/W genotype did not express the extracellular domain of W receptors, and attachment of W/W CMC to +/+ fibroblasts was significantly impaired. Fibroblasts derived from embryos of the Sl/Sl genotype did not express Sl ligands, and the attachment of +/+ CMC to Sl/Sl fibroblasts was also impaired. The Wv and W42 alleles are point mutations at the intracellular tyrosine kinase domain. Attachment of either Wv/Wv, W/Wv, or W/W42 CMC to +/+ fibroblasts was comparable with that of +/+ CMC. Moreover, the addition of monoclonal antibody against the extracellular domain of W receptors inhibited the attachment of +/+ CMC to +/+ fibroblasts. Thus, the extracellular domain of W receptors appeared to be necessary for attachment of CMC to fibroblasts.


Myeloid and erythroid progenitor cells from normal bone marrow adhere to collagen type I.

One of the mechanisms by which normal hematopoietic progenitor cells remain localized within the bone marrow microenvironment is likely to involve adhesion of these cells to extracellular matrix (ECM) proteins. For example, there is evidence that uncommitted, HLA-DR-negative progenitor cells and committed erythroid precursors (BFU-E) bind to fibronectin. However, fibronectin is not known to mediate binding of committed myeloid (granulocyte-macrophage) progenitors, raising the possibility that other ECM proteins may be involved in this process. We investigated the binding of the MO7 myeloid cell line to a variety of ECM proteins and observed significant specific binding to collagen type I (56% +/- 5%), minimal binding to fibronectin (18% +/- 4%) or to laminin (19% +/- 5%), and no binding to collagen type III, IV, or V. Similarly, normal bone marrow myeloid progenitor cells (CFU-GM) demonstrated significant specific binding to collagen type I (46% +/- 8% and 47% +/- 12% for day 7 CFU-GM and day 14 CFU-GM, respectively). The ability of collagen to mediate binding of progenitor cells was not restricted to the myeloid lineage, as BFU-E also showed significant binding to this ECM protein (40% +/- 10%). The binding of MO7 cells and CFU-GM was collagen-mediated, as demonstrated by complete inhibition of adherence after treatment with collagenase type VII, which was shown to specifically degrade collagen. Binding was not affected by anti-CD29 neutralizing antibody (anti-beta-1 integrin), the RGD-containing peptide sequence GRGDTP, or divalent cation chelation, suggesting that collagen binding is not mediated by the beta-1 integrin class of adhesion proteins. Finally, mature peripheral blood neutrophils and monocytes were also found to bind to collagen type I (25% +/- 8% and 29% +/- 6%, respectively). These data suggest that collagen type I may play a role in the localization of committed myeloid and erythroid progenitors within the bone marrow microenvironment.


Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow.

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.


Immunophenotyping and functional analysis of purified human uterine mast cells.

Human mast cells have been purified from uterine tissues, and their surface marker profile and function have been evaluated as part of ongoing studies of mast cell heterogeneity. Using a panel of antibodies, purified uterine mast cells (UMC; 81% +/- 7% purity, n = 10) were analyzed by immunofluorescence and flow cytometry for surface expression of various antigens. Consistent with previous analyses of mast cells from other tissues, UMC expressed HLA class I, IgE, c-kit receptor, CD9, CD33, CD43, CD45, and CD54, while CD11a, CD11b, CD14, CD16, CD23, and CD64 were not detected. Unlike other mast cells, UMC expressed CD11c/CD18 (p150,95) and CD32 (Fc gamma RII). Additional antigens not previously studied on mast cells included the selectin LECAM-1 (Leu-8) and several beta 1 and beta 3 integrins; expression of very late activation antigen-4 (VLA-4) (CD49d/CD29), VLA-5 (CD49e/CD29), and the vitronectin receptor (CD51/CD61) was seen. Functional studies showed that treatment of human umbilical vein endothelial cells with interleukin-1 (5 ng/mL for 4 hours) resulted in a twofold to threefold increase in adhesiveness for UMC. Purification procedures did not alter histamine release responses to anti-IgE or the calcium ionophore A23187, and treatment of UMC with an anti-CD32 monoclonal antibody (IV.3) did not induce histamine release or alter anti-IgE-induced release. These data suggest that UMC may possess unique phenotypic characteristics, and support the concept of mast cell heterogeneity.


Reactivity profiles of leukemic myeloblasts with monoclonal antibodies directed to sialosyl-Le(x) and other lacto-series type 2 chain antigens:absence of reactivity with normal hematopoietic progenitor cells.

We investigated the expression profiles of lacto-series type 2 antigens in hematopoietic cells and their progenitors, in comparison with leukemic leukocytes. Reactivity profiles of various anti-type 2 chain monoclonal antibodies (MoAbs) with leukemic blasts from 12 patients with acute myeloblastic leukemia (AML) and those from two patients with acute unclassified leukemia (AUL) show that anti-sialosyl-Le(x) MoAb SNH3 reacted strongly with greater than 95% of leukemic blast leukocyte populations from all patients (14 of 14). Another anti-sialosyl-Le(x) MoAb, FH6, showed less reactivity than SNH3 (12 of 14 patients), while anti-Le(y) MoAb AH6 showed reactivity with only 8 of 14 patients. On the other hand, none of the anti-type 2 chain MoAbs reacted with CD34+ normal adult bone marrow (BM) mononuclear cells obtained independently from three healthy volunteers. MoAb SNH3, but not FH6 or AH6, showed complement-mediated cytotoxicity to leukemic blasts from these patients, as well as to myelogenous leukemia cell line HL60. Colony-forming unit granulocyte-macrophage (CFU-GM), but not burst-forming unit-erythroid (BFU-E), was incompletely inhibited by treatment of normal BM mononuclear cells with SNH3 and complement. The absence of type 2 chain antigen expression in hematopoietic progenitor cells and in in vitro hematopoietic colonies (CFU-GM and BFU-E) strongly suggests that application of anti-carbohydrate MoAbs, particularly anti-sialosyl-Le(x) could be useful for elimination of leukemic myeloblasts infiltrating in BM, for purging of leukemic blasts in BM, and for facilitation of autologous BM transplantation.


Interactions of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, and tumor necrosis factor alpha in the priming of the neutrophil respiratory burst.

Exposure of neutrophils to a range of cytokines augments their response to subsequent agonist-induced activation of the respiratory burst. We have examined the effects of several of these factors, both singly and in combination, on the priming of f-met-leu-phe (FMLP) and complement C5a-stimulated neutrophil H2O2 production, using a whole blood flow cytometric assay designed to minimize artefactual activation. Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha) produced a similar degree of priming of the FMLP-stimulated burst in vitro (558% +/- 86%, n = 41, and 581% +/- 95%, n = 21, of the response seen with FMLP alone, respectively), but with markedly different kinetics (half-maximal response 20 minutes and 7 minutes, respectively). Preincubation with granulocyte colony-stimulating factor (G-CSF) alone caused only modest priming (202% +/- 39%, n = 14). Priming with cytokine combinations of the FMLP-stimulated burst showed that the combinations of G-CSF and TNF alpha and GM-CSF and TNF alpha are highly synergistic, with recruitment of neutrophils unresponsive to priming by single agents. Priming with the combination of GM-CSF and G-CSF was not significantly different to priming with GM-CSF alone. Similar results were obtained using C5a as the respiratory burst stimulus. Significant priming of the FMLP-stimulated respiratory burst was seen in vivo in patients receiving an infusion of GM-CSF (332% +/- 50% of preinfusion response to FMLP, P less than .005, n = 8). Priming was also seen in patients receiving G-CSF (152% +/- 58%, n = 5), although this did not reach conventional significance levels (.05 less than P less than .1). Although GM-CSF infusion caused priming in vivo, this was 48% less than predicted by preinfusion in vitro responses. This result was not due to inadequate GM-CSF levels as addition of further GM-CSF ex vivo did not correct the response. However, these neutrophils were still able to respond appropriately to ex vivo priming with TNF alpha, with a doubling in H2O2 production.


Membrane orientation of Rh(D) polypeptide and partial localization of its epitope-containing domain.

We have previously shown that the effects of various enzyme treatments on Rh antigen-containing polypeptides in situ could be monitored by an antibody preparation which recognizes only these polypeptides following Western blotting. We now have prepared antibodies that specifically react with either the N- or C-terminal ends of Rh-related proteins. Using all three, we have established that the C-terminus of Rh(D) polypeptide is at the cell surface, whereas its N-terminal domain is situated at the cytoplasmic side of the red blood cell membrane. Chymotrypsin digestion of ghosts derived from (-D-/-D-) cells that are devoid of Rh (C/c) and (E/e) antigens produces three major Rh(D)-related fragments: the 20-Kd fragment contains the molecule's C-terminal domain, the 17-Kd fragment its N-terminus, and the 13-Kd fragment neither. However, only the 17-Kd fragment forms an immune-complex with human polyclonal anti-D, indicating that it contains the Rh(D) antigenic domain. Other findings presented here provide further evidence for a unique folding of Rh(D) polypeptide within the cell membrane and suggest that Rh(C/c) and (E/e) polypeptides, when present, may form complexes with it.


Sequence variations in the 5' hypersensitive site-2 of the locus control region of beta S chromosomes are associated with different levels of fetal globin in hemoglobin S homozygotes.

We have compared the sequence of the 5' hypersensitive site-2 (5'-HS-2) of the locus control region (LCR) from a sickle cell anemia (SS) patient homozygous for haplotype 19 and with low levels of fetal hemoglobin (HbF), with the same sequence from an SS patient homozygous for haplotype 3 and with high levels of HbF. Several nucleotide variations were present in the 5'HS-2 of the haplotype 19 individual. One is the A----G at position -10905 that creates an Sp1 binding site GCCCC (A----G)CCCC. A second is the T----G at position -10924 in a sequence that binds both erythroid and ubiquitous factors and exhibits high homology to the long terminal repeat of the Moloney leukemia viruses and Friend murine leukemia virus. Other differences were in the two AT-rich stretches of DNA, and an A----T substitution at position -10390. Dot-blot analyses of amplified DNA from several SS patients showed that these variations are specific for beta S chromosomes with haplotype 19. We also examined the 5'HS-2 sequence from an SS patient who is homozygous for haplotype 19, but has abnormally high levels of HbF (greater than 20%). We observed a cross-over that has placed sequences similar to the 5'HS-2 of haplotype 3 in juxtaposition to the 5' flanking regions of haplotype 19. Thus, a beta S chromosome with haplotype 19 but having a 5'HS-2 (LCR) characteristic for haplotype 3 is associated with high gamma-chain expression. We postulate that factors produced under conditions of hematopoietic stress, together with genetic determinants on the haplotype 3-like LCR sequences, allow for high level expression of gamma-globin genes.


The human prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1 alpha,25-dihydroxyvitamin D3.

The LNCaP prostatic carcinoma cell line was examined for the presence of specific receptors for 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Whole cell binding studies identified approximately 2500 high-affinity (Kd = 1.4 x 10(-9) binding sites per cell. Competition studies revealed that these receptors are specific for the 1 alpha,25(OH)2 metabolite. Binding studies using the synthetic androgen R1881 indicate that separate androgen and vitamin D3 receptors exist in LNCaP cells. The vitamin D3 receptors sediment at approximately 3.5S on linear sucrose gradients. The sedimentation coefficient could be shifted with a monoclonal anti-vitamin D3 receptor antibody (9A7 gamma) but not with a monoclonal antibody to the androgen receptor (AN1-15). The receptor/ligand complex elutes from native DNA cellulose at 0.2 M KCl. Northern blot analysis identified an mRNA of approximately 4.6 kilobases which hybridized with a specific vitamin D3 receptor complementary DNA probe (hVDR). In the absence of androgens, 1 alpha,25(OH)2D3 stimulated growth and prostate-specific antigen production by LNCaP cells in a dose-dependent fashion. Dose-response curves indicated that at physiological concentrations (10(-9) M) 1 alpha,25(OH)2D3 was mitogenic, whereas at higher concentrations (10(-8) M) it promotes differentiation. These studies suggest that 1 alpha,25(OH)2D3 could play an important role in the natural history of and response to hormone therapy by prostatic cancer.


Expression of simple epithelial cytokeratins in mouse epidermal keratinocytes harboring Harvey ras gene alterations.

Activation of a Harvey ras (H-ras) protooncogene is a frequent event associated with mouse epidermal carcinogenesis. We report that the transfection of a human H-ras oncogene into an immortalized mouse epidermal cell line (MCA3D) induces the anomalous expression of cytokeratins (CKs) 8 and 18 characteristic of simple epithelia. The comparison of various transfectant cell clones indicated a direct correlation between the levels of CK8 expression and the mutated H-ras p21s. The expression of simple epithelial CKs is also described in cell lines derived from mouse skin carcinomas (HaCa4, CarC) and in keratinocytes transformed in vitro by a chemical carcinogen (PDV, PDVC57), all of which contain altered H-ras genes. The induction of CK8 and CK18 occurs at the mRNA level and, although both CK8 and CK18 mRNAs are expressed, CK18 protein does not accumulate whereas CK8 is incorporated into intermediate filaments. Immunofluorescence studies show that the pattern of CK8 protein expression is heterogeneous; some cells express very low amounts of CK8, whereas others synthesize relatively high levels of this protein. However, selection of strongly CK8-positive cells was found in one case where a more malignant population of cells (PDVC57) was derived by tumor transplantation of PDV. Our results suggest that activation of a H-ras gene can alter the normal differentiation program of epidermal cells and that the ability to synthesize CK8 and CK18 could be related to tumor progression.


Synthesis and evaluation of fluoromycin: a novel fluorescence-labeled derivative of talisomycin S10b.

We have synthesized fluoromycin (FLM), a novel fluorescein-labeled derivative of talisomycin S10b (TLM S10b), and used it to evaluate cellular drug accumulation and distribution in bleomycin (BLM)-sensitive and -resistant cell lines. The fluorescence intensity of FLM was 300- to 400-fold greater than that of BLM A2, TLM S10b, or the lipophilic BLM analogue, liblomycin. FLM possessed an antiproliferative potency similar to liblomycin in BLM-sensitive human A-253 squamous carcinoma cells but was less potent than BLM A2 or TLM S10b. C-10E cells, a clone of A-253 cells with 40- to 50-fold resistance to BLM A2 and TLM S10b, were 50-fold resistant to FLM. A partially revertant cell population (C-10E ND) regained sensitivity to BLM A2, TLM S10b, and FLM. FLM like BLM cleaved pGEM-3Z plasmid DNA in vitro in a concentration-dependent manner. Flow cytometric analysis of FLM content in C-10E and C-10E ND cell lines showed 4-fold and 2-fold lower fluorescence intensity, respectively, compared with A-253 cells. Similar results were seen by fluorescence spectrophotometry with cell extracts. Fluorescence microscopy indicated heterogeneous distribution among A-253 cells with at least 50% of the cells exhibiting marked nuclear fluorescence localization. In contrast, C-10E cells displayed lower cellular fluorescence and predominantly cytoplasmic localization. C-10E ND cells exhibited a mixed population of nuclear and cytoplasmic vesicular localization with fluorescence levels that were intermediate between A-253 and C-10E cells. Thus, BLM-resistant cells have reduced levels of FLM and appear to have a lower nuclear:cytoplasmic ratio of FLM. FLM may be useful in studying the intracellular fate of BLM-like drugs as well as providing a tool to detect and isolate BLM-resistant cells.


Protein-tyrosine phosphatase expression in pre-B cell NALM-6.

Protein-tyrosine phosphatase (PTP)-related complementary DNAs from NALM-6 (pre-B cell line) were amplified by reverse transcriptase polymerase chain reaction using primers corresponding to the conserved catalytic domains of PTPs. Thirty-three polymerase chain reaction products, identified as PTP related complementary DNAs, were classified to RPTP-alpha, PTP1B, and 4 novel PTPs, which were designated as BPTP-1-4. Their expressions in NALM-6 and other cell lines were confirmed by Northern blot analysis. BPTP-1 and -2 exhibited extensive homology with the first and the second catalytic domains, respectively, of leukocyte common antigen related molecule (LAR) and human PTP delta. The transcriptional sizes of BPTP-1 and BPTP-2 are the same (7.2 kilobases) as that of LAR. The expression of BPTP-1 was abundant in lymphoid cell lines TALL-1 and NALM-6 but small in colon cell line BM314, which is in sharp contrast to the expression of LAR. These data suggest that the expression levels of BPTP-1 and LAR are altered in a cell specific manner, probably making them cell type associated PTPs.


Altered expression of wild-type p53 tumor suppressor gene during murine epithelial cell transformation.

An epidermal cell model in which initiated, benign tumor-producing and carcinoma stages were derived from a cloned parental cell strain was used to examine p53 expression during multistage epithelial carcinogenesis. Increased steady-state levels of p53 RNA were detected in squamous cell carcinomas compared to papilloma and normal epidermal cells. Nontumorigenic initiated cell precursors of the carcinomas exhibited normal p53 expression, localizing altered p53 regulation to the malignant conversion stage. Immunoprecipitation and Western immunoblot analyses demonstrated elevated levels of p53 protein in the moderately differentiated carcinoma compared to normal cells, and negligible levels of p53 in the poorly differentiated carcinoma cells. Sequence analysis of p53 complementary DNA from normal and carcinoma cells revealed no mutations in the coding or 5'- and 3'-untranslated regions, suggesting a novel mechanism of p53 inactivation.


In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium.

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.


The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly.

Ligand-gated ion channels are oligomeric membrane proteins in which homologous subunits specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we used a dominant-negative assay in which truncated subunits of the mouse muscle acetylcholine receptor (AChR) were coexpressed with the four wild-type subunits in transfected COS cells. Fragments of the alpha, delta, and gamma subunits consisting solely of the extracellular N-terminal domain blocked surface expression of the AChR and the formation of alpha delta heterodimers, an early step in the assembly pathway of the AChR. Immunoprecipitation and sucrose gradient sedimentation experiments showed that an N-terminal fragment of the alpha subunit forms a specific complex with the intact delta subunit. Thus the extracellular N-terminal domain of the alpha, delta, and gamma subunits contains the information necessary for specific subunit association.


The binding site on ICAM-1 for Plasmodium falciparum-infected erythrocytes overlaps, but is distinct from, the LFA-1-binding site.

The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of three putative endothelial receptors that mediate in vitro cytoadherence of P. falciparum-infected erythrocytes. Since cytoadherence to postcapillary venular endothelium is thought to be a major factor in the virulence of P. falciparum malaria, we have examined the interaction between ICAM-1 and the P. falciparum-infected cell, and have compared it with the interaction to the physiological counter receptor, the leukocyte integrin LFA-1. Our results demonstrate that the malaria-binding site resides in the first two domains of the ICAM-1 molecule and overlaps, but is distinct from, the LFA-1 site.


Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation.

Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo B15) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo B15 appears to stop and restart translocation at several points. We have identified a sequence of amino acids in apo B15 that confers this behavior on a heterologous chimeric protein. In addition, we dissect pausing into two distinct steps, stopping and restarting, thereby trapping otherwise transient intermediates. Finally, we demonstrate the function of a second "pause transfer" sequence over 200 amino acids downstream in apo B15 that restarts translocation posttranslationally, suggesting that multiple pause transfer sequences are involved in the biogenesis of apolipoprotein B.


The role of gastric secretagogues in regulating gastric histamine release in vivo.

The effect of short-term intragastric arterial infusion of pentagastrin and methacholine on histamine and N tau-methyl histamine secretory rates was evaluated in mongrel dogs in vivo. Doses of pentagastrin and methacholine were chosen that stimulate gastric acid secretion equivalently. Histamine and N tau-methyl histamine secretory rates were evaluated by measuring the arterial and gastric venous plasma histamine and N tau-methyl histamine concentrations at several time points during the secretagogue infusion, and gastric blood flow was continuously monitored. Histamine and N tau-methyl histamine plasma concentrations were analyzed by stable isotope dilution technique using gas chromatography/negative ion-chemical ionization mass spectrometry. Histamine and N tau-methyl histamine secretory rates were calculated by subtracting the arterial from the venous plasma concentrations and multiplying the difference by gastric plasma flow. Infusion of pentagastrin resulted in large pulsed increase of histamine release from 1.5 +/- 0.7 ng/min at time 0 to 72 +/- 20 ng/min at 5 minutes, which decreased to a plateau of 20 +/- 8 ng/min at 20 minutes. N tau-Methyl histamine secretory rate increased from 6.7 +/- 1.9 ng/min at baseline to a maximum of 42.5 +/- 13.1 ng/min at 10 minutes, and the increase was maintained for the duration of the pentagastrin infusion. Methacholine infusion was associated with a small but sustained increase in histamine release, from a baseline of 1.6 +/- 0.6 ng/min to 5.9 +/- 1.7 ng/min at 5 minutes. N tau-Methyl histamine secretory rate was unchanged by methacholine. Gastric blood flow changes to pentagastrin roughly paralleled the extent of histamine release, but methacholine is a gastric vasodilator in its own right. Our data indicate that pentagastrin is a much more effective stimulator of gastric histamine release than methacholine and that the overall role of histamine in gastrin-stimulated acid output is likely to be different from the role of histamine in cholinergic-mediated gastric acid output.


Peptide-containing neurons in different regions of the submucous plexus of human sigmoid colon.

Specimens of the sigmoid colon were obtained from male and female patients (n = 11) with carcinoma of the colon or rectum and studied immunohistochemically for vasoactive intestinal polypeptide-, somatostatin-, substance P-, neuropeptide Y-, calcitonin gene-related peptide-, met- and leu-enkephalin-, 5-hydroxytryptamine-, and dopamine beta-hydroxylase-containing nerves. In the subdivisions of the submucous plexus (namely, Schabadasch's, Meissner's, and the intermediate plexuses), substance P- and vasoactive intestinal polypeptide-immunoreactive nerve fibers were the most numerous, and equal densities of these nerves were found in all three layers. In contrast, few neuropeptide Y-, met-enkephalin-, leu-enkephalin-, calcitonin gene-related peptide-, somatostatin-, 5-hydroxytryptamine-, and dopamine beta-hydroxylase-immunoreactive nerves were found in these regions. The nerve cell bodies of the submucous plexus contained vasoactive intestinal polypeptide, substance P, leu-enkephalin, somatostatin, and 5-hydroxytryptamine but not neuropeptide Y, met-enkephalin, calcitonin gene-related peptide, and dopamine beta-hydroxylase. Vasoactive intestinal polypeptide-containing nerve cell bodies were found in all three subdivisions. Substance P-, leu-enkephalin-, and somatostatin-immunoreactive nerve cell bodies were found in Schabadasch's plexus and the intermediate region of the submucous plexus, but they were absent from Meissner's plexus; 5-hydroxytryptamine-containing nerve cell bodies were only observed in Schabadasch's plexus. The possible function of the neuropeptide-, dopamine beta-hydroxylase-, and 5-hydroxytryptamine-containing neurons in the different layers of the submucous plexus is discussed.


Evidence for continuous stimulation of interleukin-6 production in Crohn's disease.

Increased serum concentrations of acute-phase proteins can be found in active inflammatory bowel disease. Because interleukin 6 (IL-6) is one of the main mediators of acute-phase protein synthesis by the liver, the serum concentrations of IL-6 and the acute-phase proteins C-reactive protein, alpha 1-antitrypsin, and alpha 1-acid glycoprotein were determined in 70 patients with Crohn's disease (CD) and 23 patients with ulcerative colitis (UC). Disease activities were determined by established clinical activity indices. Serum IL-6 concentrations were significantly (P less than 0.005) increased in patients with CD (mean +/- SEM, 6.8 +/- 0.9 U/mL) compared with patients with UC (mean, less than 4 U/mL) and healthy controls (mean, less than 4 U/mL). Of patients with CD, 68.5% had serum IL-6 concentrations of greater than or equal to 4 U/mL, compared with 21.7% of patients with UC and 0% of healthy controls. There was a tendency toward higher serum IL-6 concentrations in patients with active CD than in patients with inactive disease. However, these differences were not statistically significant. There was no correlation between IL-6 serum concentrations and clinical activity indices, possibly because of the short circulatory lifetime and rapid hepatic clearance of IL-6 from the portal venous blood. In contrast to serum IL-6, acute-phase proteins, which have a longer circulatory lifetime, were significantly correlated with clinical activity indices. Only the follow-up of individual patients with initially highly active disease showed a further increase in IL-6 levels during acute exacerbations of the inflammatory process. The results show that most patients with even moderately active CD have significantly increased serum concentrations of IL-6, most probably reflecting a continuous stimulation of IL-6-producing cells.


Enhanced mucosal cytokine production in inflammatory bowel disease.

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohn's disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.


Soybean trypsin inhibitor and cerulein accelerate recovery of cerulein-induced pancreatitis in rats.

The role of exogenous and endogenous cholecystokinin has been studied in the process of pancreatic regeneration after acute pancreatitis. A mild form of pancreatitis was induced in rats by subcutaneous cerulein at 12 micrograms.kg-1, three times a day for 2 days. After 3 days of rest, the cerulein-treated rats were divided into four groups: rats with acute pancreatitis fed 20% casein, who received no treatment; rats fed 50% casein; rats fed 20% casein supplemented with 1% soybean trypsin inhibitor (SBTI); and rats fed 20% casein who received 1 microgram.kg-1 of subcutaneous cerulein, three times a day. Controls were fed 20% casein plus saline subcutaneously. Rats were killed after 5, 10, or 20 days of treatment. Pancreatitis resulted in significant decreases in pancreatic weight and contents of protein, amylase, chymotrypsin, RNA and DNA. During the regenerative process, 1 microgram.kg-1 of cerulein increased all parameters to control values within 5 days and induced pancreatic growth thereafter. SBTI restored the pancreas to normal after 10 days with cellular hypertrophy; the 50% casein diet gave a response similar to SBTI without hypertrophy. It can be concluded that cerulein and SBTI can accelerate pancreatic regeneration after an attack of acute pancreatitis.


Expression of the Xanthomonas campestris pv. vesicatoria hrp gene cluster, which determines pathogenicity and hypersensitivity on pepper and tomato, is plant inducible.

The hrp gene cluster from Xanthomonas campestris pv. vesicatoria determines functions necessary not only for pathogenicity on the host plants pepper and tomato but also for the elicitation of the hypersensitive reaction on resistant host and nonhost plants. Transcriptional orientation and expression of the hrp loci were determined with hrp::Tn3-gus fusions. In addition, expression of the hrp loci was studied by RNA hybridization experiments. Expression of the hrp genes was not detectable after growth of the bacteria in complex medium or in minimal medium. However, high levels of induction of hrp gene expression were measured during growth of the bacteria in the plant. To search for a plant molecule responsible for this induction, we examined a variety of materials of plant origin for their ability to induce hrp gene expression. Filtrates from plant suspension cultures induced hrp genes to levels comparable to those induced in the plant. The inducing molecule(s) was found to be heat stable and hydrophilic and to have a molecular mass of less than 1,000 daltons.


The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica.

The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.


An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.


Expression of Schwann cell and peripheral T-cell activation epitopes in hereditary motor and sensory neuropathy.

To evaluate possible immune-mediated mechanisms in hereditary motor and sensory neuropathy (HMSN-I, Charcot-Marie-Tooth syndrome), we examined class II major histocompatibility complex antigen expression (MHC-II, HLA-DR) in Schwann cells and peripheral lymphocyte T-cell (Ta1, CD26) activation in five unrelated adults with HMSN-I. Evidence of increased activation expression was found in both compartments but the pattern did not suggest a general state of hyperimmunity or appear related to clinical characteristics of HMSN. Significantly increased CD26+ T-cell activation and greater than normal fluctuation of values occurred intermittently in sequential tests of eight HMSN patients and at single time points in 24 others. The combined data, consistent with repeated stimulations of an immune reaction under normal feedback control, suggest that HMSN-I expresses some characteristics also found in autoimmune polyneuropathies.


Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation.

Expression of membrane cofactor protein (CD46) on cultured human astrocytes was demonstrated by indirect immunofluorescence microscopy and flow cytometry following staining with a monoclonal antibody specific for CD46. Western transfer and immunoblotting detected a doublet of Mr 66,000 and 56,000. Analysis of astrocyte mRNA revealed the presence of multiple alternatively spliced transcripts encoding different extracellular regions or cytoplasmic tails of CD46. Astrocytes were also shown to express decay accelerating factor, but not the type 1 complement receptor. Upregulation of astrocyte CD46 occurred following cytomegalovirus infection. These results indicate that astrocytes express proteins involved in regulation of complement activation and protection against autologous complement.


Determination of the affinity of monoclonal human IgM for myelin-associated glycoprotein and sulfated glucuronic paragloboside.

We determined the association constant of eight monoclonal IgM with two of their targets, i.e. the myelin-associated glycoprotein (MAG) or the sulfated glucuronic paragloboside (SGPG). All IgM had a 10- to 100-fold higher affinity for MAG than for SGPG. The affinity of the different IgM for MAG ranged from 1.3 x 10(-6) to 7 x 10(-9) mol/liter. The Scatchard plots for MAG were curvilinear, half of the sites being of high or low affinity. In contrast, the plots were linear in the assay using SGPG. No obvious correlations were found between the fine specificity of these IgM for the glucuronyl sulfate epitope and their affinity, although most IgM with a high affinity reacted exclusively with SGPG derivatives retaining a sulfate group. There was no parallelism between the severity of the neuropathy and the affinity of the IgM for MAG or SGPG.


Astrocytes support mast cell viability in vitro.

Mast cells are normally found adjacent to blood vessels in the nervous system, and have been implicated in the development of inflammatory central nervous system (CNS) diseases such as experimental allergic encephalomyelitis. To further study mast cell-CNS interactions, we have developed a model in which viable rat peritoneal mast cells can be maintained in culture for up to 30 days on a monolayer of rat astrocytes. In this microenvironment, mast cells maintain their phenotype, morphology, and ability to degranulate in response to appropriate stimuli.


Cerebrospinal fluid myelin basic protein as predictive marker of demyelination in AIDS dementia complex.

Myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF) of patients with acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) in order to investigate the degree of white matter destruction. Results show that increased CSF levels of MBP were detected in all patients with severe ADC (10/10) and, less often, in subjects with mild (2/7) or moderate dementia (7/16). No evidence of MBP-elevated concentration was observed in 14 human immunodeficiency virus (HIV)-seropositive subjects without neurological disorders and in nine HIV-seronegative controls. Our findings suggest that the measurement of CSF MBP concentration may represent a predictive marker of myelin injury and neurologic damage during the course of ADC.


IgG monoclonal proteins from patients with axonal peripheral neuropathies bind to different epitopes of the 68 kDa neurofilament protein.

We describe three patients with a sensorimotor axonal polyneuropathy and an IgG M-protein that binds to a 68 kDa axonal protein identified as the low molecular weight neurofilament protein (NF-L). The immunological studies revealed that the M-proteins have different target epitopes: one is phosphorylated and the other two are nonphosphorylated. One of the nonphosphorylated epitopes is common to other intermediate filaments, such as desmin and vimentin.


Control of epidermal differentiation by a retinoid analogue unable to bind to cytosolic retinoic acid-binding proteins (CRABP).

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.


Effects of topical retinoids on cytoskeletal proteins: implications for retinoid effects on epidermal differentiation.

In vivo effects of retinoids on epidermal differentiation were investigated by analyzing cytoskeletal proteins in rhino mice treated topically with all-trans-retinoic acid (RA) and other retinoids (13-cis-retinoic acid, etretinate, TTNPB). Non-disulfide-linked cytoskeletal proteins, including keratins from the epidermal "living layers," were first selectively extracted using 9.5 M urea; subsequently, keratins of the stratum corneum were isolated using 9.5 M urea plus a reducing agent. Gel electrophoresis and immunoblot analysis showed that urea extracts of epidermis from vehicle-treated skin were composed predominantly of four major keratins (analogous to human epidermal keratins K1, K5, K10, and K14), and the keratin filament-associated protein filaggrin. In contrast, extracts of epidermis from retinoid-treated skin contained additional keratins (K6, K16, and K17) and almost no detectable filaggrin. Furthermore, similar analysis of stratum corneum keratins demonstrated that extracts from RA-treated skin did not contain the partially proteolyzed keratins typically observed in stratum corneum extracts of control animals. Hyperplasia-inducing agents (salicylic acid, croton oil) caused an increase in keratins K6, K16, and K17, but they did not effect filaggrin or alter proteolysis of stratum corneum keratins. The result that RA induced expression of keratins K6, K16, and K17, as commonly expressed in hyperproliferative epidermis, is consistent with the notion that retinoids increase epidermal cell proliferation in the basal and/or lower spinous layers. The findings that topical RA decreased filaggrin expression and reduced proteolysis of stratum corneum keratins, despite increased size and number of granular cells and the presence of an anucleate stratum corneum, suggest that topical RA may also modulate a later stage of epidermal differentiation involved in stratum corneum formation.


Identification of pigment cell antigens defined by vitiligo antibodies.

Patients with vitiligo have circulating antibodies to pigment cells. To characterize this response further and to identify the antigens defined by vitiligo antibodies, sera of 23 patients with vitiligo and 22 patients with unrelated conditions were analyzed by immunoprecipitation and SDS-PAGE analysis of 125I-labeled cell antigens on pigment and control cells. Antibodies to pigment cell antigens were present in 18 (78%) of the patients with vitiligo but in only three (14%) of the control patients (p less than 0.05). The antibodies were directed to one or more antigens with molecular weight (MW) in kilodaltons (kD) of approximately 35, 40-45, 75, 90, or 150. The responses were most commonly directed to the 40-45-kD, 75-kD, and 90-kD antigens. Antibodies to these antigens were present in 74%, 57%, and 35% of vitiligo patients versus in 14%, 9%, and 0% of control individuals. The 35-kD and 90-kD antigens were preferentially expressed on human pigment cells, whereas the 40-45-, 75-, and 150-kD antigens were expressed on both pigment and control cells. These antigens were labeled by the lactoperoxidase technique, suggesting that they are cell surface antigens. These results confirm that antibodies to pigment cells are associated with vitiligo. These antibodies are directed to several cell surface antigens, some of which are preferentially expressed on pigment cells.


Common pathogenetic pathways in allergic and irritant contact dermatitis.

Despite their different pathogeneses, allergic and irritant contact dermatitis show a remarkable similarity with respect to clinical appearance, histology, and immunohistology. To further analyze this apparent contradiction, our study was designed to meticulously compare cellular infiltrates in irritant and allergic patch-test reactions by immunostaining with a broad panel of monoclonal antibodies. For this purpose, skin biopsies from allergic and irritant patch-test reactions of similar inflammatory degree were obtained from the same probands. We found that after 72 h both types of reaction were characterized by an identical dermal infiltrate consisting mainly of memory T cells, many of which were activated, and macrophages. Dermal and epidermal Langerhans cell density and HLA--DR expression of keratinocytes were also virtually identical. Our results show that antigen recognition by specific memory T cells as well as irritants can finally induce the same pattern of inflammation, including activation of T cells obviously independent of exogenous antigen.


Cytokeratin polypeptide profile of Merkel cells in human fetal and adult skin: difference of expression of cytokeratins in epidermal and dermal Merkel cells.

The origin of Merkel cells is uncertain, although current evidence by immunohistochemical keratin marker studies favors an epidermal derivation. We studied the expression of keratin species in Merkel cells of human fetus and adult using 19 anti-keratin antibodies. Epidermal and dermal Merkel cells contained not only simple epithelium-type but also some stratified epithelium-type keratins. Interestingly, expression of some keratins was different between epidermal and dermal Merkel cells, for example, AN3 (50, 58 kD) and CKB1 (50 kD) recognized epidermal Merkel cells, but not dermal Merkel cells. These results suggest that surrounding keratinocytes influence the expression of cytokeratins in Merkel cells or that dermal Merkel cells undergo a modification from keratin-producing epidermal Merkel cells to a more neural cell type by the association with nerve endings in the upper dermis. On the other hand, certain cytokeratin polypeptides recognizable with Ks19.1 (40 kD), CK5 (45 kD), and CAM5.2 (52.5 kD) were expressed in both epidermal and dermal Merkel cells. The expression of simple epithelium-type keratins in Merkel cells remained even after the epidermal basal cells gradually lost their expression.


Recessive dystrophic epidermolysis bullosa phenotype is preserved in xenografts using SCID mice: development of an experimental in vivo model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a subgroup of hereditary blistering diseases characterized by repetitive wounding and healing with subsequent extensive scarring. The purpose of this study was to establish a xenograft model that retains the RDEB phenotype and thus might be used as an experimental in vivo model to explore the molecular and biochemical mechanisms of the chronically wounded phenotype of RDEB. Full-thickness, tumor-free RDEB skin tissues were grafted onto the dorsum of severe combined immunodeficiency (SCID) mice. At 4, 8, 12, and 24 weeks after grafting, the xenografts were removed for examination. Immunofluorescence studies were performed using species-specific antibodies to human class I antigen, mouse class I antigen, human type IV and VII collagens and with cross-reacting antibody against bullous pemphigoid antigen (BPA). Staining with the antibody to human class I antigen, W6/32, and with the antibody to mouse class I antigen, 20.8.4s, confirmed the species-specific results obtained with the type IV and type VII collagen and laminin antibodies. The RDEB grafts showed essentially no staining with the type VII collagen antibody. Antibodies against laminin and BPA showed normal staining patterns in RDEB grafts. There was an overall paucity of anchoring fibrils in the grafts when examined with electron microscopy. Blisters could be induced in these grafts with minor trauma and showed a sublamina densa separation by immunomapping and electron microscopy. As late as 24 weeks post-transplantation, the RDEB grafts remain human, are not significantly replaced by mouse cells, and retain the RDEB disease phenotype.


CD28 interaction with B7 costimulates primary allogeneic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes.

Engagement of the CD3/T cell antigen receptor complex on small, resting T cells is insufficient to trigger cell-mediated cytotoxicity or to induce a proliferative response. In the present study, we have used genetic transfection to demonstrate that interaction of the B7-BB1 B cell activation antigen with the CD28 T cell differentiation antigen costimulates cell-mediated cytotoxicity and proliferation initiated by either anti-CD2 or anti-CD3 monoclonal antibody (mAb). Moreover, a B7-negative Burkitt's lymphoma cell line that fails to stimulate an allogeneic mixed lymphocyte response is rendered a potent stimulator after transfection with B7. The mixed leukocyte reaction proliferative response against the B7 transfectant is inhibited by either anti-CD28 or B7 mAb. We also demonstrate that freshly isolated small, resting human T cells can mediate anti-CD3 or anti-CD2 mAb-redirected cytotoxicity against a murine Fc receptor-bearing mastocytoma transfected with human B7. These preexisting cytotoxic T lymphocytes in peripheral blood are present in both the CD4 and CD8 subsets, but are preferentially within the CD45RO+ "memory" population. While small, resting T cells apparently require costimulation by CD28/B7 interactions, this requirement is lost after T cell activation. Anti-CD3 initiates a cytotoxic response mediated by in vitro cultured T cell clones in the absence of B7 ligand. The existence of functional cytolytic T cells in the small, resting T cell population may be advantageous in facilitating rapid responses to immune challenge.


Genetic modulation of antigen presentation by HLA-B27 molecules.

In studies of antigenic peptide presentation, we have found a healthy volunteer whose lymphoblastoid cells were unable to present three different virus-derived epitopes to cytotoxic T lymphocytes (CTL) despite expressing the correct restricting HLA-B27 molecules on the cell surface. B cell lines were established from other members of the donor's family, including individuals suffering from ankylosing spondylitis and related diseases, and were tested for their ability to function as target cells in the same assay. None of the eight B cell lines that expressed HLA-B27 presented a known peptide epitope to CTL. However, cells from a family member that expressed HLA-B8 could present an epitope peptide restricted by that molecule. The B27 molecule in this family proved to be the B2702 subtype on isoelectric focusing gels, appearing in exactly the same position as B2702 from other cell lines that did present the peptide. To exclude mutations resulting in noncharged amino acid substitutions, cDNA coding for B2702 was cloned from the proband's cell line and sequenced. No coding changes were found. The cloned cDNA was transfected into HLA-A- and B-negative HMy/C1R cells, and the B2702 molecules generated in this environment rendered these cells, after incubation with peptide, susceptible to lysis by peptide-specific CTL. These data are compatible with the presence of a factor(s), possibly HLA linked, interfering with antigen presentation by otherwise normal B2702 molecules in this family.


Profilins constitute a novel family of functional plant pan-allergens.

Type I allergy is a major health problem in industrialized countries where up to 15% of the population suffer from allergic symptoms (rhinitis, conjunctivitis, and asthma). Previously, we identified a cDNA clone that encoded a birch pollen allergen as profilin. Profilins constitute a ubiquitous family of proteins that control actin polymerization in eukaryotic cells; in particular, profilin participates in the acrosomal reaction of animal sperm cells. Although profilins had been unknown in plants so far, our finding led to the assumption that profilins might have similar functions in pollens during plant fertilization and therefore represent allergenic components in almost all pollens. We show that profilins are prominent allergens that can be isolated from tree pollens (Betula verrucosa, birch), from pollens of grasses (Phleum pratense, timothy grass), and weeds (Artemisia vulgaris, mugwort). About 20% of all pollen allergic patients tested (n = 65) displayed immunoglobulin E (IgE) reactivity to recombinant birch profilin that was expressed in pKK223-3. An IgE inhibition experiment performed with recombinant birch profilin and purified natural profilins from timothy grass and mugwort indicates common IgE epitopes. Moreover, all pollen profilins purified from these far distantly related plant species, and likewise the purified recombinant birch profilin, are able to elicit dose-dependent histamine release via high affinity Fc epsilon receptor of blood basophils from profilin allergic patients. The presence of profilin and possibly related proteins as crossreacting allergenic components in various plants therefore provides an explanation as to why certain allergic patients display type I allergic reactions with pollens and even food from distantly related plants. A functional pan-allergen, like profilin, available as purified recombinant protein, may be a useful diagnostic and probably therapeutic reagent.


Mutations defining functional regions of the superantigen staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB) is both a superantigen and toxin. As a superantigen, SEB can bind to major histocompatibility complex (MHC) class II molecules to form a ligand for alpha/beta T cell receptors bearing particular V beta elements. As a toxin, SEB causes rapid weight loss in mice sometimes leading to death. We show here that both of these functions map to the NH2-terminal portion of the toxin. Three regions were identified: one important in MHC class II binding, one in T cell recognition, and one in both functions. These results support the conclusion that the toxicity of SEB is related to massive T cell stimulation and release of cytokine mediators and show that the residues interacting with MHC and the T cell receptor are intertwined.