Iron uptake and molecular recognition in Pseudomonas putida: receptor mapping with ferrichrome and its biomimetic analogs. The presence of an Fe(3+)-ferrichrome uptake system in fluorescent Pseudomonas spp. was demonstrated, and its structural requirements were mapped in Pseudomonas putida with the help of biomimetic ferrichrome analogs. Growth tests, 55Fe3+ uptake, and competition experiments demonstrated that the synthetic L-alanine derivative B5 inhibits the action of ferrichrome but does not facilitate Fe3+ transport, while the enantiomeric D-Ala derivative B6 fails to compete with ferrichrome. Contraction of the molecule's envelope by replacing L-Ala by glycine provided a synthetic carrier, B9, which fully simulates ferrichrome as a growth promoter. Sodium azide inhibited 55Fe3+ uptake of the Gly derivative B9, suggesting an active transport process. These data demonstrate the chiral discrimination of the ferrichrome receptor and its sensitivity to subtle structural changes. They further confirm that receptor binding is a necessary but not sufficient condition for Fe3+ uptake to occur and suggest that binding to the receptor and transport proteins might rely on different recognition patterns.


Characterization of the Streptococcus adjacens group antigen structure. Serological classification of bacteria requires the presence of an antigen unique to the organism of interest. Streptococci are serologically differentiated by group antigens, many of which are carbohydrates, although some are amphiphiles. This report describes the chemical characterization of the Streptococcus adjacens group antigen structure. Previous studies demonstrated that the amphiphile contained phosphorus, ribitol, galactose, galactosamine, alanine, and fatty acids. Phosphodiester bonds present in the purified group antigen were identified as part of a poly(ribitol phosphate), since ribitol phosphate was the only organic phosphate detected after acid hydrolysis. Hydrofluoric acid cleavage of the phosphodiester bonds generated oligosaccharide repeating units. Gas chromatography-mass spectrometric analysis of the methylated, acetylated oligosaccharide suggested that the repeating unit is a trisaccharide of Galp beta 1-3Galp beta 1-4GalNac with N-acetylgalactosamine attached in beta-linkage to either the number two or the number four carbon of ribitol. The lipid- and carbohydrate-substituted poly(ribitol phosphate) of the S. adjacens group antigen therefore is a unique amphiphile structure, differing in its repeating-unit structure from the polyglycerophosphate structure of the more common gram-positive amphiphile lipoteichoic acid.


Construction and application of plasmid- and transposon-based promoter-probe vectors for Streptomyces spp. that employ a Vibrio harveyi luciferase reporter cassette. Several versatile promoter-probe vectors have been constructed for Streptomyces strains which utilize the production of blue-green light as a measure of transcription activity. Three plasmid vectors (two high and one low copy number) and two transposons are described. The multicopy plasmids pRS1106 and pRS1108 contain a transcription terminator and multiple-cloning polylinker upstream of promoterless luciferase (lux) and neomycin resistance reporter genes. Plasmid pHI90 is similar in structure to the pRS vectors except that its single copy number is an advantage for regulation studies or situations in which overexpression is otherwise toxic to the cell. The two transposons carry a promoterless lux cassette cloned such that transposition into a target DNA and fusion to the target's transcription unit occur simultaneously. Tn5351 was created by inserting the luciferase genes near the right end of the viomycin resistance transposon Tn4563. Tn5353 carries the luciferase genes near the right end of a neomycin resistance transposon derived from Tn4556. The size of Tn5353 was minimized by deleting nonessential transposon sequences, making this element small enough to be cloned into phi C31 bacteriophages for efficient transposon delivery to target cells of Streptomyces strains. The two Tnlux transposons have been used to generate Streptomyces coelicolor morphological mutants and to monitor transcription from chromosomal promoters during development.


Roles of the orlA, tsE, and bimG genes of Aspergillus nidulans in chitin synthesis. Strains of Aspergillus nidulans carrying the orlA1 or tse6 allele are deficient in cell wall chitin and undergo lysis at restrictive temperatures. The strains are remediable by osmotic stabilizers or by the presence of N-acetylglucosamine (GlcNAc) in the medium. The remediation by GlcNAc suggests that the lesion(s) in chitin synthesis resides in the amino sugar biosynthetic pathway prior to the synthesis of N-acetylglucosamine-6-phosphate. orlA1 strains grown at permissive temperature exhibit an abnormally low specific activity for L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), the first enzyme unique to amino sugar synthesis. In addition, the enzyme produced is temperature sensitive in vitro. tsE6 strains grown at permissive temperature show virtually no amidotransferase activity. This finding is consistent with an extremely labile enzyme which is destroyed by cell breakage and extract preparation. The enzyme must be active in vivo at permissive temperatures since GlcNAc is not required for growth. Thus, two structural genes (orlA and tsE) are necessary for the amidotransferase activity. bimG11 strains are temperature sensitive for a type 1 protein phosphatase involved in cell cycle regulation and arrest in mitosis. Like orlA1 and tsE6 strains, conidia from bimG11 strains swell excessively when germinated and lyse; the germlings produced are deficient in chitin content. The amidotransferase from wild-type and mutant strains is sensitive to feedback inhibition by uridine diphosphate-N-acetylglucosamine. The sensitivity of the amidotransferase from bimG11 strains is dependent on growth temperature, while that from wild-type strains is independent of temperature. The enzyme can be desensitized in vitro under conditions consistent with a protein phosphatase reaction. It is proposed that amino sugar (and chitin biosynthesis) is partially regulated by phosphorylation-dephosphorylation of the amidotransferase or a protein regulator of the enzyme.


Involvement of Fis protein in replication of the Escherichia coli chromosome. We report evidence indicating that Fis protein plays a role in initiation of replication at oriC in vivo. At high temperatures, fis null mutants form filamentous cells, show aberrant nucleoid segregation, and are unable to form single colonies. DNA synthesis is inhibited in these fis mutant strains following upshift to 44 degrees C. The pattern of DNA synthesis inhibition upon temperature upshift and the requirement for RNA synthesis, but not protein synthesis, for resumed DNA synthesis upon downshift to 32 degrees C indicate that synthesis is affected in the initiation phase. fis mutations act synergistically with gyrB alleles known to affect initiation. oriC-dependent plasmids are poorly established and maintained in fis mutant strains. Finally, purified Fis protein interacts in vitro with sites in oriC. These interactions could be involved in mediating the effect of Fis on DNA synthesis in vivo.


The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids. The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.


Identification of the gene (aroK) encoding shikimic acid kinase I of Escherichia coli. DNA sequence analysis has revealed that an unidentified open reading frame (ufr1) is present immediately preceding the aroB gene of Escherichia coli. The predicted protein product of urf1 contains a consensus ATP-binding-site sequence and shows 34% amino acid homology to shikimate kinase II in a 97-amino-acid region. Inactivation of urf1 by insertion of an antibiotic resistance gene had a polar effect on aroB, indicating that these two genes constitute a transcriptional unit. The auxotrophic requirements of a strain mutant for both urf1 and aroL (encoding shikimate kinase II) are consistent with shikimate kinase deficiency. We propose that urf1 encodes shikimate kinase I and that it be designated aroK.


CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase, which is involved in the synthesis of the major wall teichoic acid in Bacillus subtilis 168, is encoded by tagF (rodC). Assays of CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (CGPTase) (EC 2.7.8.12) in membranes isolated from Bacillus subtilis 168 wild type and 11 strains bearing conditional lethal thermosensitive mutations in tagB, tagD, or tagF revealed that CGPTase deficiency was associated only with mutant tagF alleles. In vitro, thermosensitivity of CGPTase strongly suggests that the structural gene for this enzyme is tagF. We discuss apparent discrepancies between biochemical evidence favoring a membrane location for TagF and a previous report that suggested a cytoplasmic location based on sequence analysis.


Heterogeneity of c-myc expression in histologically similar infiltrating ductal carcinomas of the breast. Anti-c-myc monoclonal antibody was used to evaluate the distribution of the c-myc protein in normal and tumor cells of infiltrating ductal carcinoma. A semi-quantitative method for reporting immunohistochemical assay results (c-myc score) that enables correlations on a more quantitative basis was used in this study. HL-60 cells demonstrated the strongest nuclear staining when fixed in cold acetone (4 degrees C) for 10 min. All 24 specimens of infiltrating ductal carcinomas of the breast and 7 of 11 samples of normal breast tissues studied revealed the presence of c-myc protein. The level of expression in normal breast tissue was much lower than that in breast cancer. Heterogeneity in expression was found within individual tumors and there were substantial differences in the level of expression among different tumors. The subcellular site of staining was predominantly nuclear, occasionally nuclear and cytoplasmic in the same cell, and rarely only cytoplasmic. All four patients with tumor cells located in close proximity to the ductal basement membrane and over-expressing c-myc protein had positive lymph nodes, suggesting that these tumors are more likely to metastasize.


Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity. Conventionally in vitro cytotoxicity assays are performed as single-end-point determinations. To compensate for the diversity of growth rates among different cell lines in this report we describe a computerized kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. As a prerequisite four human breast cancer cell lines (MDA-MB-231, MCF-7, T-47-D and ZR-75-1) were characterized with regard to oestrogen and progesterone receptor content, modal chromosome number and proliferation kinetics depending on the number of passages in culture. With prolonged time in culture for ZR-75-1 exposed to various concentrations of cisplatinum a dose-related increase in drug effect was observed. Owing to a correction of the T/C values for the initial cell mass (at the time when drug is added) a sharp distinction between cytostatic and cytocidal drug effects becomes obvious in plots of corrected T/C values versus time of incubation. The influence of the untreated control on the corrected T/C values and possible time courses of theoretical inhibition profiles (reflecting cytostatic, transient cytotoxic or cytocidal drug effects as well as development of resistance) and their relationship to the corresponding growth curves of drug-treated cells are discussed. Chemosensitivity assays with diethylstilbestrol dipropionate, tamoxifen, melphalan, cisplatinum, vinblastine, Adriamycin and 5-fluorouracil prove the theoretical considerations to be true for MDA-MB-231, MCF-7, T-47-D and ZR-75-1 human breast cancer cell lines in practice.


Formation of benzo[a]pyrene-DNA adducts in blood monocytes from lung cancer patients with a familial history of lung cancer. The in vitro formation of benzo[a]pyrene-DNA adducts was determined in peripheral blood monocytes of 22 lung cancer patients with at least one first-degree relative with lung cancer and compared to results obtained in 30 healthy controls. In patients, the mean (SEM) adduct formation was 2.8 (0.3) fmol/micrograms DNA as compared to 2.1 (0.1) fmol/micrograms in controls (p less than 0.05), and it was independent of age and smoking habits. These findings support the hypothesis that carcinogen-DNA adduct formation may be one factor of a constitutionally enhanced lung cancer risk.


Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. Endothelial cell derived relaxing factor (EDRF) mediated relaxation of blood vessels is impaired in vessels exposed to lipoproteins in vitro and in arteries of hyperlipidemic humans and animals. To investigate the mechanism by which lipoproteins impair the effects of EDRF, which is likely nitric oxide (NO) or a related molecule, we have bioassayed EDRF/NO activity by measuring its ability to increase cGMP accumulation in rat fetal lung cultured fibroblasts (RFL-6 cells). Low density lipoprotein modified by oxidation (ox-LDL) induced a concentration-dependent inhibition of EDRF activity that had been released from bovine aortic endothelial cells (BAEC) stimulated with bradykinin or the calcium ionophore A23187. In addition, lipoproteins directly impaired authentic NO-induced stimulation of cGMP accumulation in the detector cells; stimulation by sodium nitroprusside was unaffected. Ox-LDL or oxidized HDL3 were highly potent in blocking NO-stimulated cGMP accumulation with EC50's of approximately 1 microgram/ml. Lipid extracted from ox-LDL blocked NO-stimulated cGMP accumulation to about the same extent as intact ox-LDL, while the protein component of ox-LDL did not inhibit the cGMP response. These results suggest that the lipid component of oxidized lipoproteins inactivate EDRF after its release from endothelial cells.


P2-purinergic receptor agonists inhibit the growth of androgen-independent prostate carcinoma cells. To develop a new approach to the treatment of advanced, hormone-refractory prostate cancer, the signal transductions regulating the growth of human androgen-independent prostate carcinoma cell lines were studied. Agonist-stimulated Ca2+ mobilization, a critical regulatory event in other secretory cell types, was studied as a means of identifying previously undescribed plasma membrane receptors that may transduce a growth inhibitory signal. In all of the cell lines tested, P2-purinergic receptor agonists, including ATP and certain hydrolysis-resistant adenine nucleotides, induced a rapid, transient increase in cytoplasmic free Ca2+ that was detectable at 50 to 100 nM ATP, was maximal at 100 microM ATP, and was inhibited approximately 50% by chelation of extracellular Ca2+. Within 8 s after addition, ATP stimulated accumulation of the polyphosphatidylinositol products inositol (1, 4, 5) trisphosphate, inositol (1, 3, 4) trisphosphate, and inositol tetrakisphosphate. In addition to stimulating phosphatidylinositol turnover and Ca2+ mobilization, ATP and hydrolysis-resistant ATP analogues induced greater than 90% inhibition of the growth of all lines tested. These data demonstrate that human androgen-independent prostate carcinoma cells express functional P2-purinergic receptors linked to phospholipase C, and that agonists of this receptor are markedly growth inhibitory, suggesting a novel therapeutic approach to this common adult neoplasm.


Double pigtail cystogastric stent in the management of pancreatic pseudocyst. We describe placement of a double pigtail cystogastric stent in 5 patients with pancreatic pseudocyst using real-time ultrasonography and fluoroscopy as guidance techniques. This was done as a two-step procedure. First, a transgastric drainage catheter was placed for a period of six days. All the patients continued to have high drainage output and the cyst size decreased, as seen sonographically. Simultaneous barium meal and sinogram were then done to measure the distance between the posterior gastric wall and the cyst. A polyurethane double pigtail stent of appropriate size was then introduced to achieve cystogastric drainage. The cyst resolved in all the patients by four weeks to six weeks, when the stent was removed endoscopically. There was no recurrence of pseudocyst during follow-up for 6 months to 13 months. None of the patients suffered any complication. Cystogastric drainage using a double pigtail stent is an easy and safe alternative to surgical cystogastrostomy.


In vivo validation of image-directed Doppler measurement of superior mesenteric artery blood flow. Image-directed Doppler measurement of superior mesenteric artery blood flow in volunteers was validated in two artificial models with either a venous or an arterial flow profile. In the "venous" model, the Doppler device overestimated the real flow velocity by 86 +/- 7%, and in the "arterial" model by 24 +/- 9%. The areas under the time-frequency curves (AUCs) from the arterial model were measured by planimetry. A correction factor of 1.47 between flow velocities calculated after planimetry and real flow was established. Correction for this factor resulted in a flow velocity of 19.5 +/- 4.7 cm/s and a blood flow rate of 377 +/- 166 ml/min in the volunteers.


Ultrasound guidance for biopsy of omental abnormalities. We review our two-year experience with ultrasound-guided fine needle aspiration biopsy of omental pathology. Eleven patients were referred for biopsy of omental abnormalities. Biopsy was performed without complication in all cases and the aspirates were positive for malignancy in 9. The discharge diagnosis was ovarian carcinoma in 6 patients, adenocarcinoma of unknown origin in 1, lymphoma in 1, and carcinoma of the colon in 1. The technique requires the use of a linear array transducer for biopsy guidance, and is safe, rapid, and easy to perform.


Echocardiographic evaluation of the valves and roots of the pulmonary artery and aorta in the developing fetus. Because of the fluid-filled lungs in the fetus, the ultrasound beam can penetrate to the root of the pulmonary artery perpendicularly. Using M-mode echocardiography, the following dimensions were measured: the pulmonary and aortic valve excursion in 19 fetuses, and the pulmonary and aortic root internal diameter in 70 fetuses, between 18 and 42 weeks, menstrual age. The measured dimensions were then correlated with the biparietal diameter. The correlation coefficients for the measurements ranged from 0.873 to 0.892. Regression analysis showed that the best fit of the data was a linear model from which the 5% and 95% confidence limits were derived for individual predictions of the arterial dimensions. The ratios of the pulmonary/aortic valve excursion and the pulmonary/aortic internal diameter were approximately 1:1. These results are of potential value in the prenatal detection of great arterial valve or root diseases.


Clinical versus ultrasound examination of the thyroid gland in common clinical practice. In a prospective series of 72 patients, clinical and ultrasonographic examination of the thyroid gland were compared in detail. Normal-sized lobes were differentiated from enlarged ones both by inspection and by palpation. When lobar size was assessed by palpation, the estimate was most clearly influenced by increase in width. The correlation between two examiners in lobe size assessment was significant. In the classification of thyroid disease as diffuse, solitary, or multinodular, clinical examination and ultrasonography correlated significantly. However, only one third of the clinically solitary nodules proved to be solitary by ultrasound examination. Of 77 separate nodules, 43 escaped detection on clinical examination. Of these 43, 14 nodules exceeded 2 cm in diameter. It is concluded that the use of ultrasonography frequently alters the primary evaluation of thyroid nodularity based on palpation.


Ophthalmic and cerebral blood flow velocities in preterm infants: influence of ambient lighting conditions. Doppler ultrasound was used to study ophthalmic and middle cerebral artery flow velocities at different ambient lighting conditions in 28 preterm infants in the first week of life. We found significant increases of ophthalmic and middle cerebral artery blood flow velocities when ambient light was increased from moderate to intense. Flow velocities in the ophthalmic artery increased significantly more than in the middle cerebral artery. Doppler ultrasound studies of ophthalmic blood flow velocity may assist in answering the intriguing question whether light-induced changes of ocular perfusion play a role in the development of retinopathy of prematurity.


Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family. The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.


Studies of CD4- CD8- alpha beta bone marrow T cells with suppressor activity. The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta receptors as compared to typical spleen T cells. The marrow T cells appear to develop directly from marrow precursors without rearranged beta chain genes during a 48 hour in vitro culture.


Hepatocellular carcinoma presenting only as portal venous tumor thrombosis: CT demonstration. We describe two cases of hepatocellular carcinoma initially presenting with portal vein tumor thrombi but with no demonstrable tumor in the liver parenchyma outside the portal vein. Both patients had cirrhosis of the liver and had tumor thrombi in the main portal vein and its branches. The tumor thrombi were demonstrated on ultrasonography, CT, angiography as expanding intraluminal masses.


Cytokine-stimulated human mesothelial cells produce chemotactic activity for neutrophils including NAP-1/IL-8. To test the hypothesis that mesothelial cells play a role in regulating inflammatory responses within the pleural space, we examined neutrophil chemotactic activity released by cytokine-stimulated mesothelial cells. Human mesothelial cells were isolated from patients with transudative pleural effusions and cultured. The purity of the cell population was assessed by morphologic, immunocytochemical, and biochemical characteristics. Confluent fourth passage mesothelial cell plates were exposed to varying concentrations of the recombinant human cytokines IL-1 alpha, TNF-alpha, or IFN-gamma, or Escherichia coli endotoxin (LPS). Polymorphonuclear neutrophil (PMN) chemotactic activity in the conditioned media was measured in microchemotaxis chambers. Although none of the cytokines demonstrated inherent chemotactic activity, each stimulated mesothelial cells to produce PMN chemotactic activity in a dose-dependent manner. TNF-alpha stimulated the release of the greatest quantity, whereas stimulation with IFN-gamma and IL-1 alpha resulted in the release of lesser but still significant quantities of PMN chemotactic activity. By contrast, LPS did not increase the basal level of chemotactic activity produced by the cells. The cytokine-induced chemotactic activity was proteinaceous, required de novo synthesis, and had a predominant m.w. of 10,000. Significant quantities of immunoreactive neutrophil-activating peptide-1 (NAP-1)/IL-8 were detected in mesothelial cell supernatants after stimulation with each of the cytokines. The neutrophil chemotactic activity of supernatants from mesothelial cells stimulated with either IL-1 alpha or IFN-gamma was completely neutralized with rabbit anti-human NAP-1/IL-8 polyclonal antiserum. The same antiserum neutralized the majority, but not all, of the neutrophil chemotactic activity in supernatants from TNF-stimulated mesothelial cells. Stimulated mesothelial cells also expressed an inducible mRNA transcript that hybridized with a specific oligonucleotide probe for human NAP-1/IL-8. These observations provide a mechanism whereby mesothelial cells could respond to inflammatory stimuli in the underlying lung and regulate inflammatory responses within the pleural space.


Sequential CT in monitoring experimental lung transplant. The criteria for diagnosis of lung rejection remain controversial. In this study early changes in lung rejection were characterized using sequential CT with the aim of developing a sensitive and safe monitoring method, which would also provide specific information on graft status. Twenty-one experimental single lung transplants (SLTs) in piglets, including unmodified rejections, immunosuppressed recipients, and autogenic reimplants, were scanned using CT on days 3, 5, 7, 10, 14, 22 up to 134 days after operation. In addition to morphological analysis, bilateral densitometric CT measurements of the peripheral lung parenchyma were used to evaluate the intensity of interstitial infiltration of lung grafts. Altogether 67 postoperative CT studies were carried out. Simultaneous transbronchial/thoracic biopsies were undertaken. The mean follow-up time was 28 days (range 0-134 days). The experience obtained was then used in relation to SLT in a male patient for chronic obstructive pulmonary disease. All piglets had a typical hilar reimplantation response, which disappeared in 10-20 days. Two distinct patterns of radiological acute rejection were found. Increasing peripheral alveolar infiltrates reflected early massive rejection in untreated animals. In immunosuppressed animals, after the initial reimplantation response, acute rejection was detected as densitometrically measured diffuse interstitial infiltration over the whole graft. Local findings, like focal infections, were seen later in the lingula and basal portions of the graft. In the SLT patient, density changes preceded clinical rejection episodes, which responded to steroid therapy. In the experimental study, the lung graft was compared to normal contralateral lung. In the SLT patient, however, this was not possible, and, therefore, the subsequent repeated CT studies formed the basis of clinical follow-up. Sequential imaging and densitometric measurements allowed objective estimation of diffuse interstitial infiltration relating to rejection.


The binding subunit of pertussis toxin inhibits IL-1 induction of IL-2 and prostaglandin production. The inhibitory effect of pertussis toxin on the action of IL-1 has been investigated. The toxin inhibited IL-1-induced production of IL-2 mRNA and protein in EL4 cells. The B oligomer of the toxin, which was shown to be devoid of ADP-ribosylating activity, proved as inhibitory as the holotoxin. The inhibition was therefore attributable to the binding subunit of the toxin and not to its ability to ADP-ribosylate G proteins. The toxin did not affect the IL-1R binding to its ligand, nor did it inhibit an early post-receptor event, the induction of the transcription factor NF kappa B. This implied that the toxin was not uncoupling IL-1R signaling. The toxin, or its B oligomer, inhibited PGE2 synthesis in human gingival fibroblasts stimulated by IL-1, but not by PMA. Assay of PG synthetic activity in the cells after addition of exogenous arachidonic acid suggested impairment by the toxin of induction of PG-synthesizing enzymes. IL-1 stimulation of IL-6 or collagenase production by fibroblasts was unaffected by pertussis toxin. The binding subunit of the toxin inhibits certain IL-1 responses by virtue of previously unrecognized actions on lymphoid and fibroblastic cells. It does not appear to block early signaling and the inhibition highly unlikely to involve inactivation of a G protein.


Transforming growth factor-beta in synovial fluids modulates Fc gamma RII (CD16) expression on mononuclear phagocytes. Mononuclear phagocytes in the synovium of patients with arthritis, in contrast to blood monocytes, were found to express a third receptor for the constant region of Ig (Fc gamma RIII), in addition to Fc gamma RI and Fc gamma RII. Previously identified on mature mononuclear phagocytes or phagocytes exposed to transforming growth factor-beta (TGF-beta) in vitro, this study documents the presence of Fc gamma RIII (CD16) expressing cells at an inflammatory site. Furthermore, the presence of CD16 on the majority of the LeuM3 (CD14) positive synovial monocytic cells could be mimicked by exposing blood monocytes to synovial fluids from patients with rheumatoid arthritis (17 of 19) and synovial fluids from patients with osteoarthritis (4 of 4). In additional studies, the soluble factor in inflammatory synovial fluids responsible for regulating CD16 expression was found to be consistent with the presence of TGF-beta. Inhibition of the activity in synovial fluids with a neutralizing antibody to TGF-beta confirmed a role for this peptide in synovial phagocytic cell CD16 expression. Moreover, signal transduction through CD16 on synovial phagocytes resulted in augmented extracellular release of superoxide anion that may contribute to tissue damage and other inflammatory sequelae. Identification of TGF-beta and its association with upregulation of CD16 at sites of chronic inflammation may provide insight into the destructive lesions associated with inflammatory arthropathies.


The mechanism of internalization of platelet-activating factor in activated human neutrophils. Enhanced transbilayer movement across the plasma membrane. Recent studies suggest that cellular internalization of platelet-activating factor (PAF), a potent ether phospholipid mediator of inflammation, is modulated by, as yet undefined cellular mechanisms. Using an albumin extraction method, the internalization of PAF and several PAF analogues was studied in the resting and stimulated human neutrophil. Our data demonstrate that internalization of these analogues is largely dependent on the state of cellular activation and that the process is not specific for certain unique structural features of the PAF molecule including the 1-position ether linkage, 2-position acetyl substitution, or choline polar head group. Furthermore, the internalization process was shown not to be dependent on the PAF receptor, metabolism of the molecule, or the process of endocytosis. Data are presented to suggest that the route of internalization of PAF is enhanced transbilayer movement (flipping) across the plasma membrane occurring as a result of changes in membrane physical properties accompanying cellular activation. It is proposed that in addition to enhanced internalization of PAF, modulation of PAF biosynthesis and net release from the stimulated neutrophil may be consequences of enhanced transbilayer movement of PAF across the activated plasma membrane.


Expression of platelet-derived growth factor-beta receptors on human fibroblasts. Regulation by recombinant platelet-derived growth factor-BB, IL-1, and tumor necrosis factor-alpha. Stimulation of human fibroblasts by platelet-derived growth factor (PDGF)-BB leads to a down-regulation of PDGF beta-receptors and a concomitant appearance of intracellular granular accumulations of receptors, as determined by stainings with the mAb PDGFR-B2. The granules contained both the ligand and PDGF beta-receptors, as revealed by double-immunofluorescence staining, and were formed in response to PDGF-BB but not in response to other cytokines tested. The formation of intracellular PDGF beta-receptor granules was dependent on PDGF-BB concentration and time of stimulation. The granular PDGF beta-receptor staining on cells treated with PDGF-BB for 1 h at 37 degrees C was used to investigate the effects of macrophage-derived cytokines on PDGF beta-receptor expression. The number of PDGF beta-receptor granules was found to be reduced in fibroblasts grown for 48 h in the presence of PDGF-BB, TNF-alpha, or IL-1; PDGF-AA under the same conditions had no effect. The reduction observed was paralleled by a decrease in cell surface expression of PDGF beta-receptors, measured as binding of 125I-PDGF-BB and of the PDGFR-B2 antibody. Furthermore, both TNF-alpha and IL-1 decreased the detergent-extractable pool of PDGF-beta receptors in the fibroblasts, as revealed by immunoblotting of detergent cell extracts. Finally, the decrease in PDGF beta-receptors after culturing of the cells in the presence of TNF-alpha and IL-1 was accompanied by a decreased incorporation of [3H]thymidine in response to PDGF-BB stimulation. In conclusion, our data suggest that certain macrophage-derived cytokines can modulate the expression of PDGF beta-receptors by cultured fibroblasts, which may contribute in part to their reduced responsiveness to PDGF.


Heterogeneity in the allosteric interaction between the gamma-aminobutyric acid (GABA) binding site and three different benzodiazepine binding sites of the GABAA/benzodiazepine receptor complex in the rat nervous system. In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.


Phosphorylation of the MARCKS protein (P87), a major protein kinase C substrate, is not an obligatory step in the mitogenic signaling pathway of basic fibroblast growth factor in rat oligodendrocytes. Basic fibroblast growth factor (bFGF) is a well-characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain-derived oligodendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c-fos in OL. The role of protein kinase C (PKC) in mediating bFGF-stimulated proliferation as well as c-fos expression in OL was investigated. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated c-fos expression but did not trigger cell proliferation. When PKC was down-regulated by pretreatment of OL with PMA for 20 h, the bFGF-mediated stimulations of OL proliferation and c-fos mRNA expression were still observed, whereas the induction of c-fos mRNA by PMA was totally inhibited. These data demonstrate that the bFGF mitogenic signaling pathway in OLs does not require PKC. On the other hand, bFGF was found to stimulate specifically the phosphorylation of a limited number of PKC substrates in oligodendroglial cells, including the MARCKS protein. The bFGF-dependent phosphorylation of MARCKS protein was totally inhibited when PKC was first down-regulated, indicating that the phosphorylation of this protein is PKC dependent. Tryptic digestion of the phosphorylated MARCKS protein revealed that bFGF stimulated specifically the phosphorylation of the MARCKS protein on a single phosphopeptide. We provide evidence that bFGF also stimulated fatty acylation of the MARCKS protein, which might explain the observed specific bFGF-dependent phosphorylation of this protein in OL. We propose that bFGF-dependent fatty acylation and phosphorylation of the MARCKS protein are not essential for the transduction of the bFGF mitogenic signal but are probably linked to differentiation processes elicited by bFGF on OL.


Biochemical responses mediated by N-methyl-D-aspartate receptors in rat cortical slices are differentially sensitive to magnesium. The effects of magnesium on the inhibition of phosphoinositide (PI) hydrolysis and the stimulation of [3H]norepinephrine release by N-methyl-D-aspartate (NMDA) in rat cortical slices were investigated. Removal of the magnesium from the buffer resulted in a small reduction of the inhibitory effect of 100 microM NMDA (34% inhibition in the absence of magnesium, compared with 51% for the control) when slices were coincubated with NMDA and carbachol. Addition of 10 mM Mg2+ also allowed the inhibitory effect of 100 microM NMDA on carbachol-stimulated PI hydrolysis to be expressed (44% inhibition) under these conditions. Concentration-effect curve analysis for the NMDA-induced inhibition of carbachol-stimulated PI hydrolysis indicated that the IC50 for NMDA was decreased from 14.9 microM for the control to 4.2 microM in the absence of magnesium. The absence of magnesium also had small effects on the concentration-effect curve for (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate reversal of the inhibitory effects of NMDA on carbachol-stimulated PI hydrolysis. The absence of magnesium also shifted slightly downward and flattened the NMDA concentration-effect curve if the cortical slices were pretreated with NMDA in the presence or absence of magnesium followed by removal of the NMDA and subsequent stimulation with carbachol. In contrast, cortical slices that had been prepared and treated similarly to the slices used in the PI experiments were very sensitive to the inhibitory effects of magnesium when using the NMDA stimulation of [3H]norepinephrine release assay in the presence or absence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)


Alterations in the gamma-aminobutyric acid-gated chloride channel following transient forebrain ischemia in the gerbil. The role of inhibitory neurotransmission in selective neuronal degeneration after transient forebrain ischemia was studied by binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to the gamma-aminobutyric acid (GABA)-gated chloride channel and measurement of GABAA receptor function in Mongolian gerbil brain. [35S]TBPS binding to the hippocampus, striatum, and cortex quantified by autoradiography and muscimol-stimulated 36Cl- uptake in synaptoneurosomes of the same regions were examined 1, 4, and 29 days after a 5-min bilateral carotid occlusion. [35S]TBPS binding was decreased in the pyramidal cell dendritic layers, stratum oriens, and stratum lacunosum-moleculare of the CA1 hippocampus, 4 and 29 days after occlusion, and in the stratum radiatum 29 days after occlusion. [35S]TBPS binding sites in the lateral striatum decreased 47% 4 days after occlusion. At the same time, there was a corresponding decrease in muscimol-stimulated 36Cl- uptake in the striatal synaptoneurosomes. Muscimol-stimulated 36Cl- uptake in the hippocampus decreased slightly 4 days after occlusion and more so after 29 days, although these decreases were not significant. No changes were observed in somatosensory cortex at any time point. These data suggest that a portion of GABAA receptors in areas sensitive to ischemic insult are associated with degenerating neurons, whereas other GABAA) receptors are spared.


Characterization of solubilized opioid receptors: reconstitution and uncoupling of guanine nucleotide-sensitive agonist binding. Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-(CHAPS). High concentrations of NaCl (0.5-1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane-bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High-affinity binding of mu, delta, and kappa agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, mu-agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin ([3H]DAGO) binding to guanosine 5'-O-(thiotriphosphate) (GTP gamma S). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTP gamma S was observed, whereas membrane-bound receptors were uncoupled only partially. Treatment with GTP gamma S, at concentrations that uncoupled the mu receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200-fold.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin receptors on cultured fetal rat diencephalic glia. The recently described family of proteins, the endothelins, are produced in neurons and bind to extravascular sites in the CNS. To characterize these receptors, we carried out studies on cultures of fetal rat diencephalic glia. Scatchard analysis of saturation binding studies was done for astrocytes (greater than 95% glial fibrillary acidic protein positive). For endothelin 3 (ET-3) and ET-1, respectively, a single receptor class of KD 0.41 +/- 0.05 and 0.62 +/- 0.04 nM and a receptor density of 42 +/- 0.8 and 58 +/- 1.1 fmol/mg of glial protein was found. Bound and cross-linked 125I-ET-3 or ET-1 showed a single predominant receptor band at Mr 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a minor band at 50,000 was also seen. At concentrations equal to the receptor KD, the major brain form of ET, ET-3, stimulated a nearly 200% increase in the incorporation of tritiated thymidine into glia. ET-3 and ET-1 significantly impaired the ability of atrial natriuretic peptide (ANP) to generate cyclic GMP, and isoproterenol to generate cyclic AMP. The ability of ET to inhibit ANP-induced cyclic GMP generation was reversed by cycloheximide and actinomycin-D, whereas the inhibition of isoproterenol-induced cyclic AMP generation was partially and significantly blocked by inhibitors of calcium influx, protein kinase C action, or G protein activation, as well. Astrocytes from this part of the brain are a potential target cell for endothelin, assuming these findings are present in vivo. This neuropeptide may serve as a growth stimulator for astrocytes and modulator of the actions of catecholamines or ANP on glia by inhibiting second messenger generation.


Effects of dopaminergic transmission interruption on the D2 receptor isoforms in various cerebral tissues. We examined the effects of an interruption of dopamine neurotransmission, by either dopamine receptor blockade or degeneration of dopamine neurons by 6-hydroxydopamine, on the levels of D2 receptor mRNAs. In addition, we evaluated by the polymerase chain reaction (PCR) the relative abundance of the two D2 receptor isoform mRNAs generated by alternative splicing. Daily injections of 4 mg/kg of haloperidol to rats elicited in striatum a rapid and progressive increase in D2 receptor mRNA levels, which reached 70% after a 15-day treatment. By contrast, there was no apparent change in D2 receptor mRNA levels in cerebral cortex and pons-medulla, in spite of an increased density of D2 receptor in the former tissue. Using the PCR with primers flanking the alternative exon, we observed that the relative proportion of the shorter receptor isoform (D2S) mRNA was slightly but significantly enhanced in cerebral cortex (17%) and pons-medulla (18%) after a 15-day haloperidol treatment. Unilateral degeneration of dopamine neurons induced by local injection of 6-hydroxydopamine resulted in a marked decrease in levels of total D2 receptor mRNAs in substantia nigra (-79%) and ventral tegmental (-63%) area, two cell body areas. In the substantia nigra, the longer isoform (D2L) mRNA was significantly more decreased in content than the D2S isoform mRNA, so that there was a large enhancement in the relative abundance of the latter (81%).(ABSTRACT TRUNCATED AT 250 WORDS)


6-Methylmercaptopurine riboside is a potent and selective inhibitor of nerve growth factor-activated protein kinase N. Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine analogs, such as 6-thioguanine and 2-aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a findings suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6-methylmercaptopurine riboside (6-MMPR) inhibits NGF-stimulated PKN activity in vitro with an apparent Ki of approximately 5 nM. This is approximately 1,000-fold lower than the Ki of the most potent purine inhibitor of PKN. Compounds similar to 6-MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. A survey of six additional purified protein kinases shows no inhibitory effect of 6-MMPR, thus indicating a good degree of specificity of this compound for PKN. In contrast to NGF-stimulated PKN, a PKN-like activity stimulated in PC12 cells in response to activation of cyclic AMP-dependent protein kinase was nearly insensitive to 6-MMPR. Application of 6-MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6-MMPR is a potent and selective agent for characterizing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action.


Quisqualate-stimulated phosphatidylinositol breakdown in rat cerebellar membranes. The effect of quisqualate, an excitatory amino acid agonist, on the breakdown of exogenously added phosphatidylinositol was investigated in a membrane preparation from the cerebellum of young rats. Quisqualate stimulated phospholipase C activity in a dose-dependent manner in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Half-maximal activation of the quisqualate response required 0.15 microM GTP gamma S and was optimal at a free Ca2+ concentration of 300 nM. Phosphoinositide breakdown was also stimulated by quisqualate using either exogenous phosphatidylinositides 4,5-bisphosphate or endogenous labeled phosphoinositides as the substrate for phospholipase C in cerebellar membranes. In the presence of guanine nucleotides, other excitatory amino acid agonists, such as L-glutamate, trans-D,L-1-aminocyclopentyl-1,3-dicarboxylic acid, and ibotenate, but not N-methyl-D-aspartate, stimulated phosphatidylinositol breakdown. However, quisqualate displayed the highest response among these excitatory amino acid agonists. These data indicate that there is a direct activation of phosphoinositide-specific phospholipase C by excitatory amino acids through a process dependent on the presence of guanine nucleotides.


4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus. Two types of spontaneous filed potentials were recorded in rat hippocampal slices after addition of 4-aminopyridine (4-AP; 50 microM). One consisted of brief, epileptiform discharges that occurred at 0.6 +/- 0.2 sec-1 in the CA3 and CA1 areas. The other type occurred less frequently (0.036 +/- 0.013 sec-1) and was recorded in CA1, CA3, and dentate areas. It corresponded in all regions to an intracellular long-lasting depolarization (LLD; duration, 300-1200 msec; peak amplitude, 2-15 mV) that was abolished by bicuculline methiodide; therefore, it was mediated by GABAA receptors. Sectioning experiments and the occurrence of propagation failures indicated that LLDs could be initiated by any area of the slice. Furthermore, the propagation of LLDs did not follow any consistent or predictable pattern along known anatomical hippocampal pathways. Finally, neither the occurrence nor the propagation of LLDs was affected when excitatory synaptic transmission was blocked by NMDA and non-NMDA receptor antagonists. In the presence of antagonists of glutamatergic receptors, LLDs disappeared after the omission of Ca2+ or the addition of Cd2+ to the perfusing solution, suggesting that synaptic transmission was required for their generation. These data indicate that 4-AP discloses both interictal epileptiform discharges and LLDs in the rat hippocampus. The first type of activity is presumably related to certain properties of CA3 pyramidal neurons and the neuronal circuit, whereas LLDs originate from the spontaneous, periodic activity of GABAergic interneurons located in any area of the hippocampus, and can propagate to the other areas by the use of nonsynaptic mechanisms. We propose that 4-AP reveals a novel type of interaction among GABAergic interneurons that is based on the accumulation and the dispersion of K+.


Partial glycinergic denervation induces transient changes in the distribution of a glycine receptor-associated protein in a central neuron. The effect of partial glycinergic denervation on the cellular distribution of the 93 kDa peripheral polypeptide associated with the glycine receptor was studied at the level of the teleost Mauthner cell, an identified neuron of the goldfish brain (Carassius auratus). Previous studies using monoclonal antibodies raised against purified glycine receptors and immunoperoxidase staining have shown that these proteins are localized in clusters on the entire surface of this neuron. Specifically, the 93 kDa polypeptide was situated only on the cytoplasmic side of the postsynaptic membrane facing active zones. Unilateral electrolytic lesions of the vestibular complex caused the degeneration of some glycinergic afferents to this neuron. When the first signs of this response appeared, 3 d after the surgery, there was also a change in the ultrastructural distribution of the 93 kDa polypeptide in the deafferented cell. The synaptic protein apposed to degenerating axons did not spread onto adjacent extrasynaptic membranes, and it disappeared a few hours after the disruption of its presynaptic element. At the same time, a cytoplasmic immunoreactivity appeared as randomly distributed clusters in the deafferented Mauthner cell; these aggregates, not seen in control preparations, were never found inside membrane-bound organelles. In some preparations these clusters were localized along arrays at a relatively constant distance from the plasma membrane. The intracellular immunoreaction product was found in the soma and the initial part of the dendrites, gradually decreasing in number and intensity toward the extremities of these processes. At later postoperative stages, 10-15 d after surgery, the 93 kDa immunoreactivity remained only at postsynaptic membranes facing intact terminals. Similar alterations following denervation were observed in reticular neurons, at the level at which degenerating presynaptic terminals were also detected. In contrast, continuous 3-d blockade of synaptic transmission by strychnine, an antagonist of the glycine receptor, had no effect on either the distribution of the surface receptor clusters, or the 93 kDa peripheral protein linked to these receptors. Taken together, our results suggest that the ultrastructural distribution of the glycine receptor complex is regulated by "trophic" factors rather than by transmitter-evoked synaptic activity.


Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel alpha-subunit expressed in mammalian cells. The rat brain IIA Na+ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na+ channel alpha-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (approximately 10 channels/microns2 in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC3H1 cells and failed to express in Ltk- cells. However, voltage-dependent Drosophila Shaker H4 K+ channels and Escherichia coli beta-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na+ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na+ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na+ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell-specific posttranslational modifications. IIA channels were blocked by approximately 90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX-resistant Na+ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence.


Cellular mechanisms underlying cholinergic and noradrenergic modulation of neuronal firing mode in the cat and guinea pig dorsal lateral geniculate nucleus. The functional properties, ionic basis, and possible convergence and interaction of postsynaptic actions mediated by muscarinic and alpha 1-adrenergic receptors were examined in cat and guinea pig dorsal lateral geniculate (LGNd) neurons maintained in thalamic slices in vitro. The possible involvement of GTP-binding proteins was also examined. Extracellular recordings from cat LGNd revealed the presence of two subpopulations of neurons. The most prevalent generated rhythmic high-frequency (300-500 Hz) bursts of two to six action potentials each, with an interburst frequency of 1-3 Hz. Intracellular recordings revealed that this activity is typical of thalamocortical relay cells in the apparent absence of neuromodulatory input. Application of ACh or noradrenaline (NA) to rhythmically oscillating neurons in the cat LGNd resulted in cessation of this activity followed by the appearance of single spike firing. Intracellular recordings revealed that this change in firing mode was associated with a depolarization of the neuron out of the range of intrinsic rhythmic oscillation and into or near the single spike firing mode. The voltage characteristics of the current underlying the cholinergic and noradrenergic slow depolarization were investigated in guinea pig LGNd neurons. Application of the muscarinic agonist acetyl-beta-methylcholine (MCh) to presumed relay neurons resulted in a hyperpolarization due to the activation of an outward K+ current. This response was followed by a slow depolarization due to reduction of a relatively non-voltage-dependent potassium current distinct from IM and IAHP. Application of NA resulted in a slow depolarization that was also associated with reduction of this relatively linear K+ current. The MCh- and NA-induced slow depolarizations displayed the property of occlusion, indicating convergence of action. However, these responses were mediated by pharmacologically distinct receptors since the MCh-induced reduction in K+ current was blocked by scopolamine while that induced by NA was blocked by the alpha 1-adrenoceptor antagonist prazosin. Intracellular diffusion of GTP-gamma-S resulted in the inward current responses to NA and MCh being irreversible, suggesting the possible involvement of a G-protein. Prior exposure to pertussis toxin did not affect the inward current response to NA and MCh, while the outward K+ current responses induced by application of MCh or the GABAB agonist baclofen were blocked. These results reveal that activation of muscarinic or alpha 1-adrenergic postsynaptic receptors in the LGNd result in a shift in firing mode from rhythmic oscillation to tonic single spike activity through a decrease in a relatively linear K+ current mediated through a pertussis toxin-insensitive G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)


Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection. Basic fibroblast growth factor (bFGF) is a potent trophic factor for neurons and astrocytes and recently has been implicated in the pathology of Alzheimer's disease. In order to better understand the role of bFGF in normal brain function and during pathology, we have analyzed its anatomical distribution and its response to injury in the CNS. Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations, and occasionally in microglial cells throughout the normal rat brain. Neuronal populations that showed bFGF immunoreactivity included septohippocampal nucleus, cingulate cortex, subfield CA2 of the hippocampus, cerebellar Purkinje cells, cerebellar deep nuclei, facial nerve nucleus, and the motor and spinal subdivisions of the trigeminal nucleus and facial nerve nucleus. The pattern of bFGF immunoreactivity in the hippocampus was examined following entorhinal cortex lesion, or fimbria-fornix transection. After entorhinal cortex lesion, bFGF immunoreactivity increased in the outer molecular layer of the dentate gyrus ipsilateral to the lesion. The lesion effect on bFGF immunoreactivity was expressed as an increase in the number of bFGF astrocytes, as an increase in the intensity of bFGF immunoreactivity within astrocytes, and as an increase of bFGF immunoreactivity in the surrounding extracellular matrix, relative to the contralateral side. The time course and pattern of reorganization paralleled the sprouting of septal cholinergic terminals in response to the same type of lesion, suggesting that bFGF may play an important role in lesion-induced plasticity. After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex and, as previously reported, prevent the death of medial septal neurons. Therefore, it appears that bFGF infusion, which has been shown to increase the synthesis of NGF by astrocytes (Yoshida and Gage, 1991), also helps enable neurons to respond to NGF. This suggests that after injury bFGF may participate in a cascade of neurotrophic events, directly and indirectly facilitating neuronal repair and/or promoting neuronal survival.


Pre- and postsynaptic inhibition by opioids in rat striatum. The physiological role of opioid peptides in the rat striatum was sought by intracellular recording in vitro. Excitatory synaptic potentials (mediated by glutamate or aspartate) and inhibitory synaptic potentials (mediated by GABA) were isolated pharmacologically and/or by positioning the stimulation electrode over the external capsule. Opioid agonists and antagonists selective for mu-, delta-, and kappa-receptors were applied by superfusion. Two main actions of opioids were observed. First, mu- and delta-selective opioids presynaptically inhibited the excitatory postsynaptic potential, whereas only delta-selective opioids decreased the inhibitory synaptic potential. Second, a small subpopulation of cells (not medium spiny neurons) were hyperpolarized by delta-selective agonists. The results indicate that the main action of opioids on striatal neurons is presynaptic inhibition of the corticostriate excitatory synaptic input.


Evidence for prenatal competition among the central arbors of trigeminal primary afferent neurons. Previous studies have shown that damage to vibrissa follicles in newborn rats and mice does not alter the brainstem representations of the remaining vibrissa as demonstrated by staining for mitochondrial enzymes such as cytochrome oxidase (CO) succinic dehydrogenase. This study asked whether this lack of effect might be due to the fact that the trigeminal primary afferents in rodents are already quite well developed at birth. We assessed this possibility by using CO staining the evaluate patterns in the brainstems of pre- and postnatal rats. A vibrissa-related pattern began to emerge in trigeminal nucleus principalis and subnucleus interpolaris (Spl) by embryonic day (E-) 19 and appeared fully developed by the day of birth (P-0). We also made partial lesions of the vibrissa pad on E-15-20 and on P-0, killed pups on P-5-7, and measured the size of the CO-stained patches in Spl on both sides of the brainstem. The correspondence between CO patches and clusters of primary afferent terminal arbors was verified in some animals by combining transganglionic horseradish peroxidase tracing and CO staining. Vibrissa pad damage on E-15-18 resulted in significant (20.1-36.9%) increases in the average area of the remaining CO patches in Spl ipsilateral to the lesion. Vibrissa pad damage on E-19, E-20, and P-0 produced small (6.2-8.9%), but insignificant, increases in patch size in Spl ipsilateral to the lesion. We used anatomical and electrophysiological methods to determine whether our lesions altered the trigeminal innervation of surviving vibrissa follicles. We recorded single trigeminal ganglion cells from 12 rats that sustained vibrissa pad lesion on E-17. As in normal rats, all of the 49 vibrissa-sensitive ganglion cells isolated in the lesioned animals were responsive to deflection of one and only one vibrissa. We also dissected 11 deep vibrissal nerves from intact follicles in adult rats that sustained fetal vibrissa pad damage on E-17, and counted numbers of myelinated axons in 1 microns plastic sections. These data were compared with counts from corresponding follicles on the intact side of the face. The average number of myelinated axons innervating follicles in the damaged vibrissa pads was 196.8 +/- 27.9, and that for the corresponding contralateral nerves was 194.6 +/- 25.7. These data suggest that competitive interactions among the central arbors of trigeminal primary afferents in fetal life may influence the development of central vibrissa representations and, further, that lesion-induced central changes need not be correlated with alterations in the peripheral innervation of undamaged follicles.


Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro. In ischemic and in inflamed tissues, pH levels down to 5.4 have been measured, and this local acidosis may contribute to pain and hyperalgesia in disease states. To evaluate the role of acid pH in nociception, we have studied identified primary afferents in a rat skin-saphenous nerve preparation in vitro where the receptive fields can be superfused at the highly permeable corium side with controlled solutions. The nerve endings were exposed to CO2-saturated synthetic interstitial fluid (SIF;pH 6.1) and to carbogen-gassed SIF phosphate buffered to different acid pH levels (5 min duration, 10 min intervals). Mechanical thresholds were repeatedly tested in a "blind" fashion by von Frey hair stimulation. Low-threshold mechanosensitive A beta- (n = 12) and A delta-fibers (n = 11) were not excited or sensitized by acid pH levels. In 24 of 96 nociceptor type C- and A delta-fibers, irregular low-frequency discharge with poor response characteristics was induced. However, a distinct subpopulation of mechanoheat sensitive, "polymodal" C-units (n = 25; 38%) showed stimulus-related responses increasing with proton concentration and encoding the time course of the pH change. Threshold levels were found to range from pH 6.9 to 6.1; mean maximum discharge was at pH 5.2. All such fibers responded to CO2 as well as to phosphate-buffered solution at the same pH 6.1. The CO2 responses, however, displayed significantly shorter latencies and more pronounced dynamic phases. The carboanhydrase blocker acetazolamide markedly delayed and reduced the CO2 responses. Prolonged application of acid pH (30 min) evoked nonadapting activity irrespective of oxygen supply. Many, but certainly not all, fibers sensitive to protons were also driven by capsaicin (10(-6) M, 10(-5) M) and vice versa. Repeated or prolonged treatment with low pH induced a significant and lasting decrease of the mechanical (von Frey) thresholds in almost all C-fibers tested (from 35 to 16 mN, on average), and this occurred whether or not a fiber was excited by protons. The sensitizing effect was more pronounced the higher the initial von Frey thresholds (0.75 rank correlation). This sensitization to mechanical stimulation was in contrast to the combined action of other inflammatory mediators, bradykinin, 5-HT, histamine and prostaglandin E2. In conclusion, we suggest that pH sensitivity of nociceptors may be an important source of pain and hyperalgesia.


Wheat bran and corn oil do not influence proliferation in the colon of healthy rats when energy intakes are equivalent. Dietary intakes of fiber (wheat bran) and fat (corn oil) by rats were quantitatively varied for 6 wk while intakes of energy and essential nutrients were constant among the diets. The influence of wheat bran and corn oil levels on colonic epithelial cell proliferation was assessed using [3H]thymidine incorporation assays. Cellular proliferation in the cecum, proximal colon and distal colon was not significantly higher when healthy rats consumed low fiber in comparison to high fiber diets, nor when healthy rats consumed high fat diets in comparison to low fat diets. Additionally, there were no significant interactions between these variables. In comparison to the low fiber diets, diets high in fiber caused a significantly lower pH of luminal contents of the cecum and distal colon, but there were no significant differences in the total short-chain fatty acid concentrations of cecal digesta. These results suggest that when energy intakes are equivalent among diets, low levels of fiber or high levels of fat in the diet do not cause biological changes in colonic mucosa that may be associated with increased risk of colonic cancer.


Human papillomavirus types 16 and 18 are not involved in human prostate carcinogenesis: analysis of archival human prostate cancer specimens by differential polymerase chain reaction. Human papilloma viruses (HPV) have been implicated in the pathogenesis of a variety of malignancies, especially in carcinomas of the female genital tract. Recently, based on observations using the polymerase chain reaction amplification assay, HPV types 16 and 18 specific DNA sequences have been detected in prostate cancer specimens obtained by transurethral resection. Since HPV types 16 and 18 have been shown to possess oncogenic potential, an association between HPV infection and prostatic carcinoma has been suggested. In order to exclude potentially HPV-colonized urethral mucosa from analysis and restrict our study to predominantly malignant tissue, cancerous areas from a series of 30 paraffin-embedded prostate adenocarcinomas were microdissected and analyzed for the presence of HPV 16 or HPV 18 specific sequences by a modification of PCR (D-PCR) and Southern blot analysis. Despite the high sensitivity of our analytical technique, we found no evidence of HPV-DNA of either type in any of the 30 primary prostate cancers. In contrast, both HPV 16 (2/8 specimens) and HPV 18 (2/8 specimens) DNA was detected in randomly chosen cervical carcinomas using the D-PCR methodology. Our data would indicate that the oncogenic HPV-types 16 and 18 are unlikely effectors of prostate carcinogenesis.


Case-mix groups for VA hospital-based home care. The purpose of this study is to group hospital-based home care (HBHC) patients homogeneously by their characteristics with respect to cost of care to develop alternative case mix methods for management and reimbursement (allocation) purposes. Six Veterans Affairs (VA) HBHC programs in Fiscal Year (FY) 1986 that maximized patient, program, and regional variation were selected, all of which agreed to participate. All HBHC patients active in each program on October 1, 1987, in addition to all new admissions through September 30, 1988 (FY88), comprised the sample of 874 unique patients. Statistical methods include the use of classification and regression trees (CART software: Statistical Software; Lafayette, CA), analysis of variance, and multiple linear regression techniques. The resulting algorithm is a three-factor model that explains 20% of the cost variance (R2 = 20%, with a cross validation R2 of 12%). Similar classifications such as the RUG-II, which is utilized for VA nursing home and intermediate care, the VA outpatient resource allocation model, and the RUG-HHC, utilized in some states for reimbursing home health care in the private sector, explained less of the cost variance and, therefore, are less adequate for VA home care resource allocation.


Proviral rearrangements and overexpression of a new cellular gene (nov) in myeloblastosis-associated virus type 1-induced nephroblastomas. Histological and anatomopathological studies performed on 152 independent myeloblastosis-associated virus type 1 (MAV1)-induced nephroblastomas allowed us to precisely define the chronology of tumor development in chickens. Three tumors representing increasing developmental stages were used to construct genomic libraries and to study both the state of proviral genomes and the sites of MAV1 integration in genomic DNA. We established that increasing levels of proviral rearrangement, eventually leading to the elimination of infectious MAV genomes, were associated with tumor progression and that 22 individual tumors, representative of different developmental stages, did not contain any common MAV1 integration site. Cloning of cellular fragments flanking the MAV1-related proviruses in tumor DNA showed that each one of eight nephroblastomas tested expressed a high level of an as yet unidentified cellular gene (nov) whose transcription is normally arrested in adult kidney cells. Cloning of the normal nov gene established that in one tumor, fused long terminal repeat-truncated nov mRNA species were expressed, indicating that at least in that case, the high level of nov expression was under the control of the MAV long terminal repeat promoter. The normal nov gene encodes a putative 32-kDa secreted polypeptide, which is a member of a new family of proteins likely to be involved in cell growth regulation. We also showed that the expression of an amino-terminal-truncated nov product in chicken embryo fibroblasts was sufficient to induce their transformation.


Oncogenes result in genomic alterations that activate a transcriptionally silent, dominantly selectable reporter gene (neo). Although oncogenes and tumor suppressor genes have been implicated in carcinogenesis and tumor progression, their relationship to the development of genomic instability has not been elucidated. To examine this role, we transfected oncogenes (polyomavirus middle [Py] and large T [MT and LT]) and adenovirus serotype 5 E1A) into two NIH 3T3-derived cell lines, EN/NIH 2-4 and EN/NIH 2-20. Both cell lines contain two stable integrants of a variant of the retrovirus vector pZipNeoSV(x)1 that has been modified by deletion of the enhancer elements from the long terminal repeats. DNA rearrangements activating the silent neomycin phosphotransferase gene (neo) present in these integrants were identified by selection of cells in the antibiotic G418. Whereas control-transfected EN/NIH cell lines do not yield G418-resistant subclones (GRSs), a fraction of oncogene-transfected EN/NIH 2-4 (8 of 19 Py MT, 5 of 17 Py LT, and 11 of 19 E1A) and 2-20 (7 of 15 Py MT) cell lines gave rise to GRSs at differing frequencies (0.33 x 10(-6) to 46 x 10(-6) for line 2-4 versus 0.11 x 10(-6) to 1.3 x 10(-6) for line 2-20) independent of cell generation time. In contrast, a distinctly smaller fraction of mutant Py MT-transfected EN/NIH cell lines (1 of 10 MT23, 1 of 10 MT1015, and 0 of 10 MT59b) resulted in GRSs. Southern analysis of DNA from selected oncogene-transfected GRSs demonstrated genomic rearrangements of neo-containing cellular DNA that varied in type (amplification and/or novel fragments) and frequency depending on the specific oncogene and EN/NIH cell line used in transfection. Furthermore, only one of the two neo-containing genomic loci present in both EN/NIH cell lines appeared to be involved in these genomic events. In addition to effects related to the genomic locus, these observations support a role for oncogenes in the development of genetic changes associated with tumor progression.


The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells. Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e., HeLa cells) but not others (i.e., Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors.


Structure of DRE, a retrotransposable element which integrates with position specificity upstream of Dictyostelium discoideum tRNA genes. Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor.


Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells. Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFR1) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFR1, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.


The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription. Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.


Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals. Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1.


Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus. The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.


Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. We have cloned a genomic region of the murine fibroblast growth factor (FGF) receptor 1 (FGFR1) gene that includes three alternative exons for the third immunoglobulinlike domain in the extracellular region of the receptor. The mRNA of one of these splice variants encodes a secreted receptor that lacks transmembrane and cytoplasmic sequences as well as a portion of the third immunoglobulinlike domain. Highest levels of mRNA encoding this variant were found in brain, skeletal muscle, and skin. We expressed this form of FGFR1 in CHO cells and showed that the recombinant secreted protein binds acidic FGF. We also discovered a novel alternative exon in the third immunoglobulinlike domain that encodes part of a transmembrane FGFR1 mRNA. This exon is highly homologous to the corresponding region of the keratinocyte growth factor receptor. Transcripts including this exon were present at highest levels in the skin. We cloned an FGFR1 cDNA which includes this exon and expressed this receptor variant in L6 rat skeletal muscle myoblasts. The new receptor variant had a 50-fold-lower affinity for basic FGF than does the published FGFR1 variant, whereas both forms of receptor bound acidic FGF with high affinity. These results show that the third immunoglobulinlike domain plays an important role in determining the binding specificities for different FGFs. Our data provide the first evidence that differential splicing in the extracellular region of a receptor gene generates receptor variants with different ligand-binding specificities.


A colony-stimulating factor 1 (CSF-1) receptor/platelet-derived growth factor-beta receptor gene fusion confers CSF-1 independence and tumorigenicity on a c-myc-immortalized monocyte cell line. Monocytes and macrophages express the receptor for the hematopoietic growth factor colony-stimulating factor 1 (CSF-1) and require this factor for growth in culture. A murine monocyte tumor cell line that lacks the usual requirement for CSF-1 was isolated. On the basis of the similarity of the structures of the CSF-1 and platelet-derived growth factor (PDGF) receptors and because monocytes normally secrete PDGF, we analyzed the tumor cell line for anomalous expression of the PDGF-R beta gene. Two different cDNAs that each contain sequences corresponding to the complete coding sequence of PDGF-R beta fused (in frame) to the amino-terminal half of the CSF-1 receptor were isolated. Introduction of these PDGF-R beta-related cDNAs into two partially transformed, CSF-1-dependent monocyte cell lines resulted in autonomous growth and cell transformation. These monocyte cell lines exhibit a novel form of growth factor receptor activation that can lead to oncogenic growth in collaboration with the c-myc oncogene.


Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers. The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.


Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces. Various species of marine Vibrio produce two distinct types of flagella, each adapted for a different type of motility. A single, sheathed polar flagellum is suited for swimming in liquid medium, and numerous unsheathed lateral flagella, which are produced only under viscous conditions, are suited for swarming over viscous surfaces. Both types of flagella are driven by reversible motors embedded in the cytoplasmic membrane. Here we report that the energy source for the polar flagellar motor of Vibrio parahaemolyticus is the sodium-motive force, whereas the lateral flagellar motors are driven by the proton-motive force. This is evidence that two distinct types of flagella powered by different energy sources are functionally active in one cell.


Common tumors.

Common tumors. Common tumors of the hand and upper extremity are, by definition, benign conditions. There is a predictable pattern of occurrence and presentation for many of these lesions. The practitioner should always bear in mind, however, that any tumor that appears anywhere in the body can appear in the hand and upper extremity. Moreover, several of the benign conditions can, in certain clinical settings, have a predilection for transformation or association with more aggressive conditions. We would emphasize that the approach to any tumor be carefully considered and include an appropriate differential diagnosis. Any suspicious lesion should be examined thoroughly, including first-order screening tests, history, physical examination, radiographic imaging, and laboratory evaluations followed by more detailed diagnostic imaging prior to surgery. Surgery should be approached as a biopsy, with the possibility of frozen section analysis, and the possibility of a two-staged operation for tumor removal with appropriate margins if necessary.


Antibody specific for the Thr-286-autophosphorylated alpha subunit of Ca2+/calmodulin-dependent protein kinase II. We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KII) autophosphorylated only at Thr-286 of the alpha subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys-289 of alpha subunit of CaM-KII)] was synthesized and phosphorylated by the CaM-KII endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase alpha subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KII both in purified and synaptic cytoskeleton-associated form. Appearance of CaM-KII subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca(2+)-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr-286- or Thr-287-autophosphorylated form of CaM-KII in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KII.


Intracellular injection of Ca2+ chelators blocks induction of long-term depression in rat visual cortex. In a variety of brain structures repetitive activation of synaptic connections can lead to long-term potentiation (LTP) or long-term depression (LTD) of synaptic transmission, and these modifications are held responsible for memory formation. Here we examine the role of postsynaptic Ca2+ concentration in the induction of LTD in the neocortex. In layer III cells of the rat visual cortex, LTD can be induced by tetanic stimulation of afferent fibers ascending from the white matter. We show that LTD induction is reliably blocked by intracellular injection of either EGTA or BAPTA [bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate], two different Ca2+ chelators. This confirms that the processes underlying the induction of LTD in neocortex are located postsynaptically and indicates that they depend on intracellular Ca2+ concentration. Thus, both LTP and LTD induction appear to involve calcium-mediated processes in the postsynaptic neuron. We propose that LTD is caused by a surge of calcium either through voltage-gated Ca2+ conductances and/or by transmitter-induced release of calcium from intracellular stores.


Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay. We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.