Solid phase synthesis of peptides containing the non-hydrolysable analog of (O)phosphotyrosine, p(CH2PO3H2)Phe. Application to the synthesis of 344-357 sequences of the beta 2 adrenergic receptor.

Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH2PO3H2)Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the beta 2 adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH2PO3H2)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc-p(CH2PO3Et2)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (approximately 60% total yield) and further deprotections provided both the p(CH2PO3H2)Phe and p(CH2PO3HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenylthiocarbamyl form (PTC).


Distribution of nerve endings and sensory neuropeptides in rat synovium, meniscus and bone.

The nervous system collects information from the outer world by specific senses and from the interior milieu by somatic senses. This information is processed and stored in memory and affects various bodily functions through the efferent arm of the nervous system. The efferent chemical neuropeptide message is transported intra-axonally to the site of action, which imparts site-specificity to the peripheral, paracrine neuropeptide effects. In the present study, immunohistochemistry using the immunoperoxidase method with nickel amplification was applied to visualize the topographical distribution of articular nerve fibres and nerve endings using the markers PGP 9.5 and synaptophysin, respectively. Furthermore, to get a comprehensive idea of the sensory innervation of the articular and para-articular tissue, antisera to calcitonin gene-related peptide (CGRP) and substance P were employed. Samples were collected after fixation by perfusion followed by immersion in fixative and decalcification by a special method, which also allows studies of the bone innervation. PGP 9.5- and synaptophysin-immunoreactive type IVa and IVb nerve fibres and endings were found in the synovial lining and sublining tissue and in the vascularized peripheral parts of the menisci. Furthermore, periosteum, bone marrow and the epiphyseal growth plates were also innervated, whereas innervation of the diaphyseal and metaphyseal bone was more sparse. PGP 9.5- and synaptophysin-immunoreactive nerves were also characterized by their CRGP, and to some extent, substance P content. Because of their distribution, the peripheral peptide-containing type IVa and IVb nerve fibres and nerve endings are in a position to participate in the pathogenesis of arthritis, including aspects of nociception, tissue remodelling and neurogenic inflammation.


Are we seeking the correct targets for therapeutic intervention?

In this plenary symposium paper, the authors review the physiological development of rheumatoid arthritis, the rationale for the manipulation of the eicosanoid pathway, the inhibition of tissue destruction, and angiogenesis. They proceed to discuss immunotherapy under the headings of intercellular adhesion, the induction of tolerance, and novel immunomodulatory agents. They conclude that the palliative treatment of the acute aspect of inflammation will continue by necessity into the foreseeable future. Whilst the selectivity and tolerability of existing and novel modulators of the acute inflammatory response are being improved, the alleviation of joint destruction in the long-term rheumatoid patient will depend on the manipulation of those mechanisms involved in the maintenance of the chronic inflammatory process. The ultimate goal for the therapy of the future, namely remission, can now be pursued with good reason for success, with the application of modern molecular biological techniques which will increase not only our understanding of autoimmunity, but also our ability to manipulate the immune system with profound selectivity. Central to the realization of these goals are the long-term chronic models of inflammatory joint disease, such as granuloma-cartilage implants and polyarticular and monoarticular arthritides, which will be essential for the assessment of the therapeutic promise of these exciting new developments.


Modified method of radical retropubic prostatectomy for localized prostatic cancer.

A modified method of retropubic radical prostatectomy was devised. The root of the penis was compressed and the bilateral internal iliac arteries were clamped before the dissection of the prostate. The urethra was divided at the junction of the prostate and bladder so that the bladder stump thus created has the size of the tip of the middle finger. Vesicourethral anastomosis was done by Vest procedure. By this method 24 patients were operated. All patients except one were alive without recurrence after the mean follow-up of 32 months. This method seems to be good enough to recommend.


In vitro models of lymphocyte transendothelial migration.

The processes of lymphocyte-endothelial cell interaction and the in vitro assays employed in their study are the subjects of this review. In motility assays in porous filters and gel matrices, it has been shown that lymphocyte migration can be modulated by interleukin-2 (IL-2), IL-3, IL-4, IL-6, and IL-8. Cytokines can also modulate lymphocyte-endothelial adhesion. Endothelial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) are induced or upregulated by IL-1 or tumor necrosis factor. In addition, interferon-gamma upregulates ICAM-1, and IL-4 can induce VCAM-1. The roles of these cytokines and adhesion molecules in transendothelial migration may be studied in assays in which lymphocytes penetrate layers of cultured endothelial cells. These models can distinguish lymphocyte adhesion from subsequent migration. Using such models, we and others have obtained evidence that both lymphocyte function-associated antigen-1 (LFA-1)/ICAM-1 and very late activation antigen 4 (VLA-4)/VCAM-1 interactions mediate lymphocyte adhesion to endothelial cells, but that LFA-1/ICAM-1 interactions play a greater role in transendothelial migration.


Cardiac anatomy instruction by ultrafast computed tomography versus cadaver dissection.

This prospective study was designed to determine to what extent an ultrafast computed tomography (UFCT) videotape of the heart could enhance or substitute for cadaver dissection in teaching anatomy to first-year medical students. A student population (n = 180) was randomized into four groups. Group 1 (control) received no instruction, group 2 viewed the videotape, group 3 participated in cardiac dissection, and group 4 performed cardiac dissection and viewed the videotape. After randomized instruction, each group was tested with 10 UFCT static cardiac images and 8 cardiac cadaver specimens. A different population consisting of nonrandomized fourth-year medical students also was tested. The results point to an interaction between instruction and the manner in which it was assessed. There was more carryover from the videotape-acquired knowledge to specimens than from dissection-acquired knowledge to UFCT images. Cardiac UFCT instruction resulted in dramatically improved image testing performance. This image-acquired knowledge was not sufficiently transferred to cardiac specimen identification; thus, videotape instruction should not replace dissection for teaching cardiac anatomy. Video provided instruction benefits beyond that gained through general clinical experience.


Management of the final 24 hours.

The management of the final 24 hours of life of 100 patients, dying in Our Lady's Hospice is reviewed. This review suggests that management might be improved by better contact between general hospitals and hospice/home care teams concerning the timing of patient transfer. The frequency of symptoms in the dying patient, even where many are semi-comatose, is highlighted. The main distressing symptoms are pain, excessive respiratory secretions and agitation. Our review confirms reliance on standard palliative medications such as morphine, however identifies the benefit of such newer preparations as hydromorphone and midazolam. Management might be improved by the earlier usage of hyoscine subcutaneously and stopping the use of intramuscular diazepam. Attention to potential hyoscine toxicity and untreated pyrexia may ease pre-terminal agitation. The dying patient's family also needs attention to complete the optimal management of the final 24 hours.


Similarity of copper and technetium binding sites in human IgG.

Kits for direct labeling of IgG with 99mTc were used without modification for the preparation of [67Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind 67Cu. The direct labeling of reduced IgG with 67Cu was highly efficient, resulting in approx. 95% 67Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of 99mTc bound to it. The results demonstrate a direct relationship between copper- and 99mTc-binding sites in IgG.


High dose rate brachytherapy in patients with local recurrences after radiotherapy of non-small cell lung cancer.

Thirty-one patients with recurrences of locally advanced Stage III lung cancer were treated with high dose rate brachytherapy. All patients had previously received a full course external beam irradiation. All treatments were performed under topical anaesthesia and took 6-14 min depending on the strength of the Iridium-192 source. The high dose rate brachytherapy was calculated as 10 Gy at one cm from the source axis for each session and this was repeated every 2 weeks to a maximum of three sessions. All treatments were well tolerated and no immediate treatment related complications were observed. Response evaluation 6 weeks after high dose rate brachytherapy showed that there was a partial response in 22 patients and nine patients were non-responders. Median survival was 7 and 3 months, respectively. All non-responders had initially presented with a T4N3 tumor. Ten patients died because of fatal pulmonary hemorrhages 2-24 weeks after brachytherapy and three others died because of a bronchial fistula. Endobronchial brachytherapy appears to be a valuable treatment alternative for local palliation. However, the relatively high number of complications at follow-up warrants further investigation to establish the optimal benefit to be derived from high dose rate brachytherapy treatment of locally advanced Stage III tumors.


Identification of surgical biopsy borders by use of india ink.

Separation of surgical biopsy borders from artifactual borders created during trimming of biopsy specimens is necessary to avoid misinterpretation of histologic borders. Misinterpretation of a contaminated trimming border as a surgical border may lead to additional surgery and excessive removal of normal tissue. Likewise, a neoplasm may regrow locally or metastasize if a surgical border infiltrated with neoplastic cells is falsely assumed to be an artifactual trimming border. The use of India ink for distinguishing between surgical biopsy borders and artifactual borders was evaluated. Ten normal tissue specimens from 8 types of tissue (skin, small intestine, urinary bladder, bone, muscle, lung, large intestine, and uterus) were obtained from freshly euthanatized dogs. The specimens were painted with India ink and examined for adherence of the ink to the cut surface of the specimen. Adherence of the ink was observed in all specimens with the exception of the cut surface of the lung. Twenty-five biopsy specimens from dogs with clinical cases of disease were similarly painted with India ink and evaluated. Twenty-two were identified as neoplastic and 3 as inflammatory lesions. Wedges of tissue were obtained from the center of the biopsy specimens to purposely create borders that contained neoplastic tissue. These positive controls were painted with India ink to evaluate the effect of the ink on the histologic appearance of the neoplastic cells. Distortion or alteration of the cellular architecture was not observed in any of the normal specimens, specimens from dogs with clinical cases, or positive controls. The use of India ink for delineation of biopsy borders is a simple technique that presents few technical difficulties.(ABSTRACT TRUNCATED AT 250 WORDS)


Non-transformed, but not ras/myc-transformed, serum-free mouse embryo cells recover from growth suppression by azatyrosine.

The anti-proliferative effect of azatyrosine, a newly discovered antibiotic from Streptomyces, was examined in Balb/c-originated serum-free mouse embryo (SFME) cells and transformed ras/myc SFME cells which have activated human c-Ha-ras genes. Azatyrosine suppressed their growth in a concentration-dependent manner. Growth suppression in both cells was detectable within 2 days after culture with 250 micrograms/ml azatyrosine. Non-transformed SFME cells, however, regained rapid growth after 6 days even in the presence of azatyrosine, whereas ras/myc SFME cells did not recover from the suppression. Despite the growth inhibition of ras/myc SFME cells, expression of human ras in the cells was not inhibited by azatyrosine. Meanwhile, SFME cells have the ability to express glial fibrillary acidic protein (GFAP). This expression is induced by serum-supplemented medium, though the serum inhibits the growth of SFME cells. Azatyrosine did not induce GFAP in ras/myc SFME cells, but inhibited growth. Furthermore, azatyrosine did not induce GFAP in SFME cells, and had no effect upon the expression of GFAP induced by serum in these cells. These results suggest that azatyrosine inhibited the growth of ras/myc SFME cells through a mechanism independent of those involved in growth inhibition and induction of GFAP expression by serum in SFME cells.


Insulin-like growth factors and binding proteins in ruminants and their nutritional regulation.

Insulin-like growth factors (IGF) are important mediators of growth, lactation, reproduction, and health. Considerable information on their role in ruminant animals has been learned in the past several years, but the precise mechanisms of their action are not known. The exact biological response of target cells is undoubtedly determined by the developmental state of the cell and synergism with other growth factors. Overall, somatomedins and their binding proteins seem to be major links between cellular developmental processes and nutrient supply. The mechanism by which nutrients control biological actions of somatomedins is not known but clearly involves the synthesis of IGF, as well as their binding proteins and receptors. In ruminants, severe feed restriction decreases circulating concentrations of IGF-I, whereas subtle alterations typical of those that occur in production systems have minimal effect. However, the responses of IGF to somatotropin are affected by modest alterations in nutritional status, including differences in nutritional status that are typically encountered in animal production systems.


Current strategies in the nursing care of infants with hypoplastic left-heart syndrome undergoing first-stage palliation with the Norwood operation.

Hypoplastic left-heart syndrome is a common congenital heart defect that carries a mortality rate of 95% in the first month of life if left untreated. In the past, surgical options were not accepted. However, recent success with the Norwood operation as part of a staged reconstructive approach has renewed interest in operative intervention for these critically ill neonates. Assessment of the balance between systemic and pulmonary blood flow is critical to maintain cardiac output and systemic saturation.


Haemagglutinins and fimbriae of soft rot Erwinias.

Strains of phytopathogenic soft rot Erwinia spp. were examined for haemagglutinin (HA) production. Mannose-sensitive HA was found only in five of 15 strains of E. carotovora subsp. carotovora. Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c. carotovora, ten of 13 strains of E.c. subsp. atroseptica and the single strain of E.c. subsp. betavasculorum, as well as all seven strains of E. chrysanthemi. MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the beta-galactoside asialofetuin. Fimbriae of ca 10 nm diameter were found on MRHA(+) bacteria E.c. carotovora and E.c. atroseptica.


Differential uptake of endothelin-1 by the coronary and pulmonary circulations.

Substantial removal of the vasoconstrictor peptide endothelin-1 (ET-1) by the pulmonary circulation has been reported to occur in perfused guinea pig and rat lungs. We examined the uptake of ET-1 by coronary and pulmonary circulations of the rabbit by measuring single-pass extraction of ET-1 in the isolated heart and lung. In separate experiments, each organ was perfused at 30 ml/min with Krebs-albumin (3%) solution. A bolus of 125I-ET-1 and [14C]dextran in 0.3 ml Krebs-albumin solution was injected, and extraction of endothelin (EET), relative to that of an intravascular reference indicator, [14C]dextran, was determined by multiple indicator-dilution technique. EET was 5 +/- 2% (SE) in the heart and 49 +/- 4% in the lung. Increasing flow rate in the lung preparation to approximate the mean transit time in the heart preparation did not significantly alter EET. Despite insignificant uptake of ET-1, the coronary circulation extracted an angiotensin-converting enzyme inhibitor (351A) and metabolized a synthetic angiotensin-converting enzyme substrate (benzoyl-phenyl-alanyl-proline), both properties of the normal pulmonary circulation. We therefore conclude that there is no significant ET-1 uptake in the coronary vascular bed.


Segmental vascular responses to voltage-gated calcium channel potentiation in rat lung.

We determined lung vascular responses to voltage-gated Ca2+ channel potentiation with BAY K 8644 (BAY). We anesthetized 46 rats (Sprague-Dawley; halothane and pentobarbital) and then excised and perfused their lungs at constant blood flow of 25 +/- 2 (SE) ml.kg-1.min-1 at constant airway and left atrial pressures of 5 and 6 cmH2O, respectively. Pulmonary arterial pressure (Ppa) increased from 13.3 +/- 0.3 cmH2O at baseline to 17.3 +/- 1.3 cmH2O after BAY (2.8 x 10(-6) M; n = 5; P less than 0.01). As determined by micropuncture, arteriolar and venular (Pven) pressures did not change. Increase of perfusate Ca2+ (16 x 10(-3) M; n = 8) similarly increased Ppa. NG-mono-methyl-L-arginine (2 x 10(-4) M), an inhibitor of endothelium-derived relaxing factor, augmented the pressor effect of BAY when given after (n = 4) but not before (n = 4) BAY (P less than 0.01). Prior cyclooxygenase blockade with indomethacin (5 mg/kg; n = 5) attenuated the Ppa response to BAY (P less than 0.01). None of these agents changed Pven. To confirm vasoactivity in veins, we induced smooth muscle depolarization with KCl (20 x 10(-3) M; n = 6) and receptor-mediated responses with histamine (3 x 10(-4) M; n = 7). Both of these agents increased Pven markedly (P less than 0.01). We interpret that, in rat lung, BAY causes arterial but not venous constriction, because the venous segment differs from the arterial with regard to Ca2+ channel potentiation.


Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)


Factor validity and norms for the aberrant behavior checklist in a community sample of children with mental retardation.

The Aberrant Behavior Checklist (ABC) is a 58-item rating scale that was developed primarily to measure the effects of pharmacological intervention in individuals living in residential facilities. This study investigated the use of the ABC in a sample of community children with mental retardation. Teacher ratings on the ABC were collected on 666 students attending special classes. The data were factor analyzed and compared with other studies using the ABC. In addition, subscales were analyzed as a function of age, sex, and classroom placement, and preliminary norms were derived. A four-factor solution of the ABC was obtained. Congruence between the four derived factors and corresponding factors from the original ABC was high (congruence coefficients ranged between .87 and .96). Classroom placement and age had significant effects on subscale scores, whereas sex failed to affect ratings. The current results are sufficiently close to the original factor solution that the original scoring method can be used with community samples, although further studies are needed to look at this in more detail.


Validity and reliability of the infant behavioral summarized evaluation (IBSE): a rating scale for the assessment of young children with autism and developmental disorders.

The Infant Behavioral Summarized Evaluation (IBSE) is a rating scale adapted from the Behavioral Summarized Evaluation (BSE) and specifically related to the assessment of behaviors of young children having autistic disorders. Content validity and reliability studies described in the paper were made from behavior ratings of videotapes for 89 children aged from 6 to 48 months. Results show a significant group of 19 items including some characteristic early autistic behaviors (communicative and social abnormalities) and some that are less commonly described in the syndrome (attentional, perceptive, and adaptive disorders). The value of the use of this scale for clinicians and professionals involved in behavioral evaluations and treatment of young children with developmental disorders and the necessity for further psychometric investigations are discussed.


A follow-up study of 201 children with autism in Kyushu and Yamaguchi areas, Japan.

A follow-up survey was conducted on 201 young adults with autism who were 18 or older (mean age, 21.5). All had participated previously in intensive therapeutic camping or had therapeutic involvement at medical consultation agencies to evaluate their outcome. Their social outcome was better than that previously reported in Japan. Although 31.5% had shown marked deterioration during adolescence, 43.2% had shown marked improvement during that period. Possible factors contributing to these results are discussed.


Postural control in children with autism.

Postural control was evaluated in samples of autistic, normal, and mentally retarded children in this pilot study using a recently developed, computerized posturographic procedure. A battery of postural positions was administered including postures involving some degree of "stress" (e.g., occluded vision or standing on pads). The postural patterns of children with autism differed from those observed in normal children, in mentally retarded children, and in adults with vestibular disorders. In comparison to normal children the autistic subjects were less likely to exhibit age-related changes in postural performance and postures were more variable and less stable with more lateral sway. Autistic subjects also exhibited a "paradoxical" response of greater stability with more "stressful" postures, putting excessive weight on one foot, one toe, or one heel. The implications for neuroanatomical models of autism are discussed.


New structural features of the antigenic extracellular polysaccharides of Penicillium and Aspergillus species revealed with exo-beta-D-galactofuranosidase.

To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.


A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.


Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes.

From the total tRNAs of Spiroplasma citri, we isolated and purified two tRNA(Trp) species by using chromatography on an RPC-5 column followed by denaturing polyacrylamide gel electrophoresis. The sequence of the two tRNAs, as well as the sequences of the corresponding genes, were determined. One of the two tRNA(Trp) species has a CCA anticodon and is able to pair with the universal UGG tryptophan codon, while the second has a U*CA (U* is a modified uridine) anticodon and is able to pair with UGA but also with UGG in accordance with the "U:N wobble" rule. Thus, in S. citri, UGA is not a stop codon but codes for tryptophan. The two tRNA(Trp) genes, together with a third tRNA gene, tRNA(Ser) (CGA), belong to a single transcription unit. The nucleotide sequences of the two tRNA(Trp) species show 82.9% similarity. The two spiroplasmal tRNA(Trp) species can be aminoacylated by using an aminoacyl-tRNA synthetase fraction from S. citri. In contrast, the enzyme fraction from Escherichia coli aminoacylates tRNA(Trp) (CCA) but not tRNA(Trp) (U*CA).


Site-specific integration of the Haemophilus influenzae bacteriophage HP1: location of the boundaries of the phage attachment site.

Plasmids containing DNA segments from the attachment region of phage HP1 were constructed and tested for the ability to replace the phage attachment site substrate in site-specific recombination reactions. The distance separating the boundaries of the functional site was 418 bp. Replacements within the 11-residue segment 5'-GGCGGTTATCG at the left boundary or within the 12-residue segment 5'-GGATTTTTTGAA at the right boundary abolished substrate activity. A segment of the 418-residue sequence preserves the integrity of an operon of three Haemophilus influenzae tRNA genes after HP1 insertion within the coding sequence.


Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956.

Carbon starvation induces the development of a starvation- and stress-resistant cell state in marine Vibrio sp. strain S14 (CCUG 15956). The starved cells remain highly responsive to nutrients during prolonged starvation and exhibit instantaneous severalfold increases in the rates of protein synthesis and RNA synthesis when substrate is added. In order to elucidate the physiological basis for the survival of cells that are starved for a long time, as well as the capacity of these cells for rapid and efficient recovery, we analyzed the ribosome content of carbon-starved Vibrio sp. strain S14 cells. By using direct chemical measurements of the amounts of ribosomal particles in carbon-starved cultures, we demonstrated that ribosomes were lost relatively slowly (half life, 79 h) and that they existed in large excess over the apparent demand for protein synthesis. After 24 h of starvation the total rate of protein synthesis was 2.3% of the rate during growth, and after 3 days this rate was 0.7% of the rate during growth; the relative amounts of ribosomal particles at these times were 81 and 52%, respectively. The ribosome population consisted of 90% 70S monoribosomes, and no polyribosomes were detected in the starved cells. The 70S monoribosomes were responsible for the bulk of the protein synthesis during carbon starvation; some activity was also detected in the polyribosome size region on sucrose density gradients. We suggest that nongrowing carbon-starved Vibrio sp. strain S14 cells possess an excess protein synthesis capacity, which may be essential for their ability to immediately initiate an upshift program when substrate is added.


Species-specific epitopes exist on the cytoplasmic amino-terminal domain of erythrocyte band 3 protein.

Monoclonal antibodies (P3-9H, P3-1F, P3-2H, P3-4A, and P3-4C) to human erythrocyte band 3 were produced using human erythrocyte membranes as the immunogen. All epitopes defined by these antibodies were found on the amino-terminal cytoplasmic domain of erythrocyte band 3. The antibodies crossreacted variously with erythrocyte band 3 of primates (chimpanzee, orangutan, Rhesus monkey, Japanese monkey, spider monkey, and capuchin monkey) in enzyme-linked immunosorbent assay. P3-9H did not crossreact with erythrocyte band 3 of any primate examined; P3-1F crossreacted only with that of chimpanzee; P3-2H crossreacted with erythrocyte band 3 of chimpanzee, spider monkey, and capuchin monkey; and P3-4A and P3-4C crossreacted with erythrocyte band 3 of all primates examined. These results suggest that evolutional changes in primates are accumulated in the amino-terminal cytoplasmic domain of band 3 and that species-specific epitopes exist on this domain.


Structural aspects of the gastric H,K-ATPase.

The gastric H,K-ATPase is an alpha, beta heterodimer. The large catalytic subunit is composed, in the case of the hog enzyme, of 1033 amino acids, whereas the beta subunit is composed of about 291 amino acids and is heavily glycosylated. The membrane topology of the alpha subunit is difficult to predict using hydropathy analysis. Tryptic hydrolysis of intact, inside out vesicles followed by cysteine labelling with fluorescein-5-maleimide provided experimental evidence for an 8 membrane spanning model for the alpha subunit, between residues 104 and 162 (M1/M2), 291 and 358 (M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/M8). No evidence was found for a pair of segments (M9/M10) towards the C terminal end of the molecule, contrary to predictions for the Na,K- and Ca-ATPases. Iodination of intact vesicles followed by carboxypeptidase Y cleavage of the C terminal tyrosines showed that the C terminal end of the alpha subunit was cytoplasmic. The epitope for antibody 146 was extracytoplasmic and located between residues 871 to 874 between M7/M8. The binding site of the K competitive imidazo-pyridine, SCH28080, was to the extracytoplasmic loop between M1 and M2, whereas the binding of the covalent SH reagent generated from acid activation of omeprazole in acid transporting vesicles was to 2 cysteines at positions 813 (or 822) and 892 predicted to be in the extracytoplasmic loops connecting M5/M6 and M7/M8, respectively. The beta subunit was only hydrolysed in broken vesicles. A fragment beginning at position 236 was liberated under these conditions only in the presence of reducing agents, showing that cysteine 210 and 263 were disulfide linked.(ABSTRACT TRUNCATED AT 250 WORDS)


Lectin domain peptides from selectins interact with both cell surface ligands and Ca2+ ions.

Selectins are receptors that mediate leukocyte adhesion to platelets or endothelial cells through Ca(2+)-dependent interactions with cell surface oligosaccharides. We found that peptides corresponding to residues 23-30, 54-63, and 70-79 of the N-terminal lectin domain of P-selectin inhibited leukocyte adhesion to P-selectin. Peptides corresponding to the homologous 23-30 and 54-63 regions of E-selectin and L-selectin also prevented cell binding to P-selectin. Immobilized albumin conjugates of the three P-selectin peptides supported adhesion of myeloid cells and certain other cells expressing fucosylated oligosaccharides. Ca2+ was required for optimal cell adhesion to the conjugates containing the 23-30 and 54-63 sequences. Furthermore, Ca2+ interacted with the 23-30 and 54-63 peptides of all three selectins, as detected by changes in intrinsic fluorescence emission intensity. These data suggest that residues contained within the 23-30 and 54-63 regions of the selectins represent contact sites for carbohydrate structures on target cells. Furthermore, binding of Ca2+ to these sequences may directly enhance their ability to interact with cell surface ligands.


Facilitated transport of methotrexate polyglutamates into lysosomes derived from S180 cells. Further characterization and evidence for a simple mobile carrier system with broad specificity for homo- or heteropeptides bearing a C-terminal glutamyl moiety.

Studies are presented further characterizing a facilitative system transporting methotrexate (MTX) polyglutamates into lysosomes derived from S180 cells. Initial influx of [3H]MTX + G1 (MTX with 1 additional glutamyl residue) exhibited a slightly alkaline pH optimum (pH 7.7) and was moderately temperature-dependent (Q10 27-37 degrees C = 3.1 +/- 0.1). An analysis of the kinetics of intralysosomal accumulation of [3H]MTX + G1 showed saturation kinetics for initial influx, but linear kinetics for the steady-state level of exchangeable [3H]MTX + G1 at different external concentrations of [3H]MTX + G1. In addition, the system exhibited substantial directional asymmetry with respect to the interaction with MTX + G1 during influx and efflux. Accelerated homo- and heteroexchange diffusion was demonstrated for influx of [3H]MTX + G1, while decelerated homoexchange diffusion was demonstrated for efflux of [3H]MTX + G1 following trans-positioning of MTX + G1 or glutamyl-gamma-glutamate in the opposite compartment. These observations were consistent with a single mobile carrier system mediating influx and efflux of this polyglutamate. Based upon an analysis of competitive interactions with [3H] MTX + G1, this system displayed specificity for MTX-gamma-glutamates, folyl-gamma-polyglutamates, alpha- or gamma-glutamyl peptides and heteropeptides bearing a C-terminal gamma-glutamate but not for MTX or glutamate, themselves. Among polyglutamates, gamma-glutamyl chain length was not a significant factor for transport except in the case of MTX polyglutamates. Overall, our results appear to delineate in the lysosomal membrane a simple mobile carrier system with broad specificity for folyl- or non-folyl-bearing peptides responsible for the transport of MTX polyglutamates.


The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.

The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.


Cloning and characterization of human cyclin D3, a cDNA closely related in sequence to the PRAD1/cyclin D1 proto-oncogene.

Cyclins regulate cell cycle progression by complexing with and activating cdc2 or related kinases. PRAD1/cyclin D1 is a recently discovered putative oncogene in several types of human tumors and may regulate G1-S phase progression. We have cloned a related human cDNA, called cyclin D3, from a placental cDNA library by cross-hybridization with PRAD1. In synchronized HeLa cells, the mRNA levels of PRAD1 and cyclin D3 were regulated reciprocally through the cell cycle: cyclin D3 mRNA levels peaked in S phase, where PRAD1 mRNA was lowest in S. In normal human mammary epithelial (70N) cells synchronized by growth factor deprivation and subsequent growth factor stimulation, PRAD1 expression peaked in G1 and declined before S phase, while cyclin D3 expression rose later in G1 and remained elevated in S. Therefore, the close relationship (53.1% identity) between PRAD1 and cyclin D3 does not necessarily imply redundant functions of these candidate G1 cyclins; they may have distinct roles in progression from G1 through S phase.


Identification of the Ca(2+)-independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linking reagent.

The Ca(2+)-independent endocytic hyaluronan (HA) receptor in rat liver sinusoidal endothelial cells (LECs) was identified using a novel cross-linking derivative of HA. The heterobifunctional, photoactivatable, reducible reagent sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) was coupled to the terminal amino group of uniquely modified HA-amine oligosaccharides (M(r) approximately 60,000) and subsequently iodinated. 125I-ASD-HA bound to cultured LECs with similar specificity and affinity as a previously characterized 125I-HA-amine/Bolton-Hunter adduct. Permeabilized LECs were incubated with 125I-ASD-HA with 10 mM EGTA and photolysed with UV light. Detergent extracts were reduced to release the HA oligosaccharides and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Two polypeptides were consistently and equally labeled at M(r) = 175,000 and 166,000. Photoaffinity labeling of these two proteins was virtually identical in cultured LECs or membranes and was competed greater than 90% with a 100-fold excess of HA. As with the previously characterized bona fide LEC HA receptor, cross-linking was also competed by chondroitin sulfate and heparin, but less efficiently by chondroitin and not with galacturonan. We conclude that the Ca(2+)-independent LEC HA receptor is composed of at least two polypeptides of M(r) approximately 175,000 and 166,000 and may exist as a heterodimer of M(r) approximately 340,000. We also conclude that the LEC HA receptor is distinct from the CD44 family of HA-binding proteins.


Escherichia coli topoisomerase III-catalyzed cleavage of RNA.

Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture. Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium. The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III. The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed. Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.


Spectral characterization of brain and macrophage nitric oxide synthases. Cytochrome P-450-like hemeproteins that contain a flavin semiquinone radical.

Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.


Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.


Functional reconstitution of a chloride channel protein from bovine trachea.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.


Heterodimeric structure of the spider toxin omega-agatoxin IA revealed by precursor analysis and mass spectrometry.

We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.


Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.


A potent enhancer made of clustered liver-specific elements in the transcription control sequences of human alpha 1-microglobulin/bikunin gene.

alpha 1-Microglobulin (A1M) and bikunin are plasma proteins which are present both as free molecules and as complexes with either IgA heavy chains for A1M or the H1, H2, and H3 heavy chains of the inter-alpha-inhibitor family for bikunin. Mature A1M and bikunin originate from the cleavage of an A1M/bikunin precursor (ABP) synthesized from a single gene with liver-specific expression. Five kilobases of the 5'-flanking region of the human ABP gene were sequenced. Deletion mutants of this region subcloned upstream of a CAT reporter gene were transfected into HepG2 hepatoma cells. A segment covering the -2.7- to -2.8-kb area is required for full activity of the ABP gene. This segment contains a cluster of six elements (boxes 1-6, 5' to 3') which are potential binding sites for the liver-enriched trans-acting factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3, and HNF-4, respectively. This cluster enhances the activity of heterologous minimal promoters in a position- and distance-independent fashion in HepG2 cells. This enhancer activity is restricted to liver cells as the cluster is unable to activate promoters in Chinese hamster ovary (CHO) or HeLa cells. By band-shift experiments we have shown that the liver-enriched transcription factors HNF-1, or HNF-3, do bind to boxes 1 and 4, or 3, respectively. The combination of a weak promoter and a strong distant and liver-specific enhancer distinguishes the ABP gene from most other plasma protein genes expressed in hepatocytes.


Apolipoprotein AI expression and high density lipoprotein distribution in transgenic mice during development.

The developmental changes in apoAI expression in human apolipoprotein AI (apoAI) transgenic and nontransgenic control mice and the effect of these changes on high density lipoprotein (HDL) sizes were investigated. Results demonstrated that both human and mouse apoAI mRNA levels sharply increased following delivery and then decreased prior to weaning. Unlike the changes in apoAI mRNA, plasma apoAI concentration and HDL mass increased following delivery and remained elevated as the animals matured. In these animals, parallel increases in plasma apoAI and HDL mass with development were accompanied by increases in HDL particle sizes. Control mice demonstrated a monodisperse population of particles that increased in particle size from 7.5 nm (at 2 days prior to birth) to 9.8 nm (at 13 days after birth). The latter is similar to the adult HDL size and distribution. Transgenic mice, unlike controls, exhibited several distinct HDL populations, which also increased in size as animals matured. These results suggest that post-transcriptional factors are important in regulating apoAI plasma levels and that developmental changes in plasma apoAI concentrations are associated with changes in HDL size distribution.


Differential sorting of lutropin and the free alpha-subunit in cultured bovine pituitary cells.

The glycoprotein hormones lutropin (LH) and follitropin (FSH) are both synthesized by gonadotrophs in the anterior pituitary but are stored in separate secretory granules prior to secretion. Despite having highly homologous beta-subunits and alpha-subunits with the identical amino acid sequence, the Asn-linked oligosaccharides on LH terminate with SO4-GalNAc while those on FSH terminate with sialic acid-Gal. In addition to LH and FSH, gonadotrophs secrete uncombined (free) alpha-subunit which bears the same sulfated oligosaccharides as LH. We have examined the synthesis and secretion of LH and free alpha-subunit in primary cultures of bovine pituitary cells in order to determine if the sulfated oligosaccharides have any impact on sorting. Our results show that newly synthesized free alpha-subunit is secreted exclusively via the constitutive pathway with a t1/2 of 1.8 h and is never found in dense-core secretory granules. In contrast, LH dimer is secreted by both the constitutive and the regulated pathways. Constitutive secretion and arrival in a dense secretory granule both occur with t1/2 values of 1-1.5 h for newly synthesized LH. Sulfation occurs immediately prior to arrival of LH in the secretory granule and is followed by a period of 1-1.5 h before the LH-containing granules become sensitive to release by gonadotropin releasing hormone. As a result the t1/2 for LH secretion in the presence of gonadotropin releasing hormone is 3.5 h. Sulfation of the free alpha-subunit oligosaccharides is not, therefore, sufficient to direct the alpha-subunit to secretory granules, and the information required for directing LH to granules must reside either in the beta-subunit or the alpha beta-complex.


Beta-adrenergic regulation of a myocardial actin gene via a cyclic AMP-independent pathway.

The skeletal alpha-actin gene encodes a major component of the embryonic cardiac sarcomere that is strongly and selectively re-induced during beta-adrenoceptor-mediated hypertrophy in neonatal rat cardiac myocytes. We present evidence that beta-adrenergic induction of this gene is mediated, not by cAMP, but by a calcium-dependent pathway involving ryanodine-sensitive calcium stores. Nifedipine-induced blockade of the plasma membrane L-type calcium entry channel prevented induction of skeletal alpha-actin mRNA by isoproterenol. Activation of calcium entry by the dihydropyridine agonist Bay K8644 independently induced skeletal alpha-actin mRNA, as did cholera toxin-mediated activation of Gs. Induction of skeletal alpha-actin mRNA by compounds that directly elevate cAMP was weak relative to their effects on other cAMP-dependent phenomena and required calcium entry. In addition, selective inhibition of protein kinase A with KT5720 did not block beta-adrenergic induction of skeletal alpha-actin. Calcium ionophore A23187 did not induce skeletal actin, but prevented its induction by isoproterenol. Ryanodine had bimodal effects: 10(-10) M ryanodine induced skeletal alpha-actin mRNA, whereas 10(-6) M ryanodine prevented skeletal actin induction by beta-adrenergic stimuli. We postulate that beta-adrenergic stimulation of skeletal alpha-actin mRNA requires G-protein-coupled calcium channel activation and compartmentalized calcium release in a manner independent of the cAMP/protein kinase A signal pathway.


Transcriptional regulation and increased functional expression of the inositol trisphosphate receptor in retinoic acid-treated HL-60 cells.

The inositol trisphosphate (InsP3) receptor is an essential regulator of intracellular calcium in many cells including chemoattractant- and cytokine-stimulated neutrophils and differentiated promyelocytic leukemic (HL-60) cells. We examined the expression and function of the InsP3 receptor and the transcriptional regulation of the InsP3 receptor gene in HL-60 cells and in HL-60 cells treated for 1-5 days with 1 microM retinoic acid. Radioligand binding studies using membranes from control and retinoic acid-treated HL-60 cells showed that the Bmax of InsP3 receptor increased progressively from 0.24 to 0.69 pmol/mg protein during 5 days retinoic acid treatment with no change in KD (19 nM). During this period, maximal InsP3-stimulated Ca2+ mobilization increased 2-3-fold. InsP3 receptor mRNA was present at low levels in HL-60 cells but was increased significantly after treatment with retinoic acid, reaching maximal levels of approximately 4-fold greater than untreated cells after 4 days treatment with retinoic acid. Nuclear run-on assays indicated that the elevated steady state level of InsP3 receptor mRNA in retinoic acid-treated HL-60 cells was primarily the result of enhanced transcription of the InsP3 receptor gene. Furthermore, the transcriptional enhancing effect of retinoic acid was seen in the presence of cycloheximide, suggesting that the InsP3 receptor gene is directly regulated by retinoic acid. The studies also demonstrate that the InsP3 receptor mRNA is rapidly degraded in HL-60 cells by a mechanism that also requires protein synthesis.


Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.


Signal transduction by the cytoplasmic domains of Fc epsilon RI-gamma and TCR-zeta in rat basophilic leukemia cells.

The gamma subunit of the high affinity IgE receptor, Fc epsilon RI, is a member of a family of proteins which form disulfide-linked dimers. This family also includes the zeta- and eta-chains of the T cell receptor. Engagement of Fc epsilon RI activates src-related protein tyrosine kinases in basophils and mast cells. However, the role of individual subunits of Fc epsilon RI in this activation is still not known. In an effort to determine the function of Fc epsilon RI-gamma, we used chimeric proteins containing the extracellular and transmembrane domains of the alpha chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domains of either T cell receptor-zeta or Fc epsilon RI-gamma. We show that while cross-linking of the Tac chimeras in the rat basophilic leukemia cell line RBL-2H3 resulted in the tyrosine phosphorylation of a subset of proteins and a portion of the degranulation normally observed after Fc epsilon RI-mediated stimulation, no detectable activation of p56lyn or pp60c-src was observed. In contrast, an apparent transient deactivation of these two kinases was observed after Tac chimera cross-linking. These observations suggest that Fc epsilon RI-gamma is responsible for some, but not all, of the signaling that occurs after engagement of its receptor, and that other receptor subunits may also play important roles in this signaling process.


Comparison of src-family cDNAs reveals distinct mechanisms underlying focus formation in transfected fibroblasts.

Despite the intensive study of both cellular transformation and src-family protein-tyrosine kinases, there have been no direct comparisons of transforming potency for normal members of this gene-family. In this study, the focus-forming activity of normal c-src, fyn, and lck cDNAs were compared in NIH 3T3 cell transfection assays. Focus formation was studied quantitatively, and individual foci were analyzed for phosphotyrosine content and expression of appropriate translational products. Each foci arising from c-src transfectants had a marked increase in phosphotyrosine content, and the majority of these foci expressed a c-src protein with an aberrant carboxyl terminus. Foci derived from lck transfectants also had a marked increase in phosphotyrosine content, and some foci expressed a lck protein with an aberrant carboxyl terminus. In contrast, foci from fyn-transfected cells were not distinguished from G418-selected mass cultures in terms of total phosphotyrosine content or expression of p59fyn. These studies support the previously published concept that overexpression of the normal fyn protein contributes to focus formation in transfected NIH 3T3 cells but suggest that the focus-forming activity observed after c-src or lck transfections is frequently attributable to mutational events. Because lck mutations have not been previously described in transformed foci, we characterized the lck transcript expressed in two foci and identified a novel point mutation that encodes a lck protein with increased in vivo kinase and focus-forming activity.


Calcitonin gene-related peptide inhibits interleukin 2 production by murine T lymphocytes.

Evidence from the literature suggests that the nervous and the immune systems closely interact via neuromediators, which affect the immune system, and cytokines, which control nerve cell growth and activity. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has been identified in numerous tissues including immune organs and inhibits the proliferation of spleen cells. We investigated whether CGRP altered the function of T lymphocytes. We present evidence that CGRP induces a dose-dependent cAMP accumulation in interleukin 2-producing TH1 cells and inhibits their production of interleukin 2. These effects are prevented by CGRP8-37, a CGRP antagonist that is missing the first 7 amino acids. This CGRP-mediated inhibition of interleukin 2 production is accompanied by a decrease in interleukin 2 mRNA accumulation. CGRP also inhibits the accumulation of mRNA coding for tumor necrosis factor-alpha and -beta and interferon-gamma. Thus, we have identified one mechanism by which CGRP inhibits the proliferation of spleen cells.


A membrane-bound monoheme cytochrome c551 of a novel type is the immediate electron donor to P840 of the Chlorobium vibrioforme photosynthetic reaction center complex.

A photosynthetic reaction center complex has been isolated from the green sulfur bacterium Chlorobium vibrioforme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 18, 15, 9, and 6 kDa. Only the 18-kDa polypeptide is stained with 3,3',5,5'-tetramethylbenzidine, a heme-specific reagent. Oxidized minus reduced difference spectra show the presence of approximately one heme/P840 and the presence of a cytochrome c551. Flash photolysis of P840 was followed by rereduction of P840+ and oxidation of cytochrome c551, both with a biphasic kinetic with t1/2 values of 7 and 50 microseconds. Using oligonucleotide probes derived from an N-terminal amino acid sequence of the 18-kDa polypeptide, a genomic clone was isolated. The sequence of the gene, which we designate cycA, predicts a single heme binding site (Cys-Asn-Lys-Cys-His). The 621-base pair open reading frame encodes an apoprotein of 22,858 Da with three predicted membrane-spanning alpha-helices. No extensive sequence similarity is found to other cytochromes. Northern blotting indicates that the cycA gene is transcribed as a monocistronic mRNA. Southern blotting shows the presence of only one cycA gene in the C. vibrioforme and Chlorobium tepidum genomes. The unique membrane-bound monoheme cytochrome c551 of C. vibriforme is assigned to a new class of c-type cytochromes. The implications for the current view of evolution of photosynthetic reaction center complexes are discussed.


Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity.

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.


Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.

In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.


Tissue distribution and cellular localization of hsp56, an FK506-binding protein. Characterization using a highly specific polyclonal antibody.

Heat shock protein 56 (hsp56) has been shown to be involved in two cellular pathways, as an immunophilin for FK506 and as a component of steroid receptor complexes. To help define its role in these cellular pathways, we have developed UPJ56, a polyclonal antibody raised against hsp56 purified from Jurkat cells. In Western blot experiments, hsp56 was highly expressed in rat thymus, liver, and spleen, with low levels in lung and muscle. In immunofluorescence experiments using untreated LLC-PK1 cells, fibrillar staining was seen in the cytoplasm, suggesting a cytoskeletal localization of hsp56. The nuclei were brightly stained, except for the nucleoli. Confocal microscopy demonstrated that the staining was present in all planes of the nucleus. These results suggest that hsp56 is expressed in tissues enriched in steroid receptors and is highly expressed in tissues involved in T cell function. Furthermore, the localization of hsp56 with the cytoskeleton and throughout the nucleus is consistent with its association with steroid receptor complexes.


Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochromocytoma cells.

Serum contains a factor that co-purifies with albumin and causes neurite retraction in PC12 cells, inhibits the proliferation of tumor cells in vitro, and activates the phosphatidylinositol/Ca2+ second messenger system in Xenopus oocytes and other cells. The activity of serum albumin depends on several lysophospholipids bound to albumin. Thin layer chromatographic analysis of the lipids extracted by methanol from serum albumin revealed over a dozen components, several of which evoked oscillatory currents in oocytes. In contrast to serum albumin, most of these lipids were absent in plasma, which lacks the biological activity. The most abundant naturally occurring active component was identified as stearoyl-lysophosphatidic acid. Synthetically prepared lysophosphatidates reproduced the biological activities of the natural serum factor. Adding synthetic lysophosphatidates to inactive fatty acid-free albumin restored activity to the albumin, making the active factor nondialyzable against aqueous solvents and protecting against digestion by various lipases. Since the biologically active lysophosphatidates were produced during blood clotting, in the presence of platelets, and lysophosphatidates have been shown previously to activate platelets, we propose that lysophosphatidates may play an important role in linking platelet activation to receptor-mediated tissue regeneration.


Ruthenium red inhibits the binding of calcium to calmodulin required for enzyme activation.

The Ca(2+)-calmodulin (CaM)-dependent activation of myosin light chain kinase is inhibited by ruthenium red competitively with respect to Ca2+, with a Ki value of 8.6 microM. The binding of Ca2+ to CaM is inhibited by micromolar concentrations of ruthenium red. In the absence of Ca2+, CaM has two binding sites for ruthenium red with the dissociation constants of 0.36 and 8.7 microM, respectively. Ca2+ antagonizes the binding of ruthenium red to the low-affinity site on CaM. Binding of ruthenium red to the high-affinity site is not affected by Ca2+. The low- and high-affinity sites for ruthenium red are shown to be located in the NH2-terminal half and the COOH-terminal half of CaM, respectively. Lower concentrations of ruthenium red are needed for enzyme inactivation than for the dissociation of enzyme-CaM-Sepharose complex, suggesting these events have different Ca2+ requirements. Moreover, ruthenium red inhibits Ca(2+)-induced contraction of depolarized vascular smooth muscle in a competitive manner with respect to Ca2+. These results suggest that ruthenium red may be a new type of CaM antagonist that inhibits the binding of Ca2+ to CaM and thereby inhibits Ca(2+)-CaM-dependent enzymes and smooth muscle contraction competitively with respect to Ca2+.


Activation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.

The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7 mM) and was blunted in the presence of the Al3+ chelator desferal mesylate suggesting that it might be due to the aluminofluoride ion, AlF-4. NaF/AlCl3 treatment did not increase acinar intracellular cAMP levels but did result in an increase in intracellular calcium concentration (from 87 +/- 5 to 181 +/- 2 nM) and in acinar cell shrinkage (12 +/- 1%). But the stimulation of the Na(+)-K(+)-2Cl- cotransporter by NaF/AlCl3 persisted in acini which had been depleted of their intracellular Ca2+ stores. In these acini no effect of NaF/AlCl3 on intracellular calcium or cell volume was observed, indicating that stimulation of the cotransporter was not secondary to either of these phenomena. The effect of NaF/AlCl3 on the cotransporter was blocked by the protein kinase inhibitor K252a indicating the involvement of a protein phosphorylation event. This result is consistent with either NaF/AlCl3-dependent protein kinase activation or phosphatase inhibition. The stimulation of the cotransporter by NaF/AlCl3 was mimicked by the protein phosphatase inhibitor calyculin A; however, this effect was not blocked by K252a suggesting that a different protein kinase from that associated with NaF/AlCl3 may be involved. The data indicate that the Na(+)-K(+)-2Cl- cotransporter in this tissue is under tight regulatory control, in all likelihood via multiple protein kinase/phosphatase systems. The physiological roles of these regulatory events in modulating acinar fluid secretion driven by the Na(+)-K(+)-2Cl- cotransporter remain to be elucidated.


Characterization of high molecular weight FK-506 binding activities reveals a novel FK-506-binding protein as well as a protein complex.

The immunoregulant FK-506 potently inhibits particular calcium-associated signal transduction events that occur early during T-lymphocyte activation and during IgE receptor-mediated exocytosis in mast cells. FK-506 binds to a growing family of receptors termed FK-506-binding proteins (FKBPs), the most abundant being a 12-kDa cytosolic receptor, FKBP12. To date, there is no formal evidence proving that FKBP12 is the sole receptor mediating the immunosuppressive effects or toxic side effects of FK-506. Using gel filtration chromatography as an assay for novel FK-506-binding proteins, we identified FK-506 binding activities in extracts prepared from calf brain and from JURKAT cells. Both of these new activities comigrated with apparent molecular masses of 110 kDa. However, further characterization of both binding activities revealed that the two are not identical. The 110-kDa activity observed in brain extracts appears to be the FKBP12.FK-506.calcineurin (CaN) complex previously reported (Liu, J., Farmer, J., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807-815) while the 110 kDa activity observed in JURKAT cells is a novel FK-506-binding protein. Our characterization of the FKBP12.FK-506.CaN complex reveals a dependence upon calmodulin (CaM) for formation of the complex and demonstrates that the peptidyl-prolyl cis-trans isomerase (PPIase) activity of FKBP12 is not required for binding of FKBP12.FK-506 to CaN or for inhibition of CaN phosphatase activity. The novel FK-506-binding protein in JURKAT cells has been purified to homogeneity, migrates with an apparent mass of 51 kDa on denaturing gels, and has been termed FKBP51. Like FKBP12, FKBP51 has PPIase activity, but, unlike FKBP12.FK-506, FKBP51.FK-506 does not complex with or inhibit the phosphatase activity of, CaN. These results indicate that complex formation with CaN may not be a general property of the FKBPs. Peptide sequencing reveals that FKBP51 may be similar, if not identical, to hsp56, a component of non-transformed steroid receptors.


Characterization of a Xenopus laevis ribonucleoprotein endoribonuclease. Isolation of the RNA component and its expression during development.

In order to facilitate studies of the assembly and transport of the site-specific RNase mitochondrial RNA processing (MRP) ribonucleoprotein, we have characterized it from Xenopus laevis cells. X. laevis RNase MRP displayed a similar spectrum of cleavage activity to that produced by previously isolated mammalian nuclear enzymes. A 277-nucleotide RNA component of the ribonucleoprotein was identified; the gene for the RNA was isolated, sequenced, and found to be 66 and 63% similar to mouse and human RNase MRP RNAs, respectively. Despite the evolutionary distance from its mammalian counterparts, X. laevis RNase MRP RNA contains five regions of homology to the mammalian RNase MRP RNA. Four of these regions correspond to those previously identified as conserved between RNase MRP and RNase P RNAs; the fifth encompasses nucleotides recently discovered to be sufficient for autoantigen binding. The expression and assembly of Xenopus RNase MRP RNA were examined in frog oocytes and developing embryos. RNase MRP RNA was expressed throughout oogenesis; it started to accumulate at stage I and reached a maximum in stage IV. During embryogenesis RNase MRP RNA expression began to elevate at approximately stage 22 and continued to rise through the swimming tadpole stage. When injected into the nucleus of mature oocytes, the X. laevis RNase MRP RNA gene was expressed accurately, and transcripts were packaged into immunoprecipitable particles.


Identification of a tissue-specific regulatory element within the murine CD14 gene.

We previously isolated and sequenced the 5'-flanking region of the mouse CD14 (mCD14) gene (Matsuura, K., Setoguchi, M., Nasu, N., Higuchi, Y., Yoshida, S., Akizuki, S., and Yamamoto, S. (1989) Nucleic Acids Res. 17, 2132). To define the regulatory elements that control expression of the mCD14 gene, we analyzed the structure of the 5' end of the gene, including a region further upstream of that determined previously. Sequentially 5'-deleted, chimeric, and point mutated clones were tested for the ability to stimulate chloramphenicol acetyltransferase. An 8-base pair sequence, TGATTCAC, at position -255, which resembled the consensus sequence of the 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE), enhanced the expression of the chloramphenicol acetyltransferase gene in macrophage (aHINS-B3) and non-macrophage (glioblastoma G203 and myeloma NS1) cells. The enhancing ability of the TRE-like sequence (TLS), however, was markedly reduced in G203 cells but not in aHINS-B3 cells when the TLS was followed by the sequence immediately downstream. The TLS and sequence immediately downstream were capable of binding nuclear proteins which were unique to aHINS-B3 cells and macrophages, suggesting that these unique protein regulate the specific expression of the mCD14 gene. Binding of AP-1 to the TLS was also found in aHINS-B3 and G203 cells. Although it is uncertain whether AP-1 is involved in expression of the mCD14 gene, the effect of AP-1 in non-macrophage cells was inhibited by a nuclear protein which binds to the sequence immediately downstream of the TLS.


Angiogenesis in healing autogenous flexor-tendon grafts.

On the basis of recent evidence that flexor tendon grafts may heal without the ingrowth of vascular adhesions, eighteen autogenous donor tendons of intrasynovial and extrasynovial origin were transferred to the synovial sheaths in the forepaws of nine dogs, and controlled passive mobilization was instituted early in the postoperative period. The angiogenic responses of the tendon grafts were determined with perfusion studies with India ink followed by cleaing of the tissues with the Spalteholz technique at two, four, and six weeks. A consistent pattern of neovascularization was noted in the donor tendons of extrasynovial origin. Vascular adhesions arising from the flexor digitorum superficialis and the tendon sheath enveloped the tendon grafts by two weeks. By six weeks, the vascularity of the tendon grafts of extrasynovial origin appeared completely integrated with that of the surrounding tissues. Examination of cross sections revealed that the segments of tendon had been completely vascularized by obliquely oriented intratendinous vessels. In contrast, the flexor tendon grafts of intrasynovial origin healed without ingrowth of vascular adhesions. Primary intrinsic neovascularization took place from the proximal and, to a lesser extent, distal sites of the sutures. Examination of cross sections revealed vessels extending through the surface layer of the tendon graft, with small vessels penetrating the interior of the tendons at regular intervals.


The EGF receptor is an actin-binding protein.

In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996.


The roles of K5 and K14 head, tail, and R/K L L E G E domains in keratin filament assembly in vitro.

Type I and type II keratins form obligatory heterodimers, which self-assemble into 10-nm intermediate filaments (IFs). Like all IF proteins, they have a central alpha-helical rod domain, flanked by nonhelical head and tail domains. The IF rod is more highly conserved than head and tail, and within the rod, the carboxy R/K L L E G E sequence is more highly conserved than most other regions. Mutagenesis studies have shed some light on the roles of the head, tail, and R/K L L E G E sequence in 10-nm filament structure. However, interpretations have often been complicated in part because many of these studies have focused on transfected cells, where filament structure cannot be evaluated. Of the few in vitro assembly studies thus far conducted, comparison of keratin mutants with other IF mutants have often been difficult, due to the obligatory heteropolymeric nature of keratin IFs. In this report, we describe in vitro filament assembly studies on headless, tailless, headless/tailless, and R/K L L E G E truncated mutants of keratin 5 and its partner keratin 14. Using varying conditions of ionic strength and pH, we examine effects of analogous K5 and K14 mutations on the stability of 10-nm filament structure. Using EM, we examine effects of mutations on the ability of subunits/protofibrils to (a) elongate and (b) laterally associate. Our results demonstrate that (a) tails of K5 and K14 are required for filament stabilization; (b) the head of K5, but not of K14, is required for filament elongation and lateral alignments; and (c) the R/K L L E G E domains are required for lateral alignments, but not for filament elongation.


Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes.

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.


Introns excised from the Delta primary transcript are localized near sites of Delta transcription.

Introns excised from the primary transcript of Delta (Dl), a Drosophila neurogenic gene, accumulate to unusually high levels in embryos. High resolution in situ hybridization reveals a striking localization of the excised introns to two foci per embryonic nucleus. The number of foci can be altered by varying the number of Dl genes present in the embryonic nucleus, suggesting that the excised introns are localized near sites of Dl transcription. This conclusion is supported by the observation that larval and imaginal disc nuclei containing two copies of Dl exhibit only one focus of intron accumulation, as expected for nuclei in which homologous chromosomes are paired. Interestingly, the excised introns do not appear to diffuse away from the foci until late prophase, at which time the foci disperse into numerous small dots of hybridization. These results suggest that the excised Dl introns may be associated with a structural element within the nucleus that is dissociated during cell division.


Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.


Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression.

The DNA sequence between position +36 and -1907 of the murine myelin basic protein gene contains the enhancer and promoter elements necessary for abundant and cell specific expression in transgenic mice. Surprisingly, the pattern of expression promoted by this DNA fragment is a subset of that exhibited by the endogenous myelin basic protein (MBP) gene. Fusion genes prepared with this promoter/enhancer and a Lac Z reporter gene are expressed only in oligodendrocytes and not in Schwann cells, whereas the endogenous MBP gene is expressed in both cell types. The level of transgene expression measured by nuclear run-on experiments is very substantial and rivals that of the endogenous MBP gene. Furthermore, this 1.9-kb DNA fragment directs transcription on the same (or very similar) developmental schedule as the endogenous gene. These results indicate that the MBP promoter/enhancer sequences are at least tripartite: a core promoter, the oligodendrocyte enhancer elements, and a third component that either expands the specificity of the oligodendrocyte enhancer to include Schwann cells or acts independently to specifically stimulate transcription in Schwann cells.


Intracellular localization of the P21rho proteins.

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.


Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.


Hyaluronan-binding protein in endothelial cell morphogenesis.

Previous studies from several laboratories have provided evidence that interaction of hyaluronan (HA) with the surface of endothelial cells may be involved in endothelial cell behavior. We have recently characterized a mAb, mAb IVd4, that recognizes and neutralizes HA-binding protein (HABP) from a wide variety of cell types from several different species (Banerjee, S. D., and B. P. Toole. 1991. Dev. Biol. 146:186-197). In this study we have found that mAb IVd4 inhibits migration of endothelial cells from a confluent monolayer after "wounding" of the monolayer. HA hexasaccharide, a fragment of HA with the same disaccharide composition as polymeric HA, also inhibits migration. In addition, both reagents inhibit morphogenesis of capillary-like tubules formed in gels consisting of type I collagen and basement membrane components. Immunocytology revealed that the antigen recognized by mAb IVd4 becomes localized to the cell membrane of migrating cells, including many of their lamellipodia. Treatment with high concentrations of HA hexamer causes loss of immunoreactivity from these structures. We conclude that HABP recognized by mAb IVd4 is involved in endothelial cell migration and tubule formation.


Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin.

The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGF-like repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.