Tyrosine phosphorylation of biliary glycoprotein, a cell adhesion molecule related to carcinoembryonic antigen. Biliary-glycoprotein (BGP), a cell adhesion molecule related to carcinoembryonic antigen (CEA), has been shown to exist as several alternatively spliced isoforms. Here we show that BGPa and BGPb are phosphorylated in the chronic myelogenous leukaemia cell line KG-1, which constitutively expresses several BGP isoforms, and Chinese hamster LR-73 cells transfected with the cDNAs encoding BGPa and BGPb. The phosphorylation can be augmented with the protein tyrosine phosphatase inhibitor ammonium vanadate and with TPA (an activator of protein kinase C). Phospho-amino acid analysis of phosphorylated BGPs demonstrated that phosphorylation occurs on serine, threonine and tyrosine residues. Phosphorylation reactions carried out in in vitro membrane preparations from KG-1 cells revealed a close association of BGP proteins with membrane associated protein tyrosine kinases. These observations suggest an association of BGP proteins with signal transduction molecules which is regulated by alternative splicing of the cytoplasmic domain.


Inhibition of human exocrine pancreatic secretion by the long-acting somatostatin analogue octreotide (SMS 201-995). The new long-acting somatostatin analogue octreotide (SMS 201-995) was investigated for its influence on secretagogue-stimulated human exocrine pancreatic secretion. Eighteen healthy volunteers participated in the study. During duodenal intubation with a background stimulation of either secretin 1 U.kg/h or secretin 1 U.kg/h + ceruletide, 120 ng.kg/h, octreotide was infused at doses of 5, 20 and 80 micrograms/h in a placebo-controlled randomized double-blind crossover trial. Duodenal juice samples were collected in 10-min intervals, and amylase, trypsin, chymotrypsin, and bicarbonate were measured in the individual fractions. During secretin stimulation, amylase was inhibited between 41 and 59%, trypsin between 28 and 72%, chymotrypsin between 55 and 70%, and bicarbonate between 0 and 31% with 5, 20 and 80 micrograms/h octreotide. During secretin and ceruletide stimulation, amylase was significantly inhibited by 84%, 78%, 81%, trypsin by 76%, 55%, 52%, chymotrypsin by 77%, 55%, 60%, and bicarbonate by 25%, 11%, 19% with 5, 20, and 80 micrograms/h octreotide, respectively (all decreases P less than 0.05). The long-acting somatostatin analogue octreotide was confirmed to be a potent inhibitor of stimulated human exocrine pancreatic secretion. The near maximal inhibitory potency of octreotide was achieved at a dose of only 5 micrograms/h. This finding may be of value in the planning of therapeutic studies with octreotide.


The genes for the inter-alpha-inhibitor family share a homologous organization in human and mouse. Inter-alpha-inhibitor (I alpha I) and related molecules in human are comprised of three evolutionarily related, heavy (H) chains and one light (L) chain, also termed bikunin. The latter originates from a precursor molecule that is cleaved to yield the bikunin and another protein designated alpha-1-microglobulin (A1m). The four H and L chains are encoded by four distinct genes designated H1, H2, H3, and L. The L and H2 genes are localized onto human chromosomes (chr) 9 and 10, respectively, whereas the H1 and H3 genes are tandemly arranged on chr 3. Mouse poly(A)+ RNAs or endonuclease-restricted mouse DNA were analyzed by standard and pulsed-field gel electrophoresis (PFGE) techniques in agarose gels and blot-hybridized with human H1, H2, H3 or L cDNA probes. The variable sized transcripts and unique restriction fragment patterns detected with each probe indicate that four genes, including one common L gene for A1m and bikunin also exist in mouse. The co-migration of H1- and H3-hybridizing fragments on PFGE suggests that the mouse H1 and H3 genes are also tandemly arranged. An Msp I restriction fragment length polymorphism (RFLP) in the mouse L gene (proposed symbol, Intin-4) links this gene to other genes already mapped at mouse Chr 4 near the brown (b) locus, a homologous region to the human chr 9q32-34 band where the human I alpha I L gene is located. Therefore, a similar number and arrangement of I alpha I genes is found in mouse and human, including the triplication of an H gene ancestor. These results point to an ancient origin of this complex set of genes.


Characterization of peripheral blood CD8/11b cells in bone marrow transplant recipients. III. Subsets of CD8/11b cells differentially regulate immunoglobulin production. Recovering bone marrow transplant (BMT) recipients have 20-70% circulating lymphocytes which co-express CD8 and CD11b (normals = 5-15%). These CD8/11b cells comprise at least two subpopulations distinguished by their expression of CD3. The CD3+ CD8/11b cells are T lymphocytes which exhibit in vitro suppressor activity (Ts); the CD3- CD8/11b cells express CD16 and are natural killer (NK) cells. In this study, we investigated whether such cells influenced circulating levels of IgA and IgM in 18 BMT recipients who each had greater than 30% circulating CD8/11b cells. We observed that in all patients whose CD8/11b cells were Ts lymphocytes (7/7) IgM and IgA levels were less than 10% of normal. Among those patients whose CD8/11b cells were NK cells (n = 11), two groups were distinguished. In one group (n = 5), less than 35% of patient NK cells expressed CD57 and serum levels of IgM and IgA were less than 10% of normal. In the second group (n = 6) greater than 60% of the NK cells expressed CD57 and serum levels of IgM and IgA were normal. In summary, our data indicate that BMT recipients have at least three distinct subsets of CD8/11b lymphocyte populations which may differentially regulate IgM and IgA production in vivo.


Plasma abnormal prothrombin (PIVKA-II): a new and reliable marker for the detection of hepatocellular carcinoma. We evaluated the clinical usefulness of a protein induced by vitamin K absence, antagonist-prothrombin (PIVKA-II), in detecting hepatocellular carcinoma (HCC) specifically in patients with liver cirrhosis, and the possible correlation between levels of PIVKA-II and pathological features of HCC. Plasma levels of PIVKA-II and alpha-fetoprotein (AFP) were measured in 628 patients with various diseases, including 253 with liver cirrhosis and 116 with HCC. PIVKA-II was detected (greater than or equal to 0.1 arbitrary unit/mL) in 54.3% of HCC and the concentration showed a positive correlation with the tumour size. As a screening test for the detection of HCC, PIVKA-II produced values comparable with those of AFP with a sensitivity, specificity and validity of 52.8, 98.8 and 51.6% respectively. Sixteen of 45 patients (37%) with HCC who had low AFP (less than 100 ng/mL) levels were positive for PIVKA-II. No apparent relationship, however, could be found between the levels of PIVKA-II and the aetiology or pathological findings of HCC. These results suggest that PIVKA-II can be a reliable marker for detecting HCC in patients with liver cirrhosis.


The pharmacology of local anesthetics--a review of the literature. In the nineteenth century, some natives of Peru noticed circumoral numbness, euphoria and analgesia after chewing the leaves of the Erythroxylen coca bush. By 1850, cocaine was isolated from the plant, marking the start of the local anesthetic era in clinical medicine. Over the past 50 years, many synthetic local anesthetics have been developed which have fewer side effects, increased specificity of action and a wider margin of safety than cocaine. Currently, local anesthetics are used topically, for local infiltration; and intravenously, for peripheral nerve blockade, for sympathetic blockade, as well as for epidural and intrathecal use. Although the route of administration may affect pharmacokinetics and pharmacodynamics, it is the purpose of this article to review the general pharmacology of this entire range of clinically useful compounds.


Subcutaneous narcotic infusions for cancer pain: treatment outcome and guidelines for use. To provide guidelines for the institution and maintenance of a continuous subcutaneous narcotic infusion program for cancer patients with chronic pain through an analysis of the narcotic requirements and treatment outcomes of patients who underwent such therapy and a comparison of the costs of two commonly used infusion systems. Retrospective study. Tertiary care facilities and patients' homes. Of 481 patients seen in consultation for cancer pain between July 1987 and April 1990, 60 (12%) met the eligibility criteria (i.e., standard medical management had failed, and they had adequate supervision at home). Continuous subcutaneous infusion with hydromorphone hydrochloride or morphine started on an inpatient basis and continued at home whenever possible. Patient selectivity, narcotic dosing requirements, discharge rate, patient preference for analgesic regimen, side effects, complications and cost-effectiveness. The mean initial maintenance infusion dose after dose titration was almost three times higher than the dose required before infusion (hydromorphone or equivalent 6.2 v. 2.1 mg/h). Eighteen patients died, and the remaining 42 were discharged home for a mean of 94.4 (standard deviation 128.3) days (extremes 12 and 741 days). The mean maximum infusion rate was 24.1 mg/h (extremes 0.5 and 180 mg/h). All but one of the patients preferred the infusion system to their previous oral analgesic regimen. Despite major dose escalations nausea and vomiting were well controlled in all cases. Twelve patients (20%) experienced serious systemic toxic effects or complications; six became encephalopathic, which necessitated dose reduction, five had a subcutaneous infection necessitating antibiotic treatment, and one had respiratory depression. The programmable computerized infusion pump was found to be more cost-effective than the disposable infusion device after a break-even point of 8 months. Continuous subcutaneous infusion of opioid drugs with the use of a portable programmable pump is safe and effective in selected patients who have failed to respond to standard medical treatment of their cancer pain. Dose titration may require rapid dose escalation, but this is usually well tolerated. For most communities embarking on such a program a programmable infusion system will be more cost-effective than a disposable system.


Making and using medical slides.

Making and using medical slides. Despite a great proliferation of new media and technologies, the traditional live lecture is still highly regarded among scientists as an effective means of teaching and communication--and the 'lantern slide' remains the most popular lecturer's aid. Here are a few thoughts on how to create good slides and how to make the best use of them.


Cultured human thymocytes lacking CD2 and CD11a/CD18 antigens are functional and adhere to endothelial cells via CD56 or CDw49d molecules. The preferential growth of CD3-CD2-CD11a/CD18- thymocytes was obtained by stimulation of CD2-CD3- thymic cells with low doses of PMA (0.5 ng/ml) and subsequent culture in the presence of recombinant interleukin-2 (100 U/ml). After 2-3 weeks, CD3-CD2-CD11a/CD18- thymocytes represented 40-60% of the total proliferating cells. Highly purified CD3-CD2-CD11a/CD18- cell populations were obtained by depletion of the CD11a/CD18+ thymocytes by immunomagnetic beads. Moreover, these populations proliferated for 2-5 weeks and did not change their surface phenotype. It is of note that these cells, despite the lack of CD2 and CD11a/CD18 adhesion molecules, could bind to umbilical vein endothelial cells as efficiently as did CD3+CD2+CD11a/CD18+ thymocytes. Furthermore we demonstrate that (a) CD56 molecule is involved in the adhesion of CD3-CD2-CD11a/CD18- thymic cells, but not of peripheral CD3-CD56+ lymphocytes, to untreated or IFN-gamma- and/or TNF-alpha-treated endothelium, (b) anti-CDw49d mAb could inhibit the adhesion of this thymus-derived population to either IFN-gamma- or TNF-alpha-treated endothelial cells but not to untreated endothelium, and (c) CD56 antigen expressed by these cultured thymocytes has a sialic acid content different from that of peripheral lymphocytes. Indeed, isoelectrofocusing analysis showed that CD56 molecule expressed on CD3-CD2-CD11a/CD18- thymocytes displayed an isoelectric point (pI 5.0) different from that of CD56 antigen expressed by peripheral NK cells (pI 4.7 and 5.4). Further, we noted that CD56 antigen showed the same pI 5.8 after desialylation obtained using neuraminidase treatment. Finally, CD3-CD2-CD11a/CD18- thymocytes mobilized Ca2+ and released TNF-alpha and IFN-gamma after treatment with lectins.


Detection of psychiatric disorders among Asian patients presenting with somatic symptoms. Patients from India and Pakistan frequently communicate their emotional distress in terms of somatic sensations and somatic metaphors. Language and cultural difficulties may prevent recognition of psychiatric disorders. A new questionnaire is described, to assist identification of psychiatric disorders among Asian patients who present with somatic symptoms.


The major rheumatoid factor cross-reactive idiotype is dominantly expressed by Staphylococcus aureus Cowan strain I-activated CD5+ control B cells. Some seropositive (RF+) and seronegative (RF-) rheumatoid arthritis (RA) patients selectively express high concentrations of the major RF cross-reactive idiotype (RCRI) in their sera and generate high frequencies of RCRI+ pokeweed mitogen (PWM)-induced plasma cells from their peripheral blood mononuclear cells (PBM). To determine if normal individuals can express RCRI in vitro, B cells from controls were activated with Staphylococcus aureus Cowan strain I (SAC) bacteria to identify RCRI and RF production. In addition, we studied the relationship of RCRI expression with the subset of B cells bearing CD5. Control CD5+ B cells are responsible for RCRI expression following SAC activation. We also observed that RCRI is dominantly expressed by control SAC-induced B cells in frequencies comparable to that expressed by some RA and juvenile rheumatoid arthritis patients' PBM activated by PWM. Therefore, the frequency of RCRI+ B cells in control and arthritis patients' PBL may be similar, or the selection and/or regulation of RCRI+ B-cell expression in vitro and in vivo may be different in arthritis patients compared to normal individuals.


Differential expression of a CD45R epitope(6B2) on murine CD5+ B cells: possible difference in the post-translational modification of CD45 molecules. Although murine peritoneal B cells were homogenously positive for an epitope Lp-2, coded for by the alternative exon 4 of the CD45 gene, they were heterogenous with respect to the expression of another CD45R epitope, 6B2, of unknown exon dependency. While the majority of 6B2-high peritoneal B cells was composed of CD5- B cells, those with low or negative 6B2 were CD5+ B cells. Both 6B2+ and 6B2- peritoneal B cells expressed mainly the same largest CD45R transcripts, with all three alternative exon (4, 5, and 6) sequences. Further, a CD5+ B lymphoma cell line, BCL-1, which was found to be Lp-2+6B2- also had the largest isoform of CD45R molecules with all three alternate structures. Although enzyme digestion studies suggested that the 6B2 epitope resides in protein, not in sugar structures, it is likely that a post-translational modification of CD45R molecules is responsible for the presence or absence of 6B2 epitope expression on peritoneal CD5+ B cells. This event may be related to the differential role of CD45R molecules in regulating lymphocyte function.


Differential effects of cyclosporin A on diverse B cell activation phenomena triggered by crosslinking of membrane IgM. Cyclosporin A (CsA) was tested for its modulatory effects on the mIgM-mediated signaling of G0*-associated increases in class II MHC expression, G1-related RNA synthesis, and S phase-related DNA synthesis in human B cells. While CsA at concentrations as low as 10-100 ng/ml could completely ablate anti-IgM-induced DNA synthesis, earlier G1-associated RNA synthesis was only partially inhibited, and signaling of increased membrane class II MHC expression was unaffected by up to 1000 ng/ml of CsA. Similar phenomena were observed in a clonal population of leukemic B lymphocytes susceptible to anti-IgM-mediated activation in the absence of T cells and T cell factors indicating (a) that the inhibitory effects are not due to CsA-mediated suppression of cytokine production by contaminating T cells, and (b) that the varying effects of CsA on the diverse activation phenomena do not reflect B cell subpopulation diversity. Pulsing studies revealed that while maximal suppression of anti-IgM-induced G1-associated RNA synthesis required CsA at culture initiation, near maximal suppression of DNA synthesis occurred when CsA, or soluble human IgM, was added up to 30 hr after the initial exposure of resting B cells to the anti-IgM ligand. These latter findings are consistent with the possibility that the CsA-mediated suppression of S phase entry is due to the inhibition of a signaling event proximal to mIgM ligation which must be repeatedly initiated throughout the first 30 hr of activation.


Fc gamma RII/CD32-negative human Langerhans cells may be responsible for the immunostimulatory activity of freshly isolated epidermal cells. Cultured murine and human epidermal Langerhans cells (LC) undergo a phenotypical and functional maturation process. In fact, they loose Fc gamma RII and Birbeck granules, increase HLA-DR expression, and become potent accessory cells for allogeneic MLR. However, resident/freshly isolated human epidermal LC represent a phenotypically heterogeneous cell population. Indeed, a subset of CD1a+ LC lacks Birbeck granules, is Fc gamma RII/CD32-, and strongly expresses HLA-DR and the RFD1 antigen that is considered to be specific for interdigitating cells. In the present study the functional capacity of this Fc gamma RII/CD32- CD1a+ LC subset was investigated in MLR assays by comparing the stimulatory activity of freshly isolated crude epidermal cells (EC) with that of freshly isolated EC depleted in CD1a+ or in Fc gamma RII+ cells. Thereby, we observed that crude EC stimulated allogeneic PBMC while the removal of CD1a+ cells abrogated this stimulation. However, crude EC depleted in Fc gamma RII/CD32+ cells still exhibited a stimulatory capacity that was at least equal to that of crude EC. Taken together, these data suggest that among resident human epidermal LC there exists a subset of phenotypically and functionally more differentiated cells that may be solely responsible for the stimulatory capacity of freshly isolated crude EC.


Changes of expression of endogenous sugar receptors by polymorphonuclear leukocytes after prolonged anaesthesia and surgery. Anaesthesia and surgery are known to depress granulocyte function in the early postoperative period, leading to deterioration of the immune defence against infection. Carbohydrate-lectin interactions may play an important role in the activities of phagocytic cells in that they facilitate initial host defence in the event of microbial antigenic challenge. A panel of biotinylated (neo)glycoproteins (chemically glycosilated carrier proteins) was used to detect endogenous carbohydrate-binding receptors /lectins/, on peripheral blood polymorphonuclear leukocytes of patients undergoing prolonged anaesthesia for replantation surgery. Four hours after induction of anaesthesia, a progressive decline of expression of endogenous sugar receptors on granulocytes was detected using the labelled (neo)glycoproteins lactose-BSA, N-acetyl-D-glucosamine-BSA, D-mannose-BSA, sialic-acid-BSA and D-xylose-BSA. Concomitant changes in peripheral white blood cell counts and the lack of depression in the absence of general anaesthetic agents suggested the existence of a possible relationship between reduced expression of (neo)glycoprotein receptors to impaired granulocyte function and anaesthetic-induced immunodepression.


High antibody levels to the mycobacterial fibronectin-binding antigen of 30-31 kD in tuberculosis and lepromatous leprosy. Immunoblot assays showed that mycobacterial fibronectin-binding antigens are important targets of the humoral immune response in tuberculosis and leprosy. Using culture filtrate antigens of Mycobacterium tuberculosis, strong reactivity with the fibronectin-binding of 30-31 kD (Fn 30-31) was demonstrated in 55.9% of tuberculosis sera and in 56.5% of lepromatous leprosy sera. Sera from patients with tuberculoid leprosy and control sera gave very weak binding. Reactivity of tuberculosis and lepromatous leprosy sera with the fibronectin-binding antigen of 58-60 kD (Fn 58-60) was less conspicuous. The ability to react with fibronectin of the antigens of 58-60 and 30-31 kD was demonstrated by parallel labelling with a fibronectin-biotin conjugate. Fn 30-31 was purified to homogeneity by a two-step procedure and used for ELISA. Positive titres were found in 63% out of 65 tuberculosis sera and in 60.5% out of 43 lepromatous leprosy sera. Antibody titres in lepromatous leprosy sera were higher than in tuberculosis sera. Our observations indicate indirectly that M. leprae possess a highly immunogenic molecule homologous to M. tuberculosis Fn 30-31, which elicits a high antibody response in lepromatous leprosy but not in tuberculoid leprosy. In this investigation, direct evidence for the presence of this antigen in M. leprae was obtained by immunochemistry of lepromatous leprosy lesions with a monospecific antibody raised against M. tuberculosis Fn 30-31.


Specific inhibition of mRNA accumulation for lymphokines in human T cell line Jurkat by mycobacterial lipoarabinomannan antigen. The immunomodulatory effect of Mycobacterium tuberculosis-derived lipoarabinomannan (LAM) on mitogen/antigen-induced expression of mRNAs for a number of cytokines in human monocytic cell line Mono-Mac-6 and in T cell line Jurkat was investigated. Interestingly, LAM exhibited a down-regulatory effect on the accumulation of mRNAs for IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-2 receptor alpha (IL-2R alpha) in T cells co-stimulated with phytohaemagglutinin-P (PHA) and 4 beta-phorbol-12-myristyl-13-acetate (PMA). In human Mono-Mac-6 cells. LAM has a weak inhibitory effect on the lipopolysaccharide (LPS)-induced mRNA accumulation for IL-1 beta, a slight stimulatory effect on mRNAs accumulation for IL-8 and tumour necrosis factor-alpha (TNF-alpha), but clearly no effect on mRNA accumulation for intercellular adhesion molecule-1 (ICAM-1). These findings imply that LAM may contribute to the immunologic defects associated with a number of mycobacterial infections by modulating these mediators.


Safety and efficacy of repeated sequential administrations of Re-186(Sn)HEDP as palliative therapy for painful skeletal metastases. Initial case reports of two patients. Single intravenous injections of 30 to 35 mCi (1,110 to 1,295 MBq) of Re-186(Sn)HEDP previously have been shown to result in palliation of painful skeletal metastases from prostate cancer. There are no reports of patients receiving repetitive Re-186(Sn)HEDP therapy. We have followed two such patients who received multiple (five to seven) injections of Re-186(Sn)HEDP at 2-month intervals. Each experienced a sustained decrease in both pain and analgesic intake. The only evident clinical or biochemical toxicity was a mild progressive decline in their total platelet counts.


Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59. CD59, the membrane regulator of autologous C5b-9 channel formation, exhibits variable sensitivity to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that releases glyco-inositolphospholipid (GPI)-anchored proteins from cell surfaces. To determine whether the GPI-anchor phospholipid of CD59 is similar to that of decay-accelerating factor (DAF) and whether variation in its structure underlies its variable enzyme susceptibility, the GPI anchors of the two proteins expressed on erythrocytes, polymorphonuclear and mononuclear leucocytes were compared in situ and after purification. Flow cytometric analyses of PI-PLC-treated cells showed parallel cell type specific release of both proteins as a function of enzyme concentration. Non-denaturing PAGE analyses of alkaline/hydroxylamine-treated proteins (affinity-purified from [125I]-surface-labelled cells) provided evidence for (i) comparable proportions of GPI-anchor acylation, and (ii) alkali-resistant rather than alkali-sensitive lipid substituents in erythrocytes. These findings argue that the differential C5b-9 sensitivity that distinguishes paroxysmal nocturnal haemoglobinuria II and III erythrocytes does not derive from expression of CD59 molecules with alternative GPI-anchor phospholipid structures.


Stimulation by TGF beta of chick embryo fibroblasts--inhibition by an IGFBP-3. The multiple effects of TGF beta on cell proliferation are not well understood. Our results show that TGF beta was a good but transient mitogen for chick embryo fibroblasts. DNA synthesis was three- to fourfold increased, even at high concentrations of TGF beta. We did not show a bimodal effect. An inhibitor of cell growth, that inhibits 100% of stimulation induced by serum in CEF, was purified to homogeneity from medium conditioned by mouse 3T3 cells. This inhibitor has been shown to be an IGF-binding protein (mIGFBP-3). In the present work, this mIGFBP-3 inhibited the TGF beta stimulation by about 50%, while the stimulation induced by PDGF or insulin was not inhibited by mIGFBP-3. Furthermore, TGF beta stimulation, in the presence of a high concentration of insulin in conditions which would saturate IGF receptors, was not significantly inhibited by mIGFBP-3. All together these results suggest that a part of the mitogenic effect of TGF beta may be through increasing IGF secretion and eventually other growth factors such as PDGF (as suggested previously).


Transdifferentiation of murine squamous vaginal epithelium in proestrus is associated with changes in the expression of keratin polypeptides. The superficial layers of the stratified squamous epithelium of the murine vagina undergo transdifferentiation into cuboidal mucinous cells during the proestrus phase of the normal estrous cycle. In contrast to their squamous progenitor cells which have the cytoskeletal characteristics of squamous epithelium, mucinous cells express keratin polypeptides typical of simple nonstratified epithelia. Accordingly, the transdifferentiation of squamous cell into mucinous cells involves not only a change in cell morphology but also a switch in the expression of keratin polypeptides. These data indicate that the stratified squamous cells of the vagina are not terminally differentiated and their phenotype can be hormonally modulated.


Induction of tyrosinase in human melanoma cells by L-tyrosine phosphate and cytochalasin D. Pigmentation of RVH 421 human melanoma cells is induced when cell division is inhibited by cytochalasin D or L-tyrosine phosphate. Increased pigmentation correlates with increased tyrosinase activity when this is monitored over a time-course. Parallel measurements show that the amount of tyrosinase mRNA correlates with enzyme activity in cells growing without these additives. In contrast, in the presence of cytochalasin D or L-tyrosine phosphate, the increase in amount of tyrosinase mRNA is not sufficient to account for the increase in enzyme activity, indicating that these compounds act mainly at a post-transcriptional level.


The role of the IGF1 receptor in the regulation of cdc2 mRNA levels in fibroblasts. The levels of cdc2 mRNA increase when quiescent cells are stimulated by growth factors. In BALB/c 3T3, both platelet-derived growth factor and insulin-like growth factor 1 (IGF-1) are required to increase cdc2 mRNA levels. In p6 cells, which constitutively overexpress the IGF-1 receptor, IGF-1 is sufficient. The importance of the IGF-1/IGF-1 receptor interaction in regulating the levels of cdc2 mRNA was further confirmed by showing that an antisense oligodeoxynucleotide to the IGF-1 receptor RNA inhibited the IGF-1-mediated increase.


Assembly and nuclear transport of the U4 and U4/U6 snRNPs. We have analyzed the assembly of the spliceosomal U4/U6 snRNP by injecting synthetic wild-type and mutant U4 RNAs into the cytoplasm of Xenopus oocytes and determining the cytoplasmic-nuclear distribution of U4 and U4/U6 snRNPs by CsCl density gradient centrifugation. Whereas the U4 snRNP was localized in both the cytoplasmic and nuclear fractions, the U4/U6 snRNP was detected exclusively in the nuclear fraction. Cytoplasmic-nuclear migration of the U4 snRNP did not depend on the stem II nor on the 5' stem-loop region of U4 RNA. Our data provide strong evidence that, following the cytoplasmic assembly of the U4 snRNP, the interaction of the U4 snRNP with U6 RNA/RNP occurs in the nucleus; furthermore, cytoplasmic-nuclear transport of the U4 snRNP is independent of U4/U6 snRNP assembly.


Kinetics of rG-CSF-induced neutrophilia in mice. To evaluate the mechanism of neutrophilia by granulocyte colony-stimulating factor (G-CSF), kinetic studies with tritiated thymidine ([3H]TdR) were performed in mice. G-CSF increased the number of circulating metamyelocytes and band and segmented neutrophils after the daily injection of G-CSF. The production of neutrophils has been estimated from the number of postmitotic neutrophils and the marrow transit time of [3H]TdR-labeled neutrophils on day 4 of daily G-CSF injection. The number of postmitotic neutrophils was determined by the radioiron dilution method and by the neutrophil and erythroid ratio in bone marrow smears. The mean values for marrow postmitotic neutrophils in the G-CSF-treated group and the controls were 71 +/- 50 x 10(7) cells and 9.3 +/- 4.2 x 10(7) cells (mean +/- SD), respectively. The mean marrow transit time derived from myelocyte metamyelocyte transition time and the emergence of [3H]TdR-labeled cells into the peripheral blood was 45 h in the G-CSF group and 116 h in the controls. Thus, the daily neutrophil turnover was estimated to be 38 x 10(7) cells/day and 1.9 x 10(7) cells/day, respectively. Our results suggest that C-CSF markedly increases the neutrophil precursors and shortens the transit time in the mitotic and postmitotic pool.


Interleukin 1-induced sequential myelorestoration: dynamic relation between granulopoiesis and progenitor cell recovery in myelosuppressed mice. The myelorestorative effect of recombinant human interleukin 1 alpha (IL-1 alpha) was studied in mice treated with anticancer drugs. The treatment of mice with 5-fluorouracil (5-FU; 250 mg/kg body weight) or cyclophosphamide (CPA; 100 mg/kg) considerably decreased bone marrow or splenic colony-forming units in culture (CFU-C) or neutrophils in blood. The daily administration of IL-1 alpha (100 ng/mouse/day) after 5-FU treatment markedly accelerated the recovery of bone marrow CFU-C. This increase was followed by the recovery of splenic CFU-C and neutrophils in blood. In the CPA-treated mice the recovery of bone marrow CFU-C over the normal level was observed regardless of the administration of IL-1 alpha 3 days after CPA treatment. The daily administration of IL-1 alpha after CPA treatment markedly increased splenic CFU-C or neutrophils over the normal level following the increase of bone marrow CFU-C. Thus, in both 5-FU- and CPA-treated mice, the increase of bone marrow CFU-C after the administration of IL-1 alpha was observed several days earlier than the increase of splenic CFU-C and neutrophils in blood. The increase of splenic CFU-C and neutrophils in blood was observed concomitantly. The experiments, in which effective timing of IL-1 alpha injection was examined, indicated that the administration of IL-1 alpha within a few days after treatment with anticancer drugs was necessary for the accelerated recovery of bone marrow progenitor cells. On the other hand, the effective recovery of splenic progenitor cells and peripheral neutrophils required the administration of IL-1 alpha after the increase of bone marrow progenitor cells. Thus, the administration of IL-1 alpha daily or every other day was the most effective for recovery from myelosuppression induced by anticancer drugs.


Recombinant interleukin 3 induces interleukin 2 receptor expression on early myeloid cells in normal human bone marrow. Human interleukin 3 (IL-3) is a multipotential cytokine that supports the growth of early hematopoietic progenitors and promotes their response to other, later-acting cytokines. We found that IL-3 was able to induce the expression of interleukin 2 (IL-2) receptor (IL-2R) (CD25) on a subset of early myeloid cells in normal human bone marrow that had been first depleted of mature hematopoietic cells and E-rosette-positive T cells by treatment with soybean lectin and sheep erythrocytes (SBA-E-BM). Immunofluorescence analysis revealed that the CD25+ cells were contained almost entirely within the lymphoblastoid gate of the IL-3-cultured marrow. CD25 was undetectable on freshly isolated marrow and less than 10% CD25+ cells could be detected following liquid culture at 37 degrees C in the presence of 10% human serum, 10% fetal calf serum, or under serum-free conditions. Addition of IL-3 (100 U/ml) significantly increased the expression of CD25 to 37%, 31%, and 24%, respectively. CD25 could also be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), but no IL-2R was detectable following exposure to granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1), interleukin 4 (IL-4), or IL-2. Expression of CD25 was dependent on the dose of IL-3 or GM-CSF added and was maximal within 24 h of exposure. Two-color immunofluorescence analysis demonstrated that CD25 was not expressed by cells of lymphoid lineage or by mature monocytes, but rather was present on cells that coexpressed CD13, CD33, CD34, MY8, and HLA-DR, and that lacked CD14 or CD11b, thus placing the CD25+ cells at or near the myeloblast stage of differentiation. An identical phenotype was found for CD25+ cells induced by GM-CSF. Cycloheximide completely inhibited the IL-3-induced expression of CD25, indicating the necessity for protein synthesis, and although most of the CD25+ cells were in G0/G1 phase, 25% of the cells were in S or G2M phase, indicating that receptor expression was not cell-cycle dependent. The p75 chain of IL-2R was not detected on the CD25+ cells. IL-3 was also found to directly induce CD25 in greater than 46% of SBA-E-BM enriched for CD34+ cells by panning. Consistent with the expression of only p55 IL-2R, the functional activity of IL-2 on enriched CD34+ cells exposed to IL-3 could not be demonstrated in either granulocyte-macrophage colony-forming unit (CFU-GM) assays or proliferation assays.(ABSTRACT TRUNCATED AT 400 WORDS)


Colony-stimulating activity in the serum of patients with hemopoietic malignancies after intensive chemotherapy/radiotherapy: its augmentation by GM-CSF in vivo and interleukin 4 in vitro. Colony-stimulating activity (CSA) in the serum of patients with hematological malignancies increased substantially after intensive therapy with cyclophosphamide/busulfan, cyclophosphamide/total body irradiation, or melphalan/total body irradiation. This was not dependent on patients receiving allogeneic bone marrow transplantation (ABMT) or autologous bone marrow rescue (ABMR). In 44 of 62 patients CSA was maximum approximately 7 days after chemotherapy/radiotherapy, whereas in 18 of 62 patients CSA was maximum between 9 and 20 days after therapy and decreased thereafter. The time course of CSA was not dependent on disease and was not affected by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) given as a continuous infusion for 14 days after therapy; however, serum from patients receiving rhGM-CSF produced significantly more colonies from donor bone marrow than serum from patients who did not receive the cytokine (p = 0.013). Despite the early peak in CSA in the majority of patients, there was no correlation between the time at which CSA was maximum and the return of patients' neutrophils to 500/microliters. Recombinant human interleukin 4 (IL-4) increased the number of granulocyte-macrophage colony-forming unit colonies, principally granulocyte colony-forming unit colonies, from normal bone marrow exposed to patients' serum after intensive therapy and antibody to GM-CSF reduced colony numbers. The results suggest that after intensive therapy granulocyte colony-stimulating factor (G-CSF) as well as GM-CSF is released into the serum and, in addition to acting directly with G-CSF, IL-4 may stimulate mononuclear cells to produce and/or release G-CSF.


Influence of combinations of cytokines on proliferation of isolated single cell-sorted human bone marrow hematopoietic progenitor cells in the absence and presence of serum. Human mast cell growth factor (MGF, a c-kit ligand) and colony stimulating factors (Epo, GM-CSF, G-CSF, IL-3) were assessed in the absence or presence of serum for stimulation in semi-solid medium of single CD34 , CD34 HLA-DR+, or CD34 HLA-DR+CD33- cells sorted per microtiter well. The % of wells containing CFU-GM and erythroid containing (BFU-E and CFU-GEMM) colonies increased in proportion to the number of cytokines added. In the presence of serum, 1, to 4 cytokine combinations resulted in respective increases in cloning efficiencies of 10 to 21.0, 19.5 to 31.5, 35.8 to 42.9, and 46.3 to 60.0%. MGF had little effect by itself, but did act in combination with CSFs to enhance numbers and size of the colonies from isolated single cells. High cloning efficiencies were also obtained in the absence of serum when multiple cytokines were used. The results demonstrate that MGF and CSFs can act directly on the proliferation of single hematopoietic progenitor cells in the absence of accessory cells and serum.


'boxA'-like sequence between the 16 S/23 S spacer in rRNA operon of mycoplasmas. We have found that a boxA-like sequence is conserved in the 16 S and 23 S rRNA intergenic spacer regions of mycoplasmas, and that it always locates on loop regions of the hypothetical secondary stem-loop structures. A nucleotide sequence similar to the '-10' box of prokaryotic promoters was identified at upstream sites of the boxA-like sequence in the 16 S/23 S spacer regions. These structures may represent an internal promoter between the 16 S and 23 S rRNA genes in mycoplasmas.


Native-type DHP-sensitive calcium channel currents are produced by cloned rat aortic smooth muscle and cardiac alpha 1 subunits expressed in Xenopus laevis oocytes and are regulated by alpha 2- and beta-subunits. Native tissue-like L-type voltage-dependent calcium channels (L-VDCC's) were expressed by in vitro transcribed cRNA injection of rat aorta or rabbit cardiac alpha 1 subunit into Xenopus laevis oocytes. Co-injection of VSM-alpha 1 with the cloned skeletal muscle beta-subunit (SK-beta) of the L-type VDCC significantly increased the expressed peak current amplitude without significant changes in kinetics. Similar results were obtained by co-injection of cardiac alpha 1 (DSHT-alpha 1) the cloned skeletal alpha 2-subunit (SK-alpha 2) or with SK-beta. The oocytes co-expressing cRNA's retained L-type VDCC pharmacology.


Mechanism and site of action of a ribosome-inactivating protein type 1 from Dianthus barbatus which inactivates Escherichia coli ribosomes. A single chain ribosome-inactivating protein with RNA N-glycosidase activity, here named Dianthin 29, was isolated from leaves of Dianthus barbatus L. Incubation of intact Escherichia coli ribosomes with Dianthin 29 and subsequent aniline treatment of the isolated rRNA releases a rRNA fragment of 243 nucleotides from 23 S rRNA. Nucleotide sequence studies showed that the site of N-glycosidic bond cleavage is at A-2660 within the universally conserved sequence 5'-AGUACGAGAGGA-3' near the 3'-end of 23/28 S rRNAs. To our knowledge, Dianthin 29 is the first ribosome-inactivating protein which is shown to inactivate intact prokaryotic ribosomes in the same manner as eukaryotic ribosomes.


Characterization of Le(x), Le(y) and A Le(y) antigen determinants in KDN-containing O-linked glycan chains from Pleurodeles waltlii jelly coat eggs. Novel acidic oligosaccharides were isolated in large amounts by reductive alkaline treatment of the jelly coat of Pleurodeles waltlii (Michah) eggs. The oligosaccharides were found to contain the newly described KDN as acidic monosaccharide and possess either the Le(x), Le(y) and A Le(y) antigenic determinants. Occurrence of Le(x) and Le(y) determinants previously recognized as tumor-associated antigen (TAA) demonstrates that mucins of lower animals may represent a rich and easily available source for preparing TAA. Moreover, it reinforces the hypothesis according to which TAA are evolution markers.


Synthesis of alpha 1-microglobulin in cultured rat hepatocytes is stimulated by interleukin-6, leukemia inhibitory factor, dexamethasone and retinoic acid. The secretion of alpha 1-microglobulin by primary cultures of rat hepatocytes was found to increase upon the addition of interleukin-6 or leukemia inhibitory factor, two mediators of acute phase response. This stimulatory effect was further enhanced by dexamethasone. alpha 1-Microglobulin is synthesized as a precursor also containing bikunin, and the precursor protein is cleaved shortly before secretion. Our results therefore suggest that both alpha 1-microglobulin and bikunin are acute phase reactants in rat hepatocytes. Furthermore, we found that retinoic acid, previously shown to be involved in the regulation of cell differentiation and development, also stimulated alpha 1-microglobulin synthesis. Only free, uncomplexed alpha 1-microglobulin (28,000 Da) was detected in the hepatocyte media, suggesting that the complex between alpha 1-microglobulin and alpha 1-inhibitor 3, found in rat serum, is formed outside the hepatocyte.


The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor. The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate-limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two-stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231-7, which was shown to be reactive with amino acid residues 51-88 of the first epidermal growth factor-like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose-dependent manner. The mechanism of this inhibition was demonstrated using a novel solid-phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231-7, again in a dose-dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the beta-galactosidase fusion proteins containing amino acid residues 51-88 (exon 4) of human factor VII. We conclude therefore that the exon 4-encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.


cDNA clones encoding leucine-zipper proteins which interact with G-CSF gene promoter element 1-binding protein. The gene expression of granulocyte colony-stimulating factor (G-CSF) is induced by lipopolysaccharide (LPS). GPE1, a cis-controlling element of the G-CSF gene, functions as an LPS-responsive element. GPE1-binding protein (GPE1-BP), a leucine-zipper protein, did not independently activate G-CSF gene expression. Protein blot analysis with biotinylated GPE1-BP revealed that there were nuclear proteins that interact specifically with GPE1-BP. Three leucine-zipper proteins were isolated from mouse cDNA expression libraries by this method: NF-IL6, ATF4, and a novel ATF4-related ATFx. The interactions of these proteins with GPE1-BP may play key roles in G-CSF gene expression.


Interaction of cholesterol-conjugated alkylating oligonucleotide derivatives with cellular biopolymers. Interactions of oligonucleotide derivatives with mammalian cells and cellular biopolymers have been investigated. The derivatives were oligonucleotides bearing an alkylating 2-chloroethylamino group at the 3'-end and a cholesterol residue at the 5'-terminal phosphate. These compounds are readily taken up by cells and react with cellular DNA, RNA and some proteins which may play a role in delivery of the compounds into cells.


Human centrosomal epitope is shared specifically with human lactate dehydrogenase-B isozyme. A rabbit serum (0013) used to identify pericentriolar proteins from isolated centrosomes (Gosti-Testu, F., Marty, M.C., Berges, J., Maunoury, R. and Bornens, M. (1986) EMBO J. 5, 2545-2550) was shown also to react through the same epitope with several non-centrosomal proteins including a major 36 kDa cytosolic antigen. This protein was identified to be human lactate dehydrogenase and the co-distribution of 0013 epitope on the centrosomal protein and on lactate dehydrogenase (LDH) was shown to be specific for human cells (Gosti, F., Marty, M.C., Courvalin, J.C., Maunoury, R. and Bornens, M. (1987) Proc. Natl. Acad. Sci. USA 84, 1000-1004). Human hepatic cells constitute, so far, the only exception to this co-distribution rule. By using this cell type which expresses only the LDH-A4 isozyme, we demonstrate that 0013 epitope is specific for the human LDH-B subunit, making serum 0013 the strongest anti-LDH-B available so far. The evolutionary and physiological significance of this situation is discussed.


Immunological modulation of lymphocyte subpopulation in cervical cancer tissue by sizofiran and OK-432. The intimate correlation between marked lymphocyte infiltration in the cancer tissue and the superior clinical prognosis has been reported in human cancers. Sizofiran (SPG), a polysaccharide consisting of beta-1,3-D-glucopyranosyl linkage with beta-1,6-D-glucopyranosyl branches, or OK-432, a heat- and penicillin-treated, lyophilized preparation of the Su strain of Streptococcus pyogenes of human origin, was administered intratumorally to nine patients with advanced uterine cervical carcinoma before radical hysterectomy to assess their immunological modulating properties to tumor-infiltrating lymphocyte subpopulations immunohistochemically. The degree of infiltration of CD3- and CD5-positive cells increased moderately or markedly in the stroma surrounding the lesion in both SPG group and OK-432 group, as well as that of CD4- and CD8-positive cells, from that before administration. The density of infiltration of CD3-positive, CD4-positive, and CD8-positive cells in the lesion increased slightly or moderately in both groups after administration. As for CD16-positive cell infiltration, both SPG and OK-432 increased its degree in the stroma lining the lesion while SPG alone augmented its degree in the lesion. This augmentation of lymphocyte infiltration in situ induced by intratumoral administration of SPG and OK-432 is expected to lead to a favorable prognosis of patients with cervical cancer.


Preliminary results of concurrent radiotherapy and chemotherapy with cis-platinum, vincristine, and bleomycin in bulky, advanced cervical carcinoma: a pilot study. Twenty-four patients with bulky (greater than 4 cm), advanced (stages IIB-IVA) carcinoma of the uterine cervix were prospectively treated with a concurrent combination of radiotherapy (RT) and chemotherapy (CT). RT consisted of 4400 cGy (22 fractions) to the whole pelvis and a 1400-cGy boost to the parametrium. This was followed by two to three intracavitary brachytherapy courses. CT consisted of one to four course (median, three) of cisplatin (50 mg/m2) on Day 1, vincristine (1 mg/m2) on Day 2, and bleomycin (25 mg/m2) on Days 2-4. CT was started on the first day of external radiation and the scheduled course interval was 21 days. Among the 20 evaluable patients who completed at least one course of chemotherapy and a full course of radiation, 13 (65%) achieved complete response and 5 (25%) had partial response. Fatal complication occurred in 1 patient with stationary disease who died of septic shock due to ruptured pyometra. The other patient with primary stage IVA disease had progressive disease with ascites appearance after two courses of CT and later expired. Transient drug fever occurred in 19 (40.4%) of the 47 bleomycin-containing CT cycles. Grade 2 or 3 hematological toxicities occurred in 16 (30.2%) of a total of 53 CT cycles. Treatment delays of 1 to 7 days occurred in 15 (28.3%) CT cycles. Except for the case of septic shock, all of the other toxicities were generally tolerable and reversible. From this preliminary result we concluded that this particular combination of RT and CT in bulky, advanced cervical carcinoma is effective in enhancing local pelvic tumor control and well tolerated if strict selection of accrued patients is applied. Further investigation to assess its impact on long-term survival is in progress.


Use of designer recombinant mitochondrial antigens in the diagnosis of primary biliary cirrhosis. The appearance of autoantibodies against mitochondria in patients with primary biliary cirrhosis has been known for more than 25 yr. In the past, based on the biochemical complexity of the mitochondrion and the use of crude extracts for immunodiagnosis, a degree of nonspecificity in assaying for antibodies to mitochondria has been present. This problem has been largely circumvented by the cloning of the mitochondrial antigens and the identification of the E2 subunits of the pyruvate dehydrogenase complex and the branched chain 2-oxo-acid dehydrogenase complex as the major and immunodominant autoantigens of primary biliary cirrhosis. More than 90% of patients with primary biliary cirrhosis have been shown to react with one or both of these enzymes using either recombinant antigen or purified native protein. Approximately 10% of patients recognize only E2 subunits of branched chain 2-oxo-acid dehydrogenase complex and not pyruvate dehydrogenase complex. Such patients would be missed by diagnostic assay that has a low sensitivity to antibodies against E2 subunits of branched chain 2-oxo-acid dehydrogenase complex. The use of recombinant and biochemically pure antigens has permitted structural and conformational analysis of epitope mapping. We have taken advantage of the antigenic mapping studies of both primary biliary cirrhosis and branched chain 2-oxo-acid dehydrogenase complex E2 subunits and designed a molecule that expresses the immunodominant epitopes of both. Using this dual-headed molecule that coexpresses the epitope of two different antigens, we report herein a sensitive and reproducible assay for antibodies to mitochondria in patients with primary biliary cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)


Polyunsaturated lecithin prevents acetaldehyde-mediated hepatic collagen accumulation by stimulating collagenase activity in cultured lipocytes. We recently found that polyunsaturated lecithin prevents ethanol from causing cirrhosis in the baboon. Because transformation of lipocytes to transitional cells plays a key role in hepatic fibrogenesis in vivo, and because this process in alcohol-fed baboons was found to be attenuated by polyunsaturated lecithin, we focused on lipocytes to study the mechanism of the protective effect. Rat lipocytes cultured on plastic undergo spontaneous activation, accompanied by expression of alpha-smooth muscle actin isoform and production of substantial amounts of type I collagen. The latter was further increased on incubation with acetaldehyde. This in vitro model was used here to study how acetaldehyde-mediated collagen production and accumulation can be turned off. Addition of polyunsaturated lecithin (10 mumols/L) was found to prevent the acetaldehyde-induced increase in collagen accumulation by 83% (p less than 0.001). By contrast, a saturated phospholipid (10 mumols/L dilauroyl phosphatidylcholine), a monounsaturated one (10 mumols/L linoleoyl-palmitoyl phosphatidylcholine) or linoleic acid (20 mumols/L bound to albumin) had no such effect. Incorporation of [3H]proline into collagen and the expression of alpha-1 (I) procollagen mRNA were increased by acetaldehyde; the latter was not significantly affected by polyunsaturated lecithin. Polyunsaturated lecithin increased lipocyte collagenase activity by 100% (p less than 0.001), whereas dilauroyl phosphatidylcholine, linoleoyl-palmitoyl phosphatidylcholine and linoleic acid had no such action. We concluded that (a) polyunsaturated lecithin selectively prevents the acetaldehyde-induced increase in collagen accumulation in lipocyte cultures, whereas other phospholipids or linoleate have no such effect; and (b) polyunsaturated lecithin does not modify the acetaldehyde-mediated increase in alpha-1 (I) procollagen mRNA, but it increases collagenase activity, suggesting that the protective effect exerted by polyunsaturated lecithin against alcohol induced fibrosis in vivo is due at least in part to stimulation of collagenase activity, which may prevent excess collagen accumulation by offsetting increased collagen production.


Improved serodiagnosis of non-A, non-B hepatitis by an assay detecting antibody to hepatitis C virus core antigen. We examined sequential serum samples from 12 patients with well-characterized posttransfusion non-A, non-B hepatitis who had an acute, resolving self-limited type of clinical course for the presence of antibody to the hepatitis C virus nucleocapsid (core) protein (p22) expressed by a recombinant baculovirus. These sera were simultaneously examined for antibody to the hepatitis C virus nonstructural protein (C100-3) that is presently used for blood screening worldwide. In three patients, both anti-p22 and anti-C100-3 antibodies were detected, but anti-p22 was detected much earlier. In four patients, only anti-p22 was detected. Two other patients were considered to be hepatitis C virus carriers who had been already infected with hepatitis C virus. In one patient, only anti-C100-3 was detected, and it was transient. In two patients, neither antibody was detected. Anti-p22 was detected in at least one of eight samples of transfused blood. Of the nine samples of donated blood that were positive for anti-p22, only four were positive for anti-C100-3. This new assay detecting the antibody to the p22 protein is thus useful for the serodiagnosis of non-A, non-B hepatitis in the acute phase and for blood screening.


Differences in the abundance of variably spliced transcripts for the second asialoglycoprotein receptor polypeptide, H2, in normal and transformed human liver. The human hepatic asialoglycoprotein receptor comprises two homologous polypeptides designated H1 and H2. Two distinct complementary DNA clones encoding these receptor subunits have been previously isolated from the human hepatoblastoma cell line HepG2. We discovered that multiple variants of H2 transcripts exist both in HepG2 cells and in the normal human liver that, at least in part, appear to be the result of alternative splicing events. We have found that (a) the complementary DNA clone for H2 previously isolated from HepG2 cells, characterized by a 57-nucleotide insertion within the 5' end of the complementary DNA that is absent from H1, represented only one third of H2-related sequences in an unamplified normal human liver complementary DNA library and less than 10% of H2 clones in HepG2 cells; (b) the predominant message for H2 expressed in the liver and HepG2 cells, designated L-H2, appeared to represent the fully processed product of the gene encoding both L-H2 and H2; and (c) a variant H2 transcript existed in HepG2 cells, designated H2', that contained a novel, 5' 88-bp nucleotide insertion. Poly(A+) RNA analysis of the normal liver and HepG2 cells by complementary RNA hybridization and ribonuclease protection corroborated the observations made during the screening of complementary DNA libraries regarding the abundance of the various messages. A striking incongruity was found between the levels of messenger RNA containing the H2-specific 57-nucleotide sequence and the levels of polypeptide expressed in the liver and HepG2 cells as recognized by antiserum specifically raised against this sequence.


Immunohistochemical study of extracellular matrix in acute galactosamine hepatitis in rats. A single injection of D-galactosamine hydrochloride induces acute self-limiting liver disease in rats that morphologically resembles drug-induced hepatitis in human beings. In this immunohistochemical study we examined the localization and expression of the hepatic extracellular matrix components fibronectin, laminin, collagen type I, collagen type III and collagen type IV and of the cell surface receptors (integrins) for fibronectin and laminin. Sections of liver tissue obtained at intervals of 6, 12, 18, 24, 30, 36, 48 and 72 hr and 7 and 21 days after galactosamine administration were immunostained with a panel of polyclonal monospecific antibodies and studied independently by two of us. Fibronectin was the first extracellular matrix component found to be increased, 12 hr after galactosamine injection, followed by collagen type III, and, in a later phase, collagen type IV, type I and laminin. Increased deposition of extracellular matrix was found in areas with liver cell necrosis and along sinusoids. Extracellular matrix immunoreactivity reached a maximum at 36 to 48 hr and decreased thereafter to preinjury levels 3 wk after galactosamine. Immunostaining for the fibronectin and laminin receptors revealed tissue localization identical to that of their ligands. However, the intensity of staining was opposite of that for the extracellular matrix, with a decrease of immunoreactivity after 24 to 48 hr. The observed sequence of changes in hepatic extracellular matrix proteins after galactosamine injection resembles the repair reaction in other tissues and may reflect the particular function that each carries out during the process of liver healing after toxic injury.


Distribution of Leu-7 antigen (HNK-1) in thyroid tumors: its usefulness as a diagnostic marker for follicular and papillary carcinomas. The reactivity of the anti-Leu-7 monoclonal antibody was tested in 39 neoplastic and nonneoplastic thyroid tissue specimens. These included eight colloid goiters, 14 follicular adenomas, nine papillary carcinomas, five follicular carcinomas, two medullary carcinomas, and one metastatic follicular carcinoma in bone. We observed strong cytoplasmic and/or cell membrane positivity in all follicular and papillary carcinomas, in both primary and metastatic tumors. However, the medullary thyroid carcinomas tested were negative. We also observed weak and only occasional staining with anti-Leu-7 antibody in colloid goiter and follicular adenomas. The staining in the benign thyroid lesions was usually focal, less than 10% of the cells; however, in cases of follicular and papillary carcinomas, both in primary and metastatic tumors, the staining was diffuse and strong. Some of the colloid material in colloid goiters and follicular adenomas showed faint homogenous staining with anti-Leu-7 antibody. Our findings suggest that anti-Leu-7 monoclonal antibody is a marker that may facilitate the differentiation between benign and malignant thyroid lesions, with the exception of medullary carcinoma. In addition, caution should be taken when using this antibody to diagnose metastatic lesions, as other metastatic carcinomas have also been reported to be positive. This antibody should be used in conjunction with a panel of antisera to complement a morphologic diagnosis.


Anti-CD34 immunoperoxidase staining in paraffin sections of acute leukemia: comparison with flow cytometric immunophenotyping. Anti-CD34 is a monoclonal antibody that reacts with bone marrow progenitor cells and leukemic blasts, and is expressed on 30% to 50% of all acute leukemias. Detection of CD34 has previously been restricted to flow cytometric studies. To expand the utility of CD34, we immunostained 46 paraffin-embedded bone marrow specimens with acute leukemia; results were compared with flow cytometric studies. CD34 reactivity was also evaluated in nine chronic leukemia cases, 27 malignant lymphoma cases (Hodgkin's disease and non-Hodgkin's lymphoma), six normal bone marrow specimens, and three benign, hyperplastic lymph node specimens. All cases that were CD34 positive by flow cytometry (11 of 19 B-cell precursor acute lymphoblastic leukemia cases, one of six T-cell acute lymphoblastic leukemia cases, and seven of 21 acute myeloblastic leukemia cases) were also CD34 positive in paraffin sections. Both cell membrane and cytoplasmic staining was seen. The positivity percentage and fluorescence intensity by flow cytometry correlated with the estimated number of stained cells and the intensity of immunoperoxidase staining in 18 of 19 CD34-positive cases. The remaining bone marrow and lymph node cases studied were CD34 negative; prominent endothelial cell staining, however, was noted. This is the first report of anti-CD34 staining of acute leukemia in paraffin-embedded sections. In contrast to other monoclonal antibodies reactive in bone marrow paraffin sections with leukemia, anti-CD34 immunoperoxidase staining is limited to leukemic blasts and may provide useful diagnostic information when flow cytometric studies are not available.


Molecular mechanisms of trenimon-induced cytotoxicity in resistant L5178Y/HBM10 cells. L5178Y/HBM10 lymphoblasts, resistant to a model quinone antitumor agent, hydrolyzed benzoquinone mustard, were approximately 2-fold more sensitive to trenimon (2,3,5-tris-ethyleneimino-1,4-benzoquinone) compared to parental cells (L5178Y). The L5178Y/HBM10 cells are reported to have a 24-fold increased level of DT-diaphorase activity over the parental cells. Inhibition of DT-diaphorase by dicoumarol markedly inhibited the cytotoxic activity of trenimon to the resistant L5178Y/HBM10 cells. Spectrophotometric analysis of the reduction of the quinone, trenimon, to its hydroquinone form was shown to occur approximately 25 times more rapidly in the L5178Y/HBM10 cells relative to the parental cells and was inhibited by discoumarol. Trenimon also induced continuous cyanide-resistant respiration in the L5178Y cells, but not in the resistant L5178Y/HBM10 cells. This suggested a one-electron reduction of trenimon to a semiquinone free radical which could then redox cycle with oxygen in the L5178Y cells. However, in the presence of dicoumarol the resistant L5178Y/HBM10 cells induced similar oxygen activation to the parental cells. Dicoumarol had no effect on trenimon-induced cyanide resistant respiration in the parental cells. These findings suggest that the two-electron reduction of trenimon to its hydroquinone derivative plays a major role in the cytotoxic activity of trenimon.


Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival. These studies tested whether WR-2721 could be used to protect hemopoietic stem cells, which after irradiation could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered WR-2721 (4 mg/mouse, i.p.) 30 min before 60Co irradiation and G-CSF (2.5 micrograms/mouse/day, s.c.) from days 1-16 after irradiation. In survival studies, saline, G-CSF, WR-2721, and WR-2721 + G-CSF treatments resulted in LD50/30 values of 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. At these LD50/30 values, the dose reduction factor (DRF) of 1.64 obtained in combination-treated mice was more than additive between the DRF's of G-CSF-treated mice (1.06) and WR-2721-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell (CFU-s) and granulocyte-macrophage progenitor cell (GM-CFC) recoveries were also accelerated most in mice treated with WR-2721 + G-CSF. In addition, mice treated with WR-2721 + G-CSF exhibited the most accelerated peripheral blood white cell, platelet, and red cell recoveries. These studies (a) demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from WR-2721-protected stem and progenitor cells, increasing the survival-enhancing effects of WR-2721 and (b) suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce risks associated with myelosuppression induced by radiation or radiomimetic drugs.


Expression of 85-kDa protein of adriamycin-resistant tumor cells during hematopoietic differentiation of THP-1 and HEL cells. An 85-kDa protein was identified in adriamycin-resistant tumor cells recognized by monoclonal antibody MRK-20. Recently, the monoclonal antibody MRK-20 was found to be reactive to human peripheral mononuclear cells. In order to investigate the molecular function of the 85-kDa protein, we carried out flow cytometric analysis of the expression of the 85-kDa protein during monocytic differentiation of the hematopoietic cells. Human myelomonocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). A maximum of 55% of the cells expressed the 85-kDa protein along with the CD-14 antigen, which is a surface marker of monocytes and macrophages. The kinetic analysis revealed that the 85-kDa protein appeared prior to the expression of the CD-14 antigen. The 85-kDa protein was also coexpressed with CD-14 in THP-1 cells that were induced to differentiate by recombinant tumor necrosis factor-alpha, which is one of the physiological inducers of monocytic differentiation. In human erythroleukemia, HEL cells, the 85-kDa protein was constitutively coexpressed with CD-14. The expression of both the 85-kDa protein and CD-14 was drastically reduced during the megakaryocytic differentiation of the HEL cells with TPA. These results suggest that the 85-kDa protein could be expressed on monocytic cells as well as CD-14 and that the expression of the 85-kDa protein might be regulated at an earlier stage of monocytic differentiation of hematopoietic cells than the expression of CD-14.


Detection of the ligand activity of the c-erbB-2 protein in calf serum. We established NIH3T3 derivatives in which wild-type c-erbB-2 or activated c-erbB-2 having a point mutation in the sequence coding for the transmembrane domain was expressed. These cell lines were termed RC and A4, respectively. A4 cells but not RC or NIH3T3 cells grew even in the presence of a low concentration (0.05%) of calf serum (CS), although the rate of their proliferation was low. In media containing 0.1% CS, both A4 cells and RC cells but not NIH3T3 cells could proliferate. Furthermore, RC cells induced foci formation when cultured in media containing 0.5% and 5% CS, while growth of the parental NIH3T3 cells was contact-inhibited under these conditions. These data suggest the presence of a factor(s) which activates protein-tyrosine kinase activity of the c-erbB-2 protein. In fact, the c-erbB-2 protein prepared from RC cells showed CS-dependent protein-tyrosine kinase activity when assayed in membrane fractions.


Okadaic acid is a potent angiogenesis inducer. Okadaic acid, which is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter and an inhibitor of protein phosphatases 1 and 2A, induced angiogenesis in the chorioallantoic membrane of the chick embryo. Its potent angiogenic activity was dose-dependent. The minimum effective dose was 5 fmol/egg and the effective dose for 50% induction was 90 fmol/egg. These results indicated that okadaic acid exhibits angiogenic activity one order of magnitude stronger than that of TPA (reported previously). Moreover, the time-course of angiogenesis induction by okadaic acid was much slower than that by TPA. The difference is consistent with the time-courses of other biochemical and biological activities and also various gene expressions induced by okadaic acid and TPA, indicating that the difference in the time-course is associated with their mechanisms of action. We conclude that okadaic acid induces angiogenesis through a different pathway than does TPA, indicating the existence of a new mechanism of angiogenesis induction.


Differential expression of three C/EBP isoforms in multiple tissues during the acute phase response. Eucaryotic organisms possess natural defense processes triggered by stress factors such as injury, infection, and inflammation. The acute phase response is an early defense mechanism during which striking changes in protein synthesis occur in the liver and other tissues. The altered expression of many acute phase protein genes is at the transcriptional level. Some of these genes have DNA binding sites for the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. We report here that in vivo expression of three isoforms of C/EBP is dramatically changed during the acute phase response. The steady-state mRNA levels of C/EBP alpha decreased significantly in the liver, lung, and fat tissues of lipopolysaccharide (LPS)-treated mice; moreover, nuclear run-off transcription assays indicated a decrease in the rate of C/EBP alpha gene transcription in isolated liver nuclei. The steady-state levels of C/EBP beta and a new isoform, C/EBP delta, were dramatically increased in many tissues within 4 h following LPS treatment. The rates of transcription of these two genes were only minimally altered in liver but significantly increased in kidney nuclei isolated from stimulated animals. Thus, the C/EBP isoforms exhibit differential mechanisms in their responses to LPS in various tissues and are likely to play an important role in mediating the transcriptional activation of genes involved in the acute phase response.


Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1. Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.


Immunochemical detection of advanced glycosylation end products in vivo. Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.


Protein kinase C and calmodulin kinase are required for endothelin-stimulated atrial natriuretic factor secretion from primary atrial myocytes. Endothelin (ET), a potent stimulator of atrial natriuretic factor (ANF) secretion in atrial myocyte cultures, has been hypothesized to act via the stimulation of protein kinase C (PKC). This study was carried out in order to determine if ET activates PKC in atrial cultures and whether this activation fully accounts for the effects of ET on ANF secretion. By monitoring the phosphorylation of p80 upon exposure to phorbol ester or ET, it was shown that ET activated PKC in atrial cultures, but to a lesser extent than phorbol ester. In contrast, ET stimulated ANF secretion to a level five times greater than phorbol ester, indicating that PKC activation alone does not fully account for the effects of ET on ANF secretion. Down-regulation of PKC or exposure to the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) resulted in a 50% decrease in ET-stimulated ANF secretion. Interestingly, increasing calcium influx with BAY K 8644 stimulated ANF secretion but did not effect the phosphorylation of p80, indicating a PKC-independent pathway of ANF secretion. Similarly, a component of ET-stimulated secretion that required calcium influx was independent of PKC activation but was sensitive to the Ca2+/calmodulin kinase (CaMK) inhibitor KN-62. Complete inhibition of ET-mediated ANF secretion was obtained only in the presence of both H7 and KN-62. These results demonstrate that ET activates PKC in atrial myocyte cultures and that the full effects of ET on ANF secretion require both PKC and Ca2+/calmodulin kinase activities.


Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones. To aid in studies of human glycoinositol-phospholipid (GPI) anchor pathway biochemistry in normal and affected paroxysmal nocturnal hemoglobinuria cells, GPI anchor-defective human K562 cell lines were derived by negative fluorescent sorting of anti-decay-accelerating factor (DAF) monoclonal antibody-stained cells either following or in the absence of ethylmethylsulfonate pretreatment. The resulting cloned cells showed deficiencies of both DAF and GPI-anchored CD59, some (designated group A) exhibiting total absence and some (designated group B) exhibiting approximately 10% levels of surface expression of the two proteins. In heterologous cell fusions, group A clones complemented defective Thy-1 expression by class A, B, C, E, and I Thy-1-negative lymphoma lines, but not H or D lines, the latter of which is defective in the Thy-1 structural gene. In contrast, group B clones complemented all previously described GPI anchor pathway-defective lymphoma classes. Immunoradiomatic assays of cells and supernatants and 35S biosynthetic labeling showed that group A cells degraded DAF protein while group B cells secreted it but failed to attach a GPI anchor structure. [3H]Man labeling of intact cells and UDP-[3H]GlcNAc and GDP-[3H]Man labeling of broken cell preparations demonstrated that group A cells failed to synthesize GlcNAc- and GlcN-PI (GPI-A and -B) as well as more polar mannolipids, whereas group B cells showed accumulation of GlcNAc-PI with approximately 10-fold diminished levels of GlcN-PI and more polar mannolipids. The failed assembly of GlcNAc-PI in group A cells and the reduced conversion of this intermediate to GlcN-PI in group B cells indicates that the former harbors a defect in UDP-GlcNAc transferase or in assembly of its PI acceptor, while the latter harbors a defect in GlcN-PI deacetylase activity.


Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein. The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.


cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide. When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.


Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro. Reconstitution of the polyoma virus middle T antigen (mT)-pp60-src complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mT-pp60c-src and PtdIns 3-kinase purified from rat liver. Both the 110- and 85-kDa subunits of the PtdIns 3-kinase associated with the mT-pp60c-src complex. The association of PtdIns 3-kinase with the mT-pp60c-src complex was dependent on the protein-tyrosine kinase activity of pp60c-src as a kinase-inactive mutant (pp60(295c-src)) still complexed with mT, but the mT-pp60(295c-src)) complex was unable to bind PtdIns 3-kinase. The mT-pp60c-src complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60c-src complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the protein-tyrosine kinase activity of pp60c-src. Comparison of 35S-labeled proteins from whole cell lysates which associated with immunopurified mT-pp60c-src and mT-pp60(295c-src) revealed proteins of 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer) containing sequences conserved between the major tyrosine phosphorylation site of murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85- and 110-kDa polypeptides with the mT-pp60c-src complex. The ability to block the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunit of PtdIns 3-kinase can associate with mT-pp60c-src. This association in vitro is a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.


Identification of a novel mammalian annexin. cDNA cloning, sequence analysis, and ubiquitous expression of the annexin XI gene. Annexins (or lipocortins) are a family of at least 10 structurally related calcium- and phospholipid-binding proteins. Each protein consists of a conserved core domain having four (or eight) repeats of a segment approximately 70 amino acids in length and a nonconserved, usually short, amino-terminal domain. To date, amino acid sequences for eight distinct mammalian annexins have been predicted from cDNAs. This report describes an additional member of this family, bovine annexin XI, identified by cDNA cloning and sequence analysis. The 503-amino acid deduced protein consists of a core domain of four annexin repeats and a long amino-terminal domain rich in glycine, proline, and tyrosine. This novel annexin gene is expressed in a wide variety of tissues and isolated cells in culture.


Fc epsilon RI-induced protein tyrosine phosphorylation of pp72 in rat basophilic leukemia cells (RBL-2H3). Evidence for a novel signal transduction pathway unrelated to G protein activation and phosphatidylinositol hydrolysis. Recently, we demonstrated that aggregation of the high affinity IgE receptor in rat basophilic leukemia (RBL-2H3) cells results in rapid tyrosine phosphorylation of a 72-kDa protein (pp72). Here we investigated the relationship of pp72 phosphorylation to guanine nucleotide-binding protein (G protein) activation and phosphatidylinositol hydrolysis. The activation of G proteins by NaF in intact cells or by guanosine 5'-O-(3-thiotriphosphate) in streptolysin O-permeabilized cells induced both phosphatidylinositol hydrolysis and histamine release without tyrosine phosphorylation of pp72. Similarly, in RBL-2H3 cells expressing the G protein-coupled muscarinic acetylcholine receptor, carbachol activated phospholipase C and induced secretion without concomitant pp72 phosphorylation. Therefore, pp72 phosphorylation was not induced by G protein activation or as a consequence of phosphatidylinositol hydrolysis. To investigate whether pp72 tyrosine phosphorylation precedes the activation of phospholipase C, we studied the effect of the tyrosine kinase inhibitor genistein. Preincubation of cells with genistein decreased, in parallel, antigen-induced tyrosine phosphorylation of pp72 (IC50 = 34 micrograms/ml) and histamine release (IC50 = 31 micrograms/ml). However, genistein at concentrations of up to 60 micrograms/ml did not inhibit phosphatidylinositol hydrolysis nor did it change the amount of the secondary messenger inositol (1,4,5)-triphosphate. Previous observations showed that there was no pp72 tyrosine phosphorylation after activation of protein kinase C or after an increase in intracellular calcium. Taken together, these results suggest that pp72 tyrosine phosphorylation represents a distinct, independent signaling pathway induced specifically by aggregation of the Fc epsilon RI.


Thrombomodulin is preferentially expressed in Balb/c lung microvessels. Previously, two rat monoclonal antibodies where developed which bind distinct epitopes on a murine glycoprotein, P112, which is expressed primarily in lung capillary endothelium. In this paper we show that P112 is identical to the endothelial anticoagulant protein, thrombomodulin (TM). Several lines of evidence support this conclusion. First, amino acid analysis of P112 shows a high degree of homology to TM, and both molecules exhibit the same mobility in gel electrophoresis. Second, P112 and TM share reactivity for two different monoclonal antibodies. Third, purified P112, like TM, acts as a cofactor for protein C activation. Finally, two cDNA clones identified with P112 polyclonal antiserum contain sequence identity with the known TM cDNA sequence. Quantitative analysis of TM (P112) expression using a two-site monoclonal antibody assay demonstrates that significantly higher levels of TM are found in lung in comparison with other highly vascularized organs, i.e. the kidney and liver. Quantitative Northern blot data coincides with the two-site assay data and demonstrates that the high level of TM expression in lung is not due to preferential binding of the monoclonal antibodies to lung TM but rather to increased production of TM mRNA in the lung relative to other highly vascularized organs. It is suggested that expression of TM is highest in cells from continuous endothelium.


Activation of Fc gamma RII induces tyrosine phosphorylation of multiple proteins including Fc gamma RII. Platelets provide a useful system for studying Fc gamma receptor-mediated signaling events because these cells express only a single class of Fc gamma receptors and because platelet aggregation and secretion can be activated through Fc gamma receptor stimulation. We report here that stimulation of platelets by cross-linking antibodies to Fc gamma RII or by treatment with an anti-CD9 monoclonal antibody, which acts through Fc gamma RII, causes an induction of tyrosine phosphorylation of multiple platelet proteins. Although the profile of tyrosine-phosphorylated proteins induced by stimulation of this Fc receptor was similar to that induced by thrombin, an additional 40-kDa phosphorylated protein was also detected. This protein co-migrated with Fc gamma RII and was immunoprecipitated with a monoclonal antibody to Fc gamma RII. In addition, after the cross-linking of Fc gamma RII in HEL cells or in COS-1 cells transfected with Fc gamma RII cDNA, the 40-kDa protein immunoprecipitated with anti-Fc gamma RII was also phosphorylated on tyrosine. These data strongly suggest that Fc gamma RII itself is a substrate for a tyrosine kinase(s) activated when Fc gamma RII is stimulated. Fc gamma RII was phosphorylated by the Src protein in vitro, suggesting that this kinase may be responsible for phosphorylation of Fc gamma RII in vivo. These studies establish that activation of platelets and human erythroleukemia cells through Fc gamma RII and CD9 involves an induction of tyrosine phosphorylation of multiple proteins including Fc gamma RII itself and suggest that these phosphorylation events may be involved in Fc gamma RII-mediated cell signaling.


Cellular differentiation is required for cAMP but not Ca(2+)-dependent Cl- secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator. The gene responsible for cystic fibrosis (CF) has recently been cloned and sequenced. When transfected into CF epithelial cells, normal transcripts of this gene correct the underlying defect in CF, i.e. cAMP-dependent Cl- secretion is restored. Thus, the protein encoded by this gene, designated "cystic fibrosis transmembrane conductance regulator" (CFTR), somehow participates in the Cl- secretory response. In this paper we have correlated CFTR gene expression with cAMP and Ca(2+)-dependent Cl- secretion in unpolarized (parental) and polarized (Cl.19A) clones of the human colonic adenocarcinoma cell line HT-29. These cell lines were found to express equally high levels of CFTR mRNA at 4 days post-passage. In addition, protein expression (determined by immunoprecipitation) was also identical. The cAMP-generating agonist forskolin had little effect on 125I efflux from the unpolarized cells. In contrast, this agonist increased 125I efflux 3-fold in polarized cells. The lack of response in the unpolarized cells was not due to the inability of forskolin to raise cAMP levels. Neurotensin, a Ca(2+)-mobilizing agonist, stimulated 125I efflux from both cell lines. In the polarized cells, the magnitude of this response was attenuated at 8 days post-seeding. At this time, the undifferentiated line attained some cAMP responsiveness. This latter effect was paralleled by the appearance of monolayers within areas of the multicell layer. Cell-attached patch-clamp recording from apical membrane patches of polarized cells revealed the presence of a forskolin-stimulated 8-pS Cl- channel; no channel activity was observed in forskolin-stimulated unpolarized cells. Ca(2+)-activated Cl- channels were found in both cell lines. In agreement with the 125I efflux data, the single-channel activation response to [Ca2+]i was smaller in the polarized cell line. From these studies, we can conclude that CFTR expression, measured both at the mRNA and protein level, does not correlate with the colonocyte's ability to secrete chloride ions in response to a cAMP-generating agonist. Cyclic AMP-dependent Cl- secretion requires cellular polarization; specifically, the delineation of an apical membrane. Differences in the cellular location of CFTR during differentiation are likely to explain our results. In contrast, Ca(2+)-stimulated Cl- secretion occurred independently of cellular polarization but was reduced when the cells formed tight junctions.


Aggrecan core protein is expressed in membranous bone of the chick embryo. Molecular and biomechanical studies of normal and nanomelia embryos. The recessive mutation nanomelia blocks the synthesis of a large aggregating proteoglycan (aggrecan) by avian embryo chondrocytes. Lack of aggrecan is associated with short stature, multiple morphological defects in cartilage, and embryo lethality. Bony defects have also been described, but were assumed to be a secondary consequence of the cartilage defect. However, two lines of evidence presented in this paper indicate that the aggrecan deficiency directly affects intramembranous bone. First, the morphology (i.e. projected area and shape) of certain membranous bones of nanomelia embryos was abnormal. Second, membranous bone from nanomelia embryos proved to be significantly stiffer in biomechanical tests that measured functional properties of the extracellular matrix. These findings were unexpected because intramembranous bones normally develop from mesenchyme and not from a cartilage intermediate, and they prompted a search for evidence of aggrecan expression in the bone of normal chick embryos. We report that: 1) aggrecan mRNA was identified by PCR analysis of total RNA isolated from day-13 chick embryo calvarium, 2) the PCR method successfully amplified aggrecan mRNA from primary chick embryo osteoblasts in culture, 3) in situ hybridization of membranous bone tissue sections demonstrated aggrecan expression by chick embryo osteoblasts in vivo, and 4) the aggrecan message was identified in Northern blots of calvarial mRNA probed at high stringency. The results of the molecular and biomechanical studies provide evidence that aggrecan is indeed expressed in membranous bone as well as cartilage. Altogether, these results suggest that aggrecan may contribute to the functional properties and the normal growth and development of avian membranous bone.


Transcriptional activation of fibroblast collagenase gene expression by a novel lymphokine, leukoregulin. Leukoregulin (LR) is a novel T-cell derived cytokine with unique anti-tumor properties. We have recently demonstrated that LR is also able to modulate the biosynthetic repertoire of normal human skin fibroblasts in culture (Mauviel, A., Rédini, F., Hartmann, D.J., Pujol, J.-P., and Evans, C.H. (1991) J. Cell Biol. 113, 1455-1462). In this study, we have examined in detail the effects of LR on collagenase gene expression in human skin fibroblast cultures. The results indicated time- and dose-dependent induction of collagenase mRNA steady-state levels, the maximum elevation being approximately 35-fold. In contrast, the mRNA levels for tissue inhibitor of metalloproteases remained unchanged in the same RNA preparations. The enhancement of collagenase mRNA levels was shown to be dependent on protein synthesis, and it could be counteracted by dexamethasone or all-trans-retinoic acid. Transient transfections of cultured fibroblasts with a human collagenase promoter/reporter gene construct indicated up-regulation of the promoter activity, which could be blocked by dexamethasone and all-trans-retinoic acid. The observation suggested regulation at the transcriptional level of collagenase gene expression. LR was also shown to induce the mRNA levels for junB, suggesting possible involvement of the AP-1 complex in the regulation. The ability of LR to selectively induce collagenase gene expression in skin fibroblasts suggests that this cytokine may significantly contribute to the degradation of the extracellular matrix in physiological situations, such as tissue development and repair, and in diseases characterized by excessive degradation and turnover of collagen.


Membrane dynamics of the phosphatidylinositol-anchored form and the transmembrane form of the cell adhesion protein LFA-3. The cell adhesion glycoprotein LFA-3 is expressed on the cell surface of nucleated cells in both a membrane-spanning form and a glycosyl phosphatidylinositol-anchored form. To determine whether distinct membrane anchors direct the dynamics of a given protein, the turnover of biosynthetically 35S-labeled and biotin surface-labeled LFA-3 molecules was followed. It is shown here that (a) expression of the two LFA-3 forms is regenerated with similar kinetics after enzymatic removal from the cell surface; (b) neither of the distinct LFA-3 molecules undergoes constitutive internalization; and (c) transmembrane and glycosyl phosphatidylinositol-anchored LFA-3 have an unusually long life span with an identical half-life of 50 h. Thus, the type of membrane anchor is not affecting turnover characteristics of a particular cell surface glycoprotein.