Binding of a major T cell epitope of mycobacteria to a specific pocket within HLA-DRw17(DR3) molecules.

CD4+ T cells recognize antigenic peptides bound to the polymorphic peptide-binding site of major histocompatibility complex (MHC) class II molecules. The polymorphism of this site is thought to dictate which peptides can be bound and thus presented to the T cell receptor. The mycobacterial 65-kDa heat-shock protein (hsp65) peptide 3-13 is an important T cell epitope: it is immunodominant in the mycobacterium-specific T cell response of HLA-DR3+ individuals but, interestingly cannot be recognized in the context of any other HLA-DR molecules. We, therefore, have tested whether the hsp65 epitope p3-13 is selected for T cell recognition in the context of only HLA-DR3 molecules by an unique binding specificity for HLA-DR3. Using biotinylated peptides and EBV-transformed BLCL comprising all known HLA class II specificities, we find that p3-13 binds to HLA-DRw17(DR3) but not to any other HLA-DR molecule. Conversely, a control peptide p307-319 influenza hemagglutinin binds to all known HLA-DR molecules but only weakly to HLA-DRw17 and HLA-DR9. Peptide binding could be inhibited by excess unbiotinylated competitor analogue as well as by anti-DR monoclonal antibodies but not by anti-class I-, anti-DP- or anti-DQ monoclonal antibodies. The amino acid sequence of DRw17 molecules differs uniquely at five positions from the other DR beta 1 sequences. Three of these five residues (positions 26, 71 and 74) are potential peptide contacting residues. These residues map closely together in the hypothetical three-dimensional model of the DR molecule and, thus, most probably form a positively charged pocket, critical for the binding of p3-13. Interestingly, p3-13 does not bind to a DR3 variant, the DRw18 molecule. The DRw18 beta 1 chain differs from DRw17 at two major positions, close to or within the DRw17-specific pocket. These substitutions drastically change the structure and charge of the pocket and thus presumably abrogate its ability to bind p3-13.


Age-associated increase in number of CD4+CD8+ intestinal intraepithelial lymphocytes in rats.

A significant number of CD4+CD8+ T cells were detected in intestinal intraepithelial lymphocytes (IEL) of various strains of rats including Wistar, WKA, BN, LEW and F344. The site of the CD4+CD8+ population in IEL increased with age in all strains we examined. Most IEL bearing CD8 expressed no CD5 antigen in young rats, while all CD4+CD8+ IEL and some of CD8+ IEL in aged rats were of CD5+CD45RB- phenotype. In germ-free Wistar rats, age-associated increase in the number of CD4+CD8+CD5+ IEL was not evident, indicating that stimulation by the intestinal microflora was important for expansion of the CD4+CD8+CD5+CD45RB- IEL. Aged athymic F344 nude rats contained appreciable numbers of CD4+ IEL and CD8+ IEL but few CD4+CD8+ IEL, suggesting that the CD4+CD8+ IEL may be derived from thymus-dependent populations. Unlike a majority of CD4+CD8+ thymocytes bearing a low intensity of CD3/T cell receptor (TcR) alpha/beta, the CD4+CD8+ T cells in IEL expressed a high intensity of CD3/TcR alpha/beta on their surface. The CD4+CD8+ IEL appear to contribute to the spontaneous proliferation of the IEL in aged rats as assessed by tritiated thymidine incorporation after in vitro culture with medium only. These results suggest that with aging a unique CD4+CD8+ IEL may expand at a local site of the intestine under the influence of intestinal microflora and may contribute to the first line of defense against various pathogens in the epithelium.


Sequence variation of cytotoxic T cell epitopes in different isolates of Epstein-Barr virus.

Previous results have identified two distinct cytotoxic T lymphocyte (CTL) epitopes encoded by Epstein-Barr virus (EBV), TETA (ORF BLRF3/BERF1 residues 329-353) and EENL (ORF BERF3/BERF4 residues 290-309). Measurement of the specificities of CTL clones (TETA-specific clone 13 and EENL-specific clone 7) directed against these epitopes indicated that the EENL epitope is conserved in all strains of EBV tested while the TETA epitope varied between individual virus strains. Sequencing of the DNA regions encoding these two CTL epitopes in different EBV isolates confirmed these interpretations and demonstrated that different TETA epitope sequences were encoded by B-type EBV strains and by the B95-8 isolate of EBV compared to the other A-type EBV strains. Titration of synthetic variants of the TETA epitope revealed that the epitope encoded by B95-8 was 15-fold less efficient as a T cell epitope than the sequence encoded by other A-type viral strains while the TETA variant encoded by the B-type strains displayed essentially no activity as a T cell epitope.


The specificity of recognition of a cytotoxic T lymphocyte epitope.

An Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) clone (LC13) was shown to recognize the minimal peptide determinant FLRGRAYGL from the EBNA 3 antigen of the BL74 strain of EBV. The equivalent epitope from the B95-8 strain (FLRGRAYGI) is not recognized when endogenously presented and the peptide is 15-fold less active than FLRGRAYGL. A replacement set of peptides was synthesized in which each residue within FLRGRAYGL was sequentially replaced with all other genetically coded amino acids. These peptides were tested for their ability to sensitize target cells to lysis by LC13. Of the 171 single-amino acid replacement peptides only 15 were more active than the peptide FLRGRAYGI. Five peptides had significantly greater activity than FLRGRAYGL and a peptide incorporating the most active of these single-amino acid substitutions (HIRGRAYSL) induced lysis at concentrations approximately 30-fold less than FLRGRAYGL. Simplified theoretical calculations based on this study suggest that CTL LC13 has a specificity for its target epitope of 1 in 4.7 x 10(10). This represents the first complete analysis of the role of single amino acids within a minimum epitope on the specificity of CTL recognition.


The CD56 adhesion molecule is the major determinant for detecting non-major histocompatibility complex-restricted cytotoxic mononuclear cells from the intestinal lamina propria.

In this study, specific antibodies against natural killer (NK) cell surface markers identify these cells to be commonly present in normal intestinal mucosa of inflammatory bowel disease (IBD) and carcinoma patients. Cells expressing the CD56 adhesion molecule were found to be far more abundant than CD16+ cells. Functional studies revealed that cells mediating non-major histocompatibility complex-restricted cytotoxicity (NK activity) in the lamina propria express the CD56 surface antigen, whereas only a minority of this activity resides in the population with CD16 expression. This is in contrast with peripheral blood NK cells, which were found to be almost exclusively both CD16+ and CD56+. Moreover, in the lamina propria of the intestine we found CD3+ T lymphocytes not to be involved in spontaneous cell-mediated killing of tumor cells. Considerably higher numbers of cells with the CD16 or CD56 surface markers were found to be present in normal mucosa of IBD patients compared with normal mucosa of carcinoma patients, which was also reflected in higher levels of cytotoxicity detected in lamina propria mononuclear cell preparations from normal IBD mucosa. Because of the disease-related localization of the mucosa studied from both patient groups, i.e. ileum vs. colon, the observed differences may be related to tissue characteristics. Within the IBD group, relatively high levels of cytotoxicity were found in cell preparations from normal mucosa of Crohn's disease patients compared with ulcerative colitis patients, which might support the current concept that Crohn's disease affects the whole of the gastrointestinal tract.


Induction of experimental autoimmune encephalomyelitis by native myelin basic protein-activated T lymphocyte lines.

Self antigens bind to MHC class II molecules in vivo. The capacity of class II molecules to bind native self antigen, namely antigen that has not been processed through an endososomal pathway, could increase susceptibility to autoimmune diseases if recognized by autoreactive T lymphocytes. To confirm this prediction we designed experiments to show that: (a) native myelin basic protein (BP) activates encephalitogenic T lymphocyte lines, and (b) these activated lines cause experimental autoimmune encephalomyelitis (EAE) in naive rats. We show that two encephalitogenic T lymphocytes lines, LEW and BN, are activated by native BP in the presence of paraformaldehyde pre-fixed antigen-presenting cells (APC). The degree of activation, as measured by T lymphocyte proliferation, was equal to that obtained in response to BP processed by APC prior to paraformaldehyde fixing. The response to native BP was confirmed by demonstrating insensitivity to the presence of the lysosomotropic agent chloroquine or protease inhibitors. Activation of T lymphocytes by BP required the presence of syngeneic APC. The activated T lymphocyte lines were injected into naive recipient rats that developed EAE within 5 days. Both disease incidence and severity were equal to that observed in rats that were treated with T lymphocytes activated by processed BP. Hence, at least in EAE, the risk of an autoimmune event could be precipitated by a complex of native antigen and class II molecules on cells that do not possess an endososomal pathway for antigen processing and presentation.


Specificity and T cell receptor beta chain usage of a human collagen type II-reactive T cell clone derived from a healthy individual.

Collagen type II (CII) is a cartilage-specific matrix compound well known as an inducer of an experimental, T cell-dependent autoimmune arthritis, a disease which shows some similarities to human rheumatoid arthritis. Here we report on an HLA-DR7-restricted human CD4 T cell clone (TC9), which was isolated from a healthy donor and recognizes human CII. After screening CNBr fragments of CII and tryptic fragments derived thereof, the T cell epitope could be mapped to amino acid residues 271-285 of the triple helical region of CII that are located within CNBr fragment 11 [alpha 1 (II) CB11]. This epitope was confirmed by a synthetic peptide stimulatory for TC9. The T cell receptor beta chain of TC9 was cloned using the polymerase chain reaction; it comprises V beta 6.7 and contains besides J beta 2.3 and C beta 2 an as yet undescribed sequence for the D segment.


American Leishmania spp. and Trypanosoma cruzi: galactosyl alpha(1-3) galactose epitope localization by colloidal gold immunocytochemistry and lectin cytochemistry.

Patients with Chagas' disease or different clinical forms of leishmaniasis (cutaneous or visceral) have elevated galactosyl alpha (1-3)galactose antibodies. Using colloidal gold immunocytochemistry--monoclonal antibody gal-13 (specific for lipid-linked galactosyl alpha (1-3)galactose residues) and anti-nidogen antibodies and lectin cytochemistry (Bandeiraea simplicifolia IB4), both techniques specific for demonstrating galactosyl alpha (1-3)galactose residues--we have found terminal disaccharide residues on the Trypanosoma cruzi external surface of Vero cell-derived trypomastigotes but not in intact epimastigotes (although disrupted epimastigotes strongly stained), in the lips of the flagellar pocket, and on the parasitic side exactly opposite to the flagellar pocket in amastigote and promastigote forms of American Leishmania. These results resemble those obtained using anti-laminin antibodies in both trypanosomatids. In addition, results obtained with anti-nidogen antibodies seem to recognize in Trypanosoma cruzi and American Leishmania culture forms another different unknown terminal disaccharide. These results confirm the presence of terminal galactosyl alpha (1-3)galactose residues in both trypanosomatids, and that rabbit anti-laminin antibodies are indeed also recognizing galactosyl alpha (1-3)galactose residues as demonstrated for human circulating antibody. The presence of abundant galactosyl alpha (1-3)galactose residues on Trypanosomatid family members suggests a specific unknown role in parasite physiology for this terminal disaccharide.


CD1-reactive leukemic cells in bone marrow: presence of Langerhans cell marker on leukemic monocytic cells.

Langerhans cells originate in bone marrow and probably belong to the monocyte-macrophage lineage. CD1 is a specific marker of Langerhans cells. By immunofluorescence and immunoelectron microscopy, CD1a antigen and myeloid markers (CD11, CD13, CD14, CD15, CD33, HLA-DR) were studied in 53 cases of acute myeloid leukemias (AML) and 3 acute lymphoblastic leukemias (ALL). The 11 ANLL without monocytic component were CD1a negative. 2/5 of acute myelomonocytic leukemias (AML4) and 9/37 of acute monocytic leukemias (AML5) were positive. All 3 ALL were negative. No correlation was found between CD1a and myeloid markers. CD1a+ AML did not differ from CD1a- AML with regard to cytogenetics or response to therapy. The CD1a positive cells may originate from an abnormal proliferation of CD1a positive cells which are present in bone marrow and which may differentiate into Langerhans cell precursors.


Altered function and surface marker expression of neutrophils induced by rhG-CSF treatment in severe congenital neutropenia.

Neutrophils from patients suffering from severe congenital neutropenia (SCN), who were receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF), were investigated in order to analyze the previously described decrease in chemotaxis. This study demonstrated the decreased chemotaxis to five well-known chemoattractants, FMLP, C5a, IL-8, LTB4 and PAF. To further investigate this impairment of patients' neutrophils, receptors and receptor turnover for chemoattractants were examined using flow cytometry. We found 1) increased FMLP receptor and decreased C5a receptor expression, 2) a normal expression of intracellular FMLP receptors after incubation with PMA, 3) increased loss and decreased re-expression of FMLP receptors after incubation with this peptide, 4) normal expression of adhesion glycoproteins CR3 (CD11b/CD18) and LFA1 (CD11a/CD18), 5) further signs of in vivo preactivation: high expression of Fc gamma-RI (CD64) and Fc gamma-RII (CD32), decreased expression of Fc gamma-RIII (CD16), increased expression of CD14, and low expression of HLA-DR. These data demonstrate that the decrease of chemotaxis of neutrophils from SCN patients is not due: a) to a decrease in the number of intra- or extracellular FMLP receptors; b) to a decrease of adhesion molecules. However, the decreased chemotaxis could result from an altered FMLP receptor turnover. The relevance of the altered Fc gamma-receptor pattern for the in vivo occurrence of side-effects, e.g. the necrotic vasculitis, of G-CSF treatment is discussed.


2-Aminopurine inhibits RNA and protein synthesis and reduces catecholamine desensitization in C6-2B rat glioma cells.

We previously proposed that intracellular cyclic AMP accumulation induces a putative, rapidly turning over protein inhibitory to further hormone activation of adenylate cyclase. In the present study, 2-aminopurine, which has been reported to selectively block c-fos gene expression, was used to test the hypothesis that c-fos protein might be involved in the desensitization to catecholamines was observed in 2-aminopurine-treated C6-2B rat glioma cells. However, we found 2-aminopurine to inhibit, in a concentration-dependent manner, total cellular RNA and protein synthesis in C6-2B, HeLa, Swiss 3T3 and BALB/c cells. mRNA synthesis was also markedly reduced in 2-aminopurine-treated cells. These unexpected findings, while supporting our hypothesis of a protein synthesis-sensitive step in the development of refractoriness, raise concern about the specificity of action of 2-aminopurine to inhibit c-fos induction and thus any cellular process, including desensitization, which might be regulated by c-fos gene expression.


Evidence for the production of a nonsteroidal endotheliotropic factor by human granulosa cells in culture.

To determine the cellular source of the angiogenic activity displayed by follicular fluid (FF). Human granulosa cells were harvested from 27 follicular aspirates obtained 34 to 36 hours after eight patients, previously treated with human menopausal gonadotropin (hMG), follicle-stimulating hormone plus hMG, or clomiphene citrate and hMG received human chorionic gonadotropin (10,000 IU intramuscularly). Granulosa cells from individual follicles were plated at 50,000 cells/cm2 in Medium 199 (Sigma, St. Louis, MO) supplemented with either 5% calf serum or 0.1% bovine serum albumin; media collected 24 hours later was assayed in vitro measuring endothelial cell migration. Fractions depleted of steroids by reversed phase C1 chromatography were assayed as well. Granulosa cell-conditioned media from 18 of 27 follicles significantly stimulated endothelial cell migration (P less than 0.05). Chemoattractant activity did not appear to be related to steroid accumulation in the media and was not diminished in steroid depleted fractions. These findings suggest that human granulosa cells are a source of (nonsteroidal) endotheliotropic-angiogenic activity in FF.


Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.


Cytotactin is involved in synaptogenesis during regeneration of the frog neuromuscular system.

The expression of cytotactin, an extracellular matrix glycoprotein involved in morphogenesis and regeneration, was determined in the normal and regenerating neuromuscular system of the frog Rana temporaria. Cytotactin was expressed in adult brain and gut as two major components of Mr 190,000 and 200,000 and a minor form of higher molecular weight, but was almost undetectable in skeletal muscle extract. However, cytotactin was concentrated at the neuromuscular junctions as well as at the nodes of Ranvier. After nerve transection, cytotactin staining increased in the distal stump along the endoneurial tubes. In preparations of basal lamina sheaths of frog cutaneous pectoris muscle obtained by inducing the degeneration of both nerve and muscle fibers, cytotactin was found in dense accumulations at original synaptic sites. In order to define the role of cytotactin in axonal regeneration and muscle reinnervation, the effect of anti-cytotactin antibodies on the reinnervation of the basal lamina sheaths preparations was examined in vivo. In control preparations, regenerating nerve terminals preferentially reinnervate the original synaptic sites. In the presence of anti-cytotactin antibodies, axon regeneration occurred with normal fasciculation and branching but with altered preterminal nerve fibers pathways. Ultrastructural observations showed that synaptic basal laminae reinnervation was greatly delayed or inhibited. These results suggest that cytotactin plays a primordial role in synaptogenesis, at least during nerve regeneration and reinnervation in the adult neuromuscular system.


Prolactin prevents the autoinduction of thyroid hormone receptor mRNAs during amphibian metamorphosis.

We have recently reported that prolactin (PRL) inhibits both morphogenesis and cell death in thyroid hormone (T3)-induced amphibian metamorphosis (Tata et al., 1991), and that the autoinduction of T3 receptor (TR alpha and beta) mRNA is among the most rapid responses of premetamorphic Xenopus tadpoles to T3 (Kawahara et al., 1991). We now demonstrate that PRL prevents the rapid T3-induced upregulation of TR alpha and beta mRNAs in stages 50-54 Xenopus tadpoles and in organ cultures of tadpole tails. This effect is followed by the inhibition of the de novo activation of 63-kDa keratin gene by T3. We present an experimentally testable model whereby PRL exerts its juvenilizing action by preventing the amplification of TR by its autoinduction by T3.


A 5'-terminal stem-loop structure can stabilize mRNA in Escherichia coli.

The 5'-untranslated region of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer capable of prolonging the lifetime in E. coli of a number of heterologous messages to which it is fused. To elucidate the structural basis of differential mRNA stability in bacteria, the domains of the ompA 5'-untranslated region that allow it to protect mRNA from degradation have been identified by mutational analysis. The presence of a stem-loop no more than 2-4 nucleotides from the extreme 5' terminus of this RNA segment is crucial to its stabilizing influence, whereas the sequence of the stem-loop is relatively unimportant. The potential to form a hairpin very close to the 5' end is a feature common to a number of stable prokaryotic messages. Moreover, the lifetime of a normally labile message (bla mRNA) can be prolonged in E. coli by adding a simple hairpin structure at its 5' terminus. Accelerated degradation of ompA mRNA in the absence of a 5'-terminal stem-loop appears to start downstream of the 5' end. We propose that E. coli messages beginning with a single-stranded RNA segment of significant length are preferentially targeted by a degradative ribonuclease that interacts with the mRNA 5' terminus before cleaving internally at one or more distal sites.


Ploidy analysis of epithelial ovarian cancers using image cytometry.

We used a computerized image analysis system to determine the DNA content of 103 epithelial ovarian cancers using touch imprints of frozen tumor samples. Similar to prior studies of ploidy using flow cytometry, we found that most ovarian cancers (78%) were aneuploid while a minority (22%) were diploid. There was no relationship between ploidy and stage, histologic grade, or the ability to perform optimal cytoreductive surgery. Also, like prior studies using flow cytometry, negative second-look laparotomy and survival were somewhat more common in advanced-stage patients with diploid cancers than in those with aneuploid cancers. We conclude that ploidy of ovarian cancers can be determined using a computerized image analysis system to quantitate feulgen staining of cells in touch imprints. Ploidy is unlikely to play a role in treatment planning for patients with advanced-stage disease. Larger studies of patients with early-stage disease are needed, however, to determine whether ploidy is a more accurate means of predicting which patients are most likely to benefit from adjuvant therapy.


Left ventricular insulin-like growth factor I increases in early renal hypertension.

Increasing interest has been directed toward the possible role of trophically acting molecules as modulators or initiators, or both, of myocardial hypertrophy. The aim of the present study was to investigate the possible role of one such molecule, namely, insulin-like growth factor I, in myocardial hypertrophy developed in response to renal artery stenosis. Two-kidney, one clip Goldblatt hypertension was induced in Wistar rats weighing 180 g, and sham-operated animals were used as controls. Blood pressure was increased as early as 2 days after clipping (133 +/- 4 versus 116 +/- 4 mm Hg, p less than 0.05), and the increase persisted 4 and 7 days after clipping (148 +/- 6 versus 129 +/- 3 mm Hg, p less than 0.01 and 171 +/- 5 versus 139 +/- 3 mm Hg, p less than 0.01, respectively). Left ventricular weight followed a similar pattern (373 +/- 7 versus 350 +/- 8 mg, NS, 415 +/- 11 versus 386 +/- 9 mg, p less than 0.01, and 466 +/- 11 versus 391 +/- 10 mg, p less than 0.01 at 2, 4, and 7 days after clipping, respectively), but no changes in body weight between the groups were observed. Insulin-like growth factor I messenger RNA (mRNA) was quantified using a solution hybridization assay. After 4 days of renal hypertension, there was a significant increase in left ventricular insulin-like growth factor I mRNA (2.0 x 10(-18) +/- 0.48 x 10(-18) versus 0.4 x 10(-18) +/- 0.07 x 10(-18) mol.microgram DNA-1), which was no longer detectable 7 days after clipping.(ABSTRACT TRUNCATED AT 250 WORDS)


Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective.


Characterization of an 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi that contains a conserved surface-exposed epitope.

Identification of antigenically conserved surface components of Haemophilus ducreyi may facilitate the development of reagents to diagnose and prevent chancroid. A hybridoma derived from a mouse immunized with nontypeable Haemophilus influenzae produced a monoclonal antibody (MAb), designated 3B9, that bound to 35 of 35 H. ducreyi strains isolated from diverse geographic regions. The MAb 3B9 bound to a non-heat-modifiable H. ducreyi outer membrane protein (OMP) whose apparent molecular weight was 18,000 (the 18K OMP), and the 3B9 epitope did not phase vary at a rate of greater than 10(-3) in H. ducreyi. In immunoelectron microscopy, the 3B9 epitope was surface exposed, and there was intrastrain and interstrain variability in the amount of 3B9 labelling of whole cells. The MAb 3B9 cross-reacted with many species of the family Pasteurellaceae and bound to the 16.6K peptidoglycan-associated lipoprotein (P6 or PAL) of H. influenzae. Unlike P6, the 18K OMP did not copurify with peptidoglycan. In Western blots (immunoblots), five of seven serum samples obtained from patients with chancroid and four of five serum samples obtained from patients with other genital ulcer diseases at the time of presentation contained antibodies that bound to the 18K OMP. In a competition enzyme-linked immunosorbent assay, four of these serum samples inhibited the binding of 3B9 to H. ducreyi by more than 50%. We conclude that members of Pasteurellaceae expressed a conserved epitope on OMPs that sometimes had different physical characteristics. Patients with chancroid usually have antibodies to the 18K OMP and the 3B9 epitope that may have resulted from infection with H. ducreyi or previous exposure to other Haemophilus or Actinobacillus sp. strains.


Myosin-cross-reactive epitope of Shigella flexneri invasion plasmid antigen B.

IpaB, invasion plasmid antigen B, of Shigella flexneri is a 62-kDa protein required for invasion of intestinal epithelial cells. IpaB is also one of several major protein antigens recognized by the humoral immune systems of most humans and monkeys after infection with shigellae. Computer analysis of the deduced IpaB amino acid sequence indicates that an alpha-helical structure is likely through much of the molecule. Homology searches with protein data banks show that one alpha-helical domain between amino acid residues 95 and 181 has a moderate level of identity with myosin and streptococcal M protein. By using a monoclonal antibody (2F1) which recognizes an epitope in the amino-terminal third of the IpaB protein, it was possible to demonstrate a cross-reactive epitope(s) on skeletal muscle myosin. Epitope mapping localized the 2F1 epitope to three noncontiguous regions of the IpaB protein within the alpha-helical domain that contains homology with myosin. Antibodies produced in rabbits immunized with synthetic peptides from one of the 2F1 epitope regions (residues 99 to 110) of IpaB were capable of reacting with IpaB as well as myosin. Furthermore, sera from several monkeys previously infected with S. flexneri 2a contained antibodies to IpaB pep 101-116 (IpaB peptide 101-116) and also myosin. Sera from animals with antibodies against other IpaB peptides did not contain antibodies against myosin.


Helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric Helicobacter.

Helicobacter mustelae has been isolated from stomachs of ferrets with chronic gastritis and ulcers. When H. mustelae is inoculated orally into H. mustelae-negative ferrets, the animals become colonized and develop gastritis, a significant immune response, and a transient hypochlorhydria. All of these features mimic Helicobacter pylori-induced gastric disease in humans. Because the epidemiology of H. pylori infection is poorly understood and its route of transmission is unknown, the feces of weanling and adult ferrets were cultured for the presence of H. mustelae. H. mustelae was isolated from the feces of 11 of 36 ferrets by using standard helicobacter isolation techniques. H. mustelae was identified by biochemical tests, ultrastructural morphology, reactivity with specific DNA probes, and 16S rRNA sequencing. H. mustelae was not recovered from 20-week-old ferrets which had been H. mustelae positive as weanlings, nor was H. mustelae recovered from 1-year-old ferrets. Isolation of H. mustelae from feces may correspond to periods of transient hypochlorhydria, or H. mustelae may be shed in feces intermittently. The H. mustelae-colonized ferret provides an ideal model for studying the pathogenesis and transmission of H. pylori-induced gastric disease.


Genetic control of immune responses in mice to synthetic peptides of a Streptococcus mutans surface protein antigen.

The immune responses to a cell surface protein antigen (PAc) of Streptococcus mutans and a peptide corresponding to residues 301 to 319 of the protein antigen [PAc(301-319)] in various strains of mice were studied, with attention being given to the haplotype of major histocompatibility complex (MHC) class II genes. Subcutaneous immunization of mice carrying the MHC class II I-Ad gene [BALB/c, B10.D2, B10.GD, and (B10.D2 x B10.G)F1 mice] with the peptide induced strong serum immunoglobulin G (IgG) responses to recombinant PAc (rPAc) and the peptide. Subcutaneous immunization of mice carrying the haplotype k or b of the H-2 I-A gene (C3H/HeN, C57BL/6, B10.BR, B10.A, or B10 mice) with the peptide induced intermediate serum IgG responses to rPAc and the peptide, and subcutaneous immunization of mice carrying the haplotype s or q of the H-2 I-A gene (DBA/1, B10.S, or B10.G mice) induced weak serum IgG responses to rPAc and the peptide compared with the responses of mice carrying the I-Ad gene. PAc(301-319) strongly induced PAc(301-319)-specific T-cell proliferation in B10.D2 mice but not in B10.G mice. The T-cell proliferation in B10.D2 mice was inhibited by treatment of antigen-presenting cells with anti-I-Ad monoclonal antibody but not with anti-I-Ab monoclonal antibody. These results indicate that the immune responses to the peptide in mice are genetically restricted or dominated by the MHC class II gene (I-Ad). To map antigenic epitopes in PAc(301-319) and PAc in mice bearing different H-2 haplotypes, 10 overlapping decapeptides covering PAc(301-319) and 153 decapeptides covering the entire mature PAc were synthesized. Of 10 decapeptides covering PAc(301-319), 6, 7, 1, and 1 decapeptides showed strong reactions with anti-PAc(301-319) sera from B10.D2 (H-2d), B10.GD (H-2g2), B10.BR (H-2k), and B10.A (H-2a) mice, respectively. None of these overlapping decapeptides reacted with anti-PAc(301-319) sera from B10.S (H-2s) and B10.G (H-2q) mice. Epitope-scanning analyses of the mature PAc molecule showed that antigenic epitopes scattered throughout the molecule and that antigenic epitope patterns differed in mice with different H-2 haplotypes. In addition, there was little overlap of immunogenic peptides among the mice with different haplotypes.


Residues 20, 22, and 26 determine the subtype specificities of staphylococcal enterotoxins C1 and C2.

Nonconserved residues of staphylococcal enterotoxin C1 (SEC1) were converted to their counterparts in SEC2. The mutants that resulted were examined for reactivity with monoclonal antibodies (MAbs). Substitution at position 20, 22, or 26 interfered with binding of an SEC1-specific MAb. SEC1 mutants with substitutions at all three positions reacted only with an SEC2-specific MAb. Antibody-binding patterns were not associated with isoelectric point differences. All mutants retained biological activity.


Expression of insulin-like growth factors I and II and their receptor mRNAs in primary human astrocytomas and meningiomas; in vivo studies using in situ hybridization and immunocytochemistry.

These studies demonstrate the expression of IGF-1, IGF-11, and their respective receptor mRNAs in primary human astrocytomas and meningiomas. In situ hybridization and immunocytochemistry have localized a strong expression of both IGF-1 and IGF-11 mRNAs and of their protein products in the tumor cells of astrocytomas and meningiomas. The expression of IGF-1 and IGF-11 mRNAs in the tumor cells was accompanied by the co-expression of their respective type-1 and type-11 IGF receptor mRNAs. Control, non-malignant human brain expressed IGF-1 mRNA and IGF-1 and IGF-11 receptor mRNAs. There was no significant expression of IGF-11 MRNA in the control brain specimens. Control pachymeninges (dura mater) expressed low levels of IGF-1 mRNA and IGF-1 receptor mRNA. There was no significant expression of IGF-11 and IGF-11 receptor mRNAs in pachymeninges. The co-expression of IGFs and their receptors in brain tumors may contribute in their development and maintenance. The strong inappropriate expression of IGF-11 mRNA and its protein product in the tumor cells of astrocytomas and meningiomas, but not in normal brain specimens, may serve as molecular markers for the early detection of these tumors.


Biphasic effect of cAMP-elevating agents on ICAM-1 expression stimulated by retinoic acid and interferon gamma.

Incubation of the human glioma cell line HS 683 in the presence of IFN-gamma or retinoic acid strongly stimulates the cell-surface expression of the intercellular adhesion molecule ICAM-1. We have investigated the role of the cAMP-mediated signal transduction pathway in this process and report that pharmacological agents which increased the intracellular levels of cAMP exhibited a biphasic action on ICAM-1 expression in human glioma cell line HS 683. Treatment for 1 hr with 25 microM forskolin or 1 mM isobutylmethylxanthine, or for 12 hr with 100 ng/ml pertussis toxin or 50 micrograms/ml cholera toxin transiently stimulated ICAM-1 expression with a maximal level of expression 8 hr post treatment, after which time ICAM-1 expression returned to the basal level. On the other hand, such pretreatments inhibited the inducing effects of either retinoic acid or IFN-gamma. Indeed, 24 hr after treatment with cAMP-elevating agents, both the retinoic-acid- and the IFN-gamma-induced ICAM-1 expression were inhibited by 60 to 80%, with a maximal 90 to 100% inhibition 72 hr post treatment. This inhibition of the cell-surface expression of ICAM-1 was confirmed at the mRNA level. The intracytoplasmic levels of cAMP were also quantified following treatments with forskolin, retinoic acid or IFN-gamma. In response to forskolin, cAMP levels increased 30-fold within 5 min, whereas a 10-fold increase occurred 60 min following treatment with 10 microM retinoic acid. Interferon gamma, in contrast, did not induce cAMP accumulation. These results were also correlated with an in vitro activation of adenylyl cyclase activity by retinoic acid and inhibition of this activity by IFN-gamma, in a dose-dependent and a GTP-dependent manner. Our results suggest that the suppression of IFN-gamma-induced ICAM-1 expression, obtained upon pre-treatment with cAMP-elevating agents, is due to direct antagonism with IFN-gamma action on adenylyl cyclase. However, the inhibition of retinoic-acid-induced ICAM-1 expression cannot be explained by the same mechanisms. The timing of adenylyl cyclase stimulation and cAMP accumulation, as well as the levels of cAMP accumulation, are probably involved in this inhibition. Our results also emphasize the fact that the induction of ICAM-1 expression is a multi-step process implicating different transductional signals among which cAMP might be involved as a second messenger.


The relationship between multi-drug resistance and resistance to natural-killer-cell and lymphokine-activated killer-cell lysis in human leukemic cell lines.

Results concerning a possible link between susceptibility to natural-cell-mediated immune cytolysis and the multi-drug resistance (MDR) phenotype are conflicting. We evaluated in human acute lymphocytic leukemia the relationship between acquired drug resistance and susceptibility to cytolysis mediated by endogenous, interferon-activated, and interleukin-2-activated natural cytotoxic cells. Eight human leukemia drug-resistant/sensitive cell line pairs were evaluated; drug-resistant sub-lines included those selected for primary resistance to adriamycin, etoposide, teniposide, vincristine, and vinblastine. A majority of P-glycoprotein-positive MDR sub-lines displayed slight but statistically significant resistance to endogenous and/or interferon-activated natural-killer(NK)-cell-mediated lysis, as compared with the drug-sensitive parental type. P-glycoprotein-negative sub-lines displayed variable NK susceptibility; within this group, the variants selected for primary etoposide resistance were more susceptible to NK cytolysis than parental cells. Results of cold-target-inhibition experiments suggest that altered NK susceptibility does not arise solely from modulation of NK target recognition and adherence structures. IL2-activated killer (LAK) cells lysed both drug-sensitive and drug-resistant lines. Two MDR lines selected for primary etoposide resistance displayed enhanced LAK susceptibility. In contrast, the 2 variants selected for resistance to adriamycin exhibited partial resistance to LAK-mediated killing, which could be overcome at high effector-to-target ratios. Our results support the development of interleukin-2/LAK immunotherapy for the treatment of leukemias with acquired drug resistance.


Activation of NK cells in mice following corneal infection with herpes simplex virus type-1.

Natural killer (NK) cells are large granular lymphocytes that mediate antigen nonspecific, non-major histocompatibility complex (MHC) restricted lysis of virus infected cells. They are thought to play a role in innate resistance to herpes simplex virus (HSV) infections. In most animal studies reported to date, the virus was injected intraperitoneally, not a natural route of infection. Using a murine model of acute HSV-1 ocular infection, we demonstrate that increased splenic NK activity is induced in BALB/c mice following corneal infection with four different strains of HSV-1. The kinetics of NK cell activation depended on the strain of virus and was associated with virulence of the strain and with the ability of the viruses to grow in vivo. We also assessed the role of interferon-alpha/beta, IFN-gamma, and interleukin-2 (IL-2) in the HSV-1 induced NK cell activation by treating mice with antisera against these lymphokines prior to infection. Treatment with anti-IFN-alpha/beta or anti-IFN-gamma significantly reduced NK cell cytotoxicity, suggesting that these lymphokines were involved in the activation of NK cells following HSV-1 ocular infection. Treatment with anti-IL-2 resulted in increased NK cell activity, suggesting that in vivo, IL-2 is involved in the suppression of NK cell activity in infected mice. Treatment with a combination of anti-IL-2 and anti-IFN also increased NK cytotoxicity. Despite the induction of high levels of NK activity, mice developed severe ocular disease or died of encephalitis.


Clonal growth and differentiation of rabbit meibomian gland epithelium in serum-free culture: differential modulation by EGF and FGF.

We have established a serum-free clonal culture system to study the growth and differentiation of individual progenitor epithelial cells of the meibomian gland independent of other cell types and undefined serum factors. Single meibomian gland epithelial cells were obtained by subjecting whole meibomian glands to a brief EDTA treatment, needle aspiration, and nylon mesh filtration. The cells had been isolated by a previously described method using enzymatic and microsurgical techniques. Four to five hundred cells obtained were seeded on a 35 mm or 60 mm dish with serum-free MCDB 151 medium containing insulin, transferrin, selenium, ethanolamine, o-phosphorylethanolamine, dimethyl sulfoxide, and calcium. Control cultures with this basic medium did not show continuous clonal growth. Addition of epidermal growth factor (EGF) from 1 to 5 and 10 ng/ml enhanced clonal growth with a decreasing effect as measured by colony forming efficacy and colony size. Clonal growth was associated with the occurrence of two types of colony morphology and an increase in intracellular lipid production in the BrdU-labelled cells, shown by Nile red fluorescent staining. In contrast, the clonal growth stimulated by addition of acidic fibroblast growth factor (aFGF) from 1 to 100 ng/ml exhibited a pattern of an initial increase followed by a decrease, with the maximum noted at 10 ng/ml. Moreover, the aFGF-stimulated clonal growth was associated with a uniform colony morphology and minimal lipid synthesis even in the non-BrdU-labelled cells. These results indicate that clonal growth of meibomian gland epithelium can be achieved in a serum-free culture by adding either of these two peptide growth factors. Furthermore, the clonal growth stimulated by EGF was associated with progressive cellular differentiation more so than that of aFGF. Further exploration of such a differential regulation of proliferation and differentiation by EGF and aFGF may provide a better understanding of normal meibomian gland function and pathogenesis of various meibomian gland disorders.


Characteristics of a glycoprotein in the ocular surface glycocalyx.

A monoclonal antibody has been produced that binds to the apical squames (flattened cells) of the rat ocular surface epithelium and to the goblet cells of the conjunctiva. Immunoelectron microscopic localization of the antigen indicates that in apical cells it is present along the apical-microplical membrane in the region of the glycocalyx. In subapical squames, the antigen is in cytoplasmic vesicles. In some goblet cells, the antigen is in the Golgi network, and in others, it is located primarily in the membrane of the mucous granules. SDS-PAGE and immunoblot analysis demonstrate that the molecular weight of the antigen is greater than 205 kD, and the electrophoretic band stains with Alcian blue followed by silver stain. Periodate oxidation of immunoblots and cryostat sections removes antibody binding. Neuraminidase treatment of cryostat sections does not remove antibody binding, whereas N-glycanase does. Taken together, these data indicate that the antigen recognized by the monoclonal antibody is a carbohydrate epitope on a high-molecular-weight, highly glycosylated glycoprotein in the glycocalyx of the ocular surface epithelium and goblet cell mucin granule membrane. The antigen appears to be stored within cytoplasmic vesicles and reaches the glycocalyx when cells differentiate to the apical-most position where the glycocalyx interfaces with the mucin layer of the tear film.


Retinal pigment epithelial cells secrete interleukin-6 in response to interleukin-1.

Interleukin-6 (IL-6) is a peptide whose properties include the ability to activate T-lymphocytes, stimulate the secretion of immunoglobulin, induce neuronal differentiation, and trigger the release of acute phase proteins. We have detected IL-6-like activity in conditioned medium from cultured human retinal pigment epithelial (RPE) cells with a bioassay based on the ability of IL-6 to induce the proliferation of murine B-9 plasmacytoma cells. Biologic activity increased approximately 90-fold when the cells were cultured in the presence of IL-1 alpha (30 units/ml). Western blot analysis confirmed that conditioned medium from IL-1 alpha-stimulated RPE cells contained peptides with molecular weights ranging between 19,000 and 30,000 and reactive with antibody to IL-6. Finally, Northern blot analysis indicated that cells cultured in the presence of interleukin-1 contained a 1.2 kilobase transcript that hybridized to a cDNA probe specific for IL-6 messenger RNA. IL-6 peptide on Western blots and mRNA on Northern blots were undetectable unless cells were cultured in the presence of IL-1 alpha. Although IL-6 is synthesized by a variety of cell types, this report is the first to detect its synthesis by an eye-specific cell type. Furthermore, these observations indicate that retinal pigment epithelial cells respond to IL-1, a cytokine that previously has been implicated in ocular inflammation.


Expression of fibroblast growth factor receptors by embryonal carcinoma cells and early mouse embryos.

We have previously shown that differentiation of embryonal carcinoma (EC) cells leads to both increased binding of FGF (fibroblast growth factor) and suppression of k-FGF expression. In the current study, we examined the expression of FGF receptors by EC cells, EC-derived differentiated cells and early mammalian embryos using the technique of reverse transcription-polymerase chain reaction (RT-PCR). We determined that both mouse, F9, and human, NT2/D1, EC cells as well as their differentiated counterparts express transcripts for two forms of FGF receptors, bek (bacterially expressed kinase) and flg (fms-like gene). In addition, we determined that mouse blastocysts express flg transcripts. The presence of FGF receptor transcripts in early embryos and the previous finding of FGF-related activity in medium conditioned by mouse blastocysts argue that the FGF family plays important roles during early mammalian development.


Thyroid hormone up-regulates NGFI-A gene expression in rat brain during development.

NGFI-A is an immediate-early response gene induced by signals that initiate growth and differentiation. Its mRNA encodes a sequence-specific transcriptional activator possibly implicated in the control of brain developmental processes. Due to the essential role of thyroid hormone for a correct brain development, we have now investigated the possible regulation by 3,5,3'-triiodo-L-thyronine (T3) of NGFI-A gene expression during maturation of the central nervous system. Our results indicate that expression of mRNA encoding NGFI-A transcription factor is about 8-fold decreased in the brain of neonatal hypothyroid rats. No changes were seen when hypothyroidism was induced in adult life. T3 treatment increased NGFI-A mRNA within 1 h, suggesting that thyroid hormone effect is likely to be a direct one. These data indicate a strong regulation by thyroid hormone of the expression of the growth factor inducible gene NGFI-A during brain development, making this gene a suitable model to study T3 action in the early developing nervous system.


The binding of a novel bisheteroarylpiperazine mediates inhibition of human immunodeficiency virus type 1 reverse transcriptase.

The bisheteroarylpiperazines (BHAPs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and specifically block HIV-1 replication (Romero, D. L., Busso, M., Tan, C.-K., Reusser, F., Palmer, J. R., Poppe, S. M., Aristoff, P. A., Downey, K. M., So, A. G., Resnick, L., and Tarpley, W. G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8806-8810). Here we show that the radiolabeled BHAP [3H]U-88204 binds specifically to HIV-1 RT with high affinity (KD of 50 nM) and a stoichiometry of 1 mol of U-88204 per 1 mol of p66/p51 RT heterodimer. Binding of [3H]U-88204 to RT is unaffected by the presence of saturating poly(rC).oligo (dG)12-18 template-primer. Direct measurement of competition between [3H]U-88204 and other RT inhibitors for binding to RT reveals mutually exclusive competition between [3H]U-88204 and the non-nucleoside RT inhibitor BI-RG-587 (Kopp, E. B., Miglietta, J. J., Shrutkowski, A. G., Shih, C.-K., Grob, P. M. and Skoog, M.T. (1991) Nucleic Acids Res. 19, 3035-3039), indicating that both share the same binding site. Phosphonoformate in concentrations up to 50 microM shows no competition with [3H]U-88204 for binding to RT either alone or in the presence of template-primer. Dideoxynucleotide RT inhibitors affect the binding of [3H]U-88204 to RT when complementary template-primer is present. [3H]U-88204 and the dideoxynucleotide ddGTP can bind RT simultaneously, but the presence of one ligand decreases the affinity of RT for the second. Inasmuch as ddGTP approximates the nucleotide substrate of RT, the direct demonstration of an RT-dideoxynucleotide-[3H]U-88204 complex validates the use of indirect kinetic methods to assess the strength of BHAP interaction with RT and suggests that RT inhibition by U-88204 is achieved via effects on nucleotide substrate binding.


cDNA to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine.

A 1.9-kb cDNA clone to chick lumican (keratan sulfate proteoglycan) was isolated by screening an expressing vector library made from chick corneal RNA with antiserum to chick corneal lumican. The cDNA clone contained an open reading frame coding for a 343-amino acid protein, Mr = 38,640. Structural features of the deduced sequence include: a 18-amino acid signal peptide, cysteine residues at the N- and C-terminal regions, and a central leucine-rich region (comprising 62% of the protein) containing nine repeats of the sequence LXXLXLXXNXL/I, where X represents any amino acid. Lumican contains three variations of this sequence that are tandemly linked to form a unit and three units tandemly linked to form the leucine-rich region. The sequential arrangement of these repeats and their spacing suggest that this region arose by duplication. The deduced sequence shows five potential N-linked glycosylation sites, four of which are in the leucine-rich region. These sites are also potential keratan sulfate attachment sites. The cDNA clone to lumican hybridizes to a 2.0-kb mRNA found in tissues other than cornea, predominantly muscle and intestine. Radiolabeling and immunoprecipitation studies show that lumican core protein is also synthesized by these tissues. The primary structure of lumican is similar to fibromodulin, decorin, and biglycan, which indicates it belongs to the small interstitial proteoglycan gene family. The expression of lumican in tissues other than cornea indicates a broader role for lumican besides contributing to corneal transparency.


The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.


Nuclear signaling in human neutrophils. Stimulation of RNA synthesis is a response to a limited number of proinflammatory agonists.

At inflammatory sites, neutrophils are stimulated by a range of proinflammatory molecules which elicit a number of cellular responses. Considerable information on the cytoplasmic events that occur following activation of neutrophils at the cell membrane level already exists. In this study, we have focused on the ability of neutrophil agonists to initiate nuclear signaling events by investigating the induction of de novo RNA synthesis. Of a total of 14 different known potent leukocyte agonists, only three had a significant effect on the induction of RNA synthesis in neutrophils; the formylated oligopeptide formyl-methionyl-leucylphenylalanine (fMet-Leu-Phe), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. All three agonists induced de novo RNA synthesis in neutrophils at concentrations known to be optimal for the activation of a number of other cellular responses occurring in inflammation. Of significance was the observation that activation of RNA synthesis in neutrophils is a G-protein-mediated event, is also dependent on tyrosine phosphorylation, but is not influenced by cAMP. Finally, we have demonstrated that all three agonists also induce de novo synthesis of a limited number of proteins, with granulocyte-macrophage colony-stimulating factor and fMet-Leu-Phe having the most potent effect. These studies define the effects of neutrophil agonists on de novo RNA and protein synthesis in a proinflammatory context and suggest that these events in neutrophils occur in a restricted fashion, highly dependent on the stimuli present at sites of inflammation.


Structural analysis of low TCR-CD3 complex expression in T cells of an immunodeficient patient.

Normal membrane expression of the T cell receptor (TCR) depends on the coordinated synthesis and assembly of all seven proteins composing the complex, i.e. the TCR alpha and beta chains, the CD3 gamma, delta, and epsilon chains, and the zeta-zeta or zeta-eta dimer. In an experimental TCR membrane-defective T cell variant (Sussman, J. L., Bonifacino, J. S., Lippincott-Schwartz, J., Weissman, A. M., Saito, T., Klausner, R. D., and Ashwell, J. D. (1988) Cell 52, 85-95) and in two siblings whose lymphocytes express only a low level of the TCR/CD3 complex (Alarcon, B., Berkhout, B., Breitmeyer, J., and Terhorst, C. (1988) N. Engl. J. Med. 319, 1203-1208), a defect in zeta chain synthesis and/or assembly was thought to account for the defective membrane synthesis of the whole complex. We report on another immunodeficient patient whose T lymphocytes express the T cell receptor at one-tenth of normal fluorescence intensity and are not triggered to proliferate in vitro by anti-CD3 or anti-CD2 antibodies. Biochemical analysis of the patient's surface-iodinated peripheral blood lymphocytes failed to detect TCR alpha and beta, or CD3 gamma, delta, and epsilon proteins but revealed the presence of the zeta homodimer (probably as a result of the high proportion of natural killer cells). In the cytoplasm, TCR alpha and beta proteins and the zeta chain were detected, but, using monoclonal anti-CD3 antibodies, the CD3 gamma, delta, and epsilon chains were not. In addition, the CD3 epsilon chain was not detected with polyclonal antiserum in a very sensitive Western blotting detection system. The zeta chain was shown to be synthesized by the patient's T and natural killer cells. Northern blot analysis revealed normal levels of normal-size TCR beta and CD3 gamma, delta gene-specific mRNAs and decreased levels of TCR alpha mARN; CD3 epsilon gene transcripts were of abnormal size and present in lower than normal amounts. These findings suggest that this defect in T cell receptor-CD3 expression involves a mutation in the CD3 epsilon gene leading to the synthesis of an abnormal and unstable CD3 epsilon subunit.


Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.

Tau (tau) is a major constituent of paired helical filaments (PHF) found in Alzheimer's disease. The current study examines the possibility that the distinct properties of PHF-associated tau proteins (tau PHF) result from post-translational modifications of normal soluble tau (tau s). Following hydrofluoric acid (HF) treatment, tau PHF proteins are heat- and acid-stable, soluble in 2-(N-morpholino)ethanesulfonic acid buffers and display the same molecular weight, pI, and immunochemical properties as normal tau s. Alkaline phosphatase treatment of dissociated PHF results in similar, although less extensive, electrophoretic changes and a reduction in PHF-1 immunoreactivity. Therefore, phosphorylation of normal tau s appears to be responsible for the distinct properties of tau PHF. Although our results suggest that all of the normal tau isoforms are in PHF, the relative abundance of individual tau species differs in HF-treated PHF and tau s samples. Moreover, the loss of PHF following HF treatment suggests that post-translational modifications contribute to the structural stability of PHF.


Structural determinants for binary and ternary complex formation between insulin-like growth factor-I (IGF-I) and IGF binding protein-3.

Structural analogs of recombinant human insulin-like growth factor-I (IGF-I), with alterations to each of the B, C, A, and D domains, have been tested for their ability to form binary complexes with IGF-binding protein-3 (IGFBP-3) and ternary complexes with IGFBP-3 and the acid-labile subunit (alpha-subunit). Two functionally distinct regions of IGF-I have been identified. The first, involving residues 3 and 4 and the alpha-helix between residues 8 and 18 of the B-domain, as well as residues 49-51 in the A-domain, appears important for IGFBP-3 binding, such that substitution of these residues results in decreased binary complex available for alpha-subunit binding. The second region, distal to the IGFBP-3-binding epitope and primarily involving the D-domain and B-domain near residue 24, with some involvement of the C-domain, appears slightly inhibitory to binary complex formation, such that analogs with a truncated D-domain or with a Gly4 bridge substituted for the C-domain show enhanced binding to IGFBP-3. However, binary complexes formed from these analogs bind the alpha-subunit with reduced affinity, the effect being most marked when substitution of the C-domain, or replacement of Tyr24, is superimposed on D-domain truncation. It is concluded that although the alpha-subunit does not itself bind IGF-I, its interaction with IGFBP-3 in the ternary complex is dependent on structural determinants on IGF-I distal to the IGFBP-3 binding domain.


Antibodies against synthetic peptides used to determine the topology and site of glycosylation of the cGMP-gated channel from bovine rod photoreceptors.

Peptides corresponding to amino acids 321-339 (peptide GS21) and 416-431 (peptide GS31) of the cGMP-gated channel from bovine rod photoreceptors were synthesized and used as antigens for the preparation of polyclonal antibodies. After affinity purification, both antipeptide antibodies were found to bind specifically to the channel protein after Western blotting, but only the antibody against GS21 gave satisfactory results on enzyme-linked immunosorbent assay and electron microscopy. Using immunocytochemistry, we were able to localize amino acids 321-339 to the extracellular side of the rod photoreceptor plasma membrane. By synthesizing heptapeptides corresponding to amino acids 324-330 (peptide GS2s) and 420-426 (peptide GS3s), we were able to affinity purify antibodies specific for two N-glycosylation consensus sites in the channel protein. As assessed by Western blotting, antibodies against GS3s were found to bind to both the glycosylated and deglycosylated channel proteins, whereas antibodies against GS2s only bound to the channel protein after enzymatic deglycosylation. Together, these results allow the refinement of folding models for the cGMP-gated channel and implicate Asn-327 as being the sole site of N-glycosylation.


Cloning of a Na(+)- and Cl(-)-dependent betaine transporter that is regulated by hypertonicity.

Many hypertonic bacteria, plants, marine animals, and the mammalian renal medulla are protected from the deleterious effects of high intracellular concentrations of electrolytes by accumulating high concentrations of the nonperturbing osmolyte betaine. When kidney-derived Madin-Darby canine kidney (MDCK) cells are cultured in hypertonic medium, they accumulate betaine to 1,000 times its medium concentration. This results from induction by hypertonicity of high rates of betaine transport into cells. We have isolated a cDNA (BGT-1) encoding a renal betaine transporter by screening an MDCK cell cDNA library for expression of a betaine transporter in Xenopus oocytes. The cDNA encodes a single protein of 614 amino acids, with an estimated molecular weight of 69 kDa. The deduced amino acid sequence exhibits highly significant sequence and topographic similarity to brain gamma-amino-n-butyric acid (GABA) and noradrenaline transporters, suggesting that the renal BGT-1 is a member of the brain GABA/noradrenaline transporter gene family. Expression in oocytes indicates that the BGT-1 protein has both betaine and GABA transport activities that are Cl(-)- as well as Na(+)-dependent and functionally similar to betaine and GABA transport in MDCK cells. Northern hybridization indicates that transporter mRNA is localized to the kidney medulla and is induced in MDCK cells by hypertonicity.


The delta'-subunit of higher plant six-subunit mitochondrial F1-ATPase is homologous to the delta-subunit of animal mitochondrial F1-ATPase.

Mitochondrial F1-ATPases purified from several dicotyledonous plants contain six different subunits of alpha, beta, gamma, delta, delta' and epsilon. Previous N-terminal amino acid sequence analyses indicated that the gamma-, delta-, and epsilon-subunits of the sweet potato mitochondrial F1 correspond to the gamma-subunit, the oligomycin sensitivity-conferring protein and the epsilon-subunit of animal mitochondrial F1F0 complex (Kimura, T., Nakamura, K., Kajiura, H., Hattori, H., Nelson, N., and Asahi, T. (1989) J. Biol. Chem. 264, 3183-3186). However, the N-terminal amino acid sequence of the delta'-subunit did not show any obvious homologies with known protein sequences. A cDNA clone for the delta'-subunit of the sweet potato mitochondrial F1 was identified by oligonucleotide-hybridization selection of a cDNA library. The 1.0-kilobase-long cDNA contained a 600-base pair open reading frame coding for a precursor for the delta'-subunit. The precursor for the delta'-subunit contained N-terminal presequence of 21-amino acid residues. The mature delta'-subunit is composed of 179 amino acids and its sequence showed similarities of about 31-36% amino acid positional identity with the delta-subunit of animal and fungal mitochondrial F1 and about 18-25% with the epsilon-subunit of bacterial F1 and chloroplast CF1. The sweet potato delta'-subunit contains N-terminal sequence of about 45-amino acid residues that is absent in other related subunits. It is concluded that the six-subunit plant mitochondrial F1 contains the subunit that is homologous to the oligomycin sensitivity-conferring protein as one of the component in addition to five subunits that are homologous to subunits of animal mitochondrial F1.


Cloning, functional expression, and developmental regulation of a neuropeptide Y receptor from Drosophila melanogaster.

Neuropeptide Y, peptide YY, and pancreatic polypeptide are homologous 36-amino acid peptides that differ from most other peptide transmitters by having a relatively rigid conformation in aqueous solutions, defined as the pancreatic polypeptide fold, and a critical C-terminal tyrosine amide. These peptides serve as gastrointestinal hormones and neurotransmitters. A cDNA encoding a novel G protein-coupled receptor activated by neuropeptide Y was cloned from Drosophila by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed early in embryogenesis. The cDNA encodes a protein of 449 amino acids with the characteristics of a G protein-coupled receptor and shares significant amino acid identity with mammalian tachykinin receptors. When expressed in Xenopus oocytes, the PR4 protein is activated by mammalian neuropeptides in the order: peptide YY greater than neuropeptide Y much greater than pancreatic polypeptide. Northern analysis showed that PR4 receptor is expressed at equivalent levels in adult Drosophila head and body and that the expression of the PR4 receptor is regulated during development. The molecular characterization of this receptor should lead to a better understanding of the functional role of this important family of hormone receptors in adult organisms and during development.


Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.


Secondary structure of the mRNA for ribosomal protein S20. Implications for cleavage by ribonuclease E.

A synthetic RNA transcript containing the entire sequence of one of the two natural mRNAs for Escherichia coli ribosomal protein S20 is a substrate for specific cleavage by an endonuclease which is or depends on ribonuclease E (Mackie, G. A. (1991) J. Bacteriol. 173, 2488-2497). Partial cleavage with ribonucleases T1 or CL3 and limited modification with dimethyl sulfate have been employed to identify residues that are likely to be single stranded in the S20 mRNA's native state. The data show that the 5' one-third of the mRNA is relatively unstructured whereas the 3' one-third is extensively folded. The latter property can account for the previously observed accumulation of a 147-residue product co-terminal with the 3' end of the S20 mRNA (Mackie, G. A. (1989) J. Bacteriol. 171, 4112-4120). Sites of cleavage by the ribonuclease E-dependent activity map to single-stranded regions of the RNA. In addition, denaturation of the RNA substrate results in loss of susceptibility to the ribonuclease E-dependent activity and simultaneous loss of the single-stranded character of the two most prominent cleavage sites. It is proposed that ribonuclease E is a single-strand-specific enzyme with few primary structural constraints but a preference for an AU dinucleotide 3' to the site of cleavage.


Molecular cloning and characterization of v-mos-activated transformation-associated proteins.

Using monoclonal antibodies we previously detected two forms of transformation-associated proteins, a 64-kDa protein and a 68-kDa protein, in temperature-sensitive 110-Moloney murine sarcoma virus-mutant-transformed rat kidney 6m2 cells. The identity and functions of the transformation-associated proteins were previously unknown. By molecular cloning techniques and immunoscreening, we have isolated two cDNA clones (34A and 79B3) that were found by Western blot analysis to code for a monoclonal anti-transformation-associated protein antibody-reactive polypeptide of approximately 58 kDa. Limited restriction enzyme mapping indicated 34A and 79B3 are two different cDNA clones. The nucleotide sequence of 34A cDNA was determined, and a search of GenBank revealed that it is identical to that of rat transin-2. The deduced amino acid sequence of 34A shares 71% sequence identity with rat transin and 41-76% identity with six human metalloproteinases. The limited restriction enzyme mapping and partial nucleotide sequencing data indicated that 79B3 may be the rat transin gene. When either 34A cDNA or 79B3 cDNA was used as a probe in Northern blot analysis, one mRNA band of approximately 1.9 kilobases was detected in 6m2 cells grown at the permissive temperature of 33 degrees C, at which the cells exhibited transformation properties, and a much lower level in 6m2 cells grown at the nonpermissive temperature of 39 degrees C, at which the cells reverted to normal phenotypes. These results suggest that at 39 degrees C, these two genes were not transcribed at the same level as at 33 degrees C. Zymogram and Western blot analysis of 6m2 cells further confirmed that the 64- and 68-kDa proteins have metalloproteinase activities and that the synthesis of metalloproteinases was also temperature-sensitive. Apparently, the two proteins we formerly designated transformation-associated proteins are members of the rat transin gene family. Therefore, within v-mos transformed 6m2 cells, the absence of transformation-associated protein (metalloproteinase) synthesis at the nonpermissive temperature was due to the absence of transcription of two rat transin genes.


The mouse gastric H,K-ATPase beta subunit. Gene structure and co-ordinate expression with the alpha subunit during ontogeny.

The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is composed of two subunits, alpha and beta. The structure of the gene encoding the mouse beta subunit and the expression of both subunits during ontogeny are reported. The gene spans approximately 12 kilobase pairs and contains 7 exons. The positions at which introns interrupt the coding regions of the mouse H,K-ATPase beta subunit and mouse Na,K-ATPase (EC 3.6.1.37) beta 2 subunit genes are identical. The alternative beta subunit isoform of the Na,K-ATPase, beta 1, has a similar but not identical gene structure. Primer extension and S1 nuclease analysis of RNA isolated from mouse stomachs aged between 2 and 25 days indicated that major transcription-initiation sites are between 22 and 25 base pairs 5' of the translation initiation site at all ages. The expression of the H,K-ATPase alpha and beta subunit genes during ontogeny (day 1-40) was found to be co-ordinated. Protein levels of both the ATPase alpha and beta subunits were very low until day 15 and then increased to adult levels by day 30. In any mucosal cell throughout ontogeny, expression of the beta subunit gene invariably coincided with the expression of the alpha subunit gene. Cells detected by anti-H,K-ATPase beta subunit antibodies in sections from 10- and 30-day-old mice all had typical morphology of parietal cells and were arranged in glandular structures. Co-ordinate expression of the two subunit genes suggest that the regulatory mechanisms will be similar and that the beta subunit may be required for localization and function of the catalytic alpha subunit.


Effect of glycoinositolphospholipid anchor lipid groups on functional properties of decay-accelerating factor protein in cells.

The inositol ring in the glycoinositolphospholipid (GPI) anchor of human decay-accelerating factor (DAF) is unmodified in nucleated cells, whereas it is fatty acid acylated in erythrocytes (Ehu). To assess the effect of this and of the glycerol sn-2-associated acyl substituent on the abilities of DAF to cell membrane incorporate and function, 1) endogenous (physiologically anchored) DAF proteins bearing three- and two-"footed" GPI anchors were purified from Ehu and HeLa cells and 2) synthetic DAF variants bearing alternative one- "footed" anchors (retaining either the sn-1 glycerol- or inositol-associated lipid) were prepared by alkaline hydroxylamine treatment and phosphatidylinositol-specific phospholipase D digestion of Ehu DAF, respectively. The different DAF species were added to antibody-sensitized sheep erythrocytes (EshA) and their abilities to insert into the plasma membranes of the cells and control subsequent complement activation on their surfaces were compared. DAF proteins bearing all four GPI anchor structures adhered to the Esh hemolytic intermediates and inhibited expression of C3 convertase (C4b2a) activity. However, mixing of DAF-treated EshA with untreated EshAC142 and stripping of cell-associated DAF proteins with vesicles showed that only the physiologically anchored proteins remained stably associated with the lipid bilayer and functioned intrinsically. Both three- and two-"footed" Ehu and HeLa DAF proteins exhibited comparable ability to incorporate and function in the intermediates as well as to accumulate to levels 1000-fold higher/cell in Schistosoma mansoni schistosomula. These findings indicate that 1) an intact inositolphospholipid-containing GPI anchor is necessary for stable membrane integration and intrinsic function, 2) endogenous GPI anchors (with either unsubstituted and acylated inositol) incorporate and function with comparable efficiency, and 3) the transfer of either endogenous DAF form can account for the previously described circumvented uptake of human C3b by blood stage schistosomula.


The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.

Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.


Human transcription factor GATA-2. Evidence for regulation of preproendothelin-1 gene expression in endothelial cells.

Previously, we showed that the promoter of the gene encoding preproendothelin-1 (PPET-1) contains a GATA motif that is essential for activity and interacts with a nuclear factor similar in size and binding specificity to the erythroid transcription factor GATA-1. To identify this endothelial GATA-binding protein, a human endothelial cell cDNA library was screened with oligonucleotide probes for a portion of the zinc finger domain of GATA-1. A 2.6-kilobase cDNA encoding a 470 amino acid protein was obtained. Sequence analysis revealed a predicted protein which is the human counterpart of a related chicken protein, designated GATA-2. Human GATA-2 is expressed by a variety of cells, including erythroid, HeLa, and endothelial cells. A complex of a GATA-containing probe and recombinant GATA-2 expressed in COS cells comigrates with that present in gel shift experiments with nuclear extract derived from endothelial cells. In addition, expressed human GATA-2 protein transactivates reporter gene constructs containing either minimal GATA promoter elements or the native PPET-1 promoter in a cotransfection assay. Retinoic acid treatment of endothelial cells results in down-regulation of GATA-2 expression as well as down-regulation of PPET-1 gene expression. Human homologs of other known GATA-binding transcription factors are either absent from endothelial cells (in the case of GATA-1) or made in small quantities and not significantly affected by retinoid acid in these cells (in the case of GATA-3), making it unlikely that they regulate the PPET-1 gene. We propose that GATA-2 is the GATA-binding protein required for PPET-1 gene expression in endothelial cells.


The effects of cysteine mutations on the reverse transcriptases of human immunodeficiency virus types 1 and 2.

Chemical modification of HIV-1 and HIV-2 (human immunodeficiency virus, types 1 and 2) reverse transcriptases (RT) with three thiol reactive compounds selectively inhibits the RNase H function of the enzyme. HIV-1 RT has 2 cysteines (at positions 38 and 280); HIV-2 RT has 3 (38, 280, 445). Both of the cysteines in HIV-1 RT are in the polymerase domain. To investigate the role of the cysteines in the structure and function of the HIV RTs, we have converted each cysteine to serine and made combinations of the mutations. Since HIV-1 RT has alanine at position 445, we have also substituted alanine for serine at this position in HIV-2 RT. Neither of the single mutations in HIV-1 RT nor the double mutation mimics the effects of the chemical modification. The serine 280 mutation has little effect on either polymerase or RNase H; the serine 38 mutation affects both activities, as does the 38/280 double mutant. The 38 and 280 serine mutations in HIV-2 RT resemble the equivalent mutations in HIV-1 RT. Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity. The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue. We suggest that the reason the mutation (cysteine 280 to serine) does not mimic the chemical modification is because the chemical modification produces a greater change in the structure of the protein. We also suggest that position 280 lies at or near the important points of contact between the RNase H and polymerase domains, so that chemical modification of this position, which lies within the polymerase domain, distorts the RNase H domain.


NKD, a developmentally regulated tachykinin receptor in Drosophila.

A number of neuropeptides have been described which are present in the insect nervous system. The physiological role of these neuropeptides has not yet been clarified. We have characterized a Drosophila melanogaster cDNA coding for a protein, NKD, whose sequence resembles that of mammalian G protein-coupled neuropeptide receptors. This protein shows 38% homology with the mammalian tachykinin NK3 receptor within the transmembrane domain region. Stable cell lines expressing this cDNA are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. The expression of this gene is detected principally in adult fly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila embryonic development (3 h) and reaches the highest level of expression at 12-16 h, a time which correlates with the period of major neuronal development. In situ hybridization experiments demonstrate that NKD is expressed in the central nervous system, as well as in subsets of neurons in each segment of the developing ventral ganglia. The cytological localization of this gene is at position 86C on the Drosophila third chromosome.


Characterization of a novel tumor necrosis factor-alpha-induced endothelial primary response gene.

The response of endothelial cells to the cytokine tumor necrosis factor-alpha (TNF) is complex, involving the induction and suppression of multiple genes and gene products. Differential screening of a TNF-stimulated, cycloheximide-treated human umbilical vein endothelial cell library has resulted in the cloning of several novel cDNAs whose protein products are involved in the primary response of the endothelium to TNF. One of these cDNAs, designated B12, is further characterized here. B12 is encoded by a 3.5-kilobase transcript and is induced rapidly and transiently by TNF. Transcript expression is found to be developmentally regulated in a tissue-specific manner, with B12 message being differentially expressed in the heart and liver during mouse embryogenesis. The open reading frame of B12 predicts a 316-amino acid sequence rich in charged residues, particularly at the carboxyl terminus, and has neither significant homology to other known proteins nor to any extent sequence motifs. B12 is found to be a highly conserved single-copy gene which is located in the q22----q23 region of human chromosome 17. Polyclonal antibodies raised against a large portion of the B12 open reading frame immunoprecipitate a 36-kilodalton polypeptide from wheat germ lysates programmed to translate in vitro transcribed B12 mRNA. The B12 protein is further shown to be induced in human umbilical vein endothelial cells by TNF, and the protein is shown to be rapidly degraded.


MARCKS protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells.

Activation of protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.


A possible mechanism of inverse developmental regulation of alpha-fetoprotein and albumin genes. Studies with epidermal growth factor and phorbol ester.

Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically suppress activities of the promoter and enhancer of the human alpha-fetoprotein (AFP) gene in HuH-7 human hepatoma cells as analyzed by transient transfection assays using the chloramphenicol acetyltransferase gene as a reporter. In contrast to the AFP gene, albumin promoter and enhancer activities were not affected by EGF and TPA. Unexpectedly, however, Northern blot analysis revealed that the albumin mRNA level as well as the AFP mRNA level were reduced by treatment with EGF and TPA. We propose that in HuH-7 cells, the AFP enhancer stimulates the albumin promoter as well as the AFP promoter; and consequently, inhibition of the AFP enhancer by EGF and TPA results in reduction of both AFP and albumin mRNA levels. The significance of the involvement of the AFP enhancer in albumin transcription is discussed in relation to the inverse pattern of expression of the AFP and albumin genes in neonatal growth.


An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.


The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.


Reaction of Co(II)bleomycin with dioxygen.

The reaction of Co(II)bleomycin with dioxygen has been investigated. Dioxygen binds to the Co(II) complex within the time of mixing according to electron spin resonance and uv-visible spectroscopy and dioxygen analysis. Then, two dioxygenated cobalt centers react, releasing 1 mol of O2 and forming an intermediate characterized by a few highly shifted 1H NMR resonances and loss of the ESR spectrum. This is thought to be a dioxygen-bridged dimer of cobalt bleomycin molecules. Time-dependent absorbance and dioxygen measurements yield the same second order rate constant for this step of the reaction. According to uv-visible and NMR spectral analysis, the intermediate decays into diamagnetic products in a first order rate process. High performance liquid chromatography and 1H NMR studies demonstrate that the product contains two bleomycin species of equal concentration. One component is Co(III)bleomycin, designated Form II. The other is the peroxide adduct of Co(III)bleomycin, Form I, as determined by direct determination of hydrogen peroxide, which is slowly released from the product at low pH. In contrast, hydrogen peroxide is readily detected during the reaction of Co(II)Blm with O2. In isolation, Form I is unstable at pH 7 and is converted within 24 h into a mixture of Form I and Form II.


Reaction of DNA-bound Co(II)bleomycin with dioxygen.

The aerobic oxidation of Co(II)bleomycin bound to calf thymus DNA has been investigated in relation to the mechanism of reaction in solution in the absence of DNA. Kinetics of dioxygenation of the Co(II) complex were followed by spectrophotometric and electron spin resonance spectroscopy as well as dioxygen analysis. The reaction is slower than when carried out in solution; its rate is inversely related to the ratio of DNA base pairs to Co(II)bleomycin. The subsequent oxidation reaction, observed spectrophotometrically and by dioxygen analysis, is second order in cobalt complex. The calculated second order rate constant is also inversely related to the base pair to metal complex ratio. Once this ratio exceeds three, the reaction rate slows significantly with each additional increment of DNA added to the starting reaction mixture. Taking advantage of the high stability of O(2)-Co(II)bleomycin bound to greater than a 3-fold excess of DNA base pairs, it could be demonstrated that the rate constant for oxidation of two O(2)-Co(II)bleomycin molecules is much slower than that for O(2)-Co(II)bleomycin plus Co(II)bleomycin. With the same technique it was observed that the metal centers of O(2)-Co(II)bleomycin and Fe(II)bleomycin also undergo oxidation. The binding to DNA of both solution products of the oxidation of Co(II)bleomycin by O2 was examined by 1H NMR spectroscopy. Peroxy-Co(III)bleomycin, Form I, binds with higher affinity than Co(III)bleomycin, Form II. At lower ionic strength, the size of the DNA binding site for each form is about 2 base pairs/molecule of drug.


Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits.

Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low density lipoprotein (LDL) receptors, that we have used as a model for developing liver-directed gene therapies. Our strategy is to reconstitute hepatic LDL receptor expression in vivo by administering a DNA-protein complex that is capable of targeting the delivery of functional LDL receptor genes to hepatocytes. Infusion of this DNA-protein complex into the peripheral circulation of a rabbit animal model for familial hypercholesterolemia resulted in hepatocyte-specific gene transfer and a temporary amelioration of hypercholesterolemia. This noninvasive approach to gene therapy should have applications in the treatment of a wide spectrum of human diseases.


Characterization of guanosine diphospho-D-mannose dehydrogenase from Pseudomonas aeruginosa. Structural analysis by limited proteolysis.

Alginate is believed to be a major virulence factor in the pathogenicity of Pseudomonas aeruginosa in the lungs of patients suffering from cystic fibrosis. Guanosine diphospho-D-mannose dehydrogenase (GDPmannose dehydrogenase, EC 1.1.1.132) is a key enzyme in the alginate biosynthetic pathway which catalyzes the oxidation of guanosine diphospho-D-mannose (GDP-D-mannose) to GDP-D-mannuronic acid. In this paper, we report the structural analysis of GMD by limited proteolysis using three different proteases, trypsin, submaxillary Arg-C protease, and chymotrypsin. Treatment of GMD with these proteases indicated that the amino-terminal part of this enzyme may fold into a structural domain with an apparent molecular mass of 25-26 kDa. Multiple proteolytic cleavage sites existed at the carboxyl-terminal end of this domain, indicating that this segment may represent an exposed region of the protein. Initial proteolysis also generated a carboxyl-terminal fragment with an apparent molecular mass of 16-17 kDa which was further digested into smaller fragments by trypsin and chymotrypsin. The proteolytic cleavage sites were localized by partial amino-terminal sequencing of the peptide fragments. Arg-295 was identified as the initial cleavage site for trypsin and Tyr-278 for chymotrypsin. Catalytic activity of GMD was totally abolished by the initial cleavage. However, binding of the substrate, GDP-D-mannose, increased stability toward proteolysis and inhibited the loss of enzyme activity. GMP and GDP (guanosine 5'-mono- and diphosphates) also blocked the initial cleavage, but NAD and mannose showed no effect. These results suggest that binding of the guanosine moiety at the catalytic site of GMD may induce a conformational change that reduces the accessibility of the cleavage sites to proteases. Binding of [14C]GDP-D-mannose to the amino-terminal domain was not affected by the removal of the carboxyl-terminal 16-kDa fragment. Furthermore, photoaffinity labeling of GMD with [32P]arylazido-beta-alanine-NAD followed by proteolysis demonstrated that the radioactive NAD was covalently linked to the amino-terminal domain. These observations imply that the amino-terminal domain (25-26 kDa) contains both the substrate and cofactor binding sites. However, the carboxyl-terminal fragment (16-17 kDa) may possess amino acid residues essential for catalysis. Thus, proteolysis had little effect on substrate binding, but totally eliminated catalysis. These biochemical data are in complete agreement with amino acid sequence analysis for the existence of substrate and cofactor sites of GMD. A linear peptide map of GMD was constructed for future structure/functional studies.


The NusA and NusG proteins of Escherichia coli increase the in vitro readthrough frequency of a transcriptional attenuator preceding the gene for the beta subunit of RNA polymerase.

The genes for the beta (rpoB) and beta' (rpoC) subunits of Escherichia coli RNA polymerase are the distal members of a complex transcriptional unit that contains four upstream ribosomal protein genes. The RNA polymerase subunit genes are transcribed at a lower frequency than the ribosomal protein genes as a result of termination at an attenuator preceding rpoB. A purified in vitro transcription system was developed using linear DNA templates that carry the attenuator. The ability of known termination and antitermination proteins to modulate termination at the attenuator was tested. Both NusA and NusG increase the frequency of transcriptional readthrough at the attenuator whereas NusB, S10, and Rho had no significant effect in this system.