Thymotaxin, a chemotactic protein, is identical to beta 2-microglobulin.

Thymotaxin, an 11-kilodalton protein chemotactic for rat bone marrow hematopoietic precursors, was purified from media conditioned by a rat thymic epithelial cell line. The NH2-terminal sequence of thymotaxin was identical to that of rat beta 2-microglobulin (beta 2m). Antibodies to beta 2m removed thymotaxin activity from the fraction containing the 11-kilodalton protein. Chemotactic activity was observed with rat plasma beta 2m, human beta 2m, and mouse recombinant beta 2m, further supporting the identity of thymotaxin with beta 2m. The directional migration, as opposed to random movement, of the cells was also confirmed. The only rat bone marrow cells that migrated toward beta 2m were Thy1+ immature lymphoid cells devoid of T cell, B cell, and myeloid cell differentiation markers.

Humoral hypercalcemia of malignancy. Release of a prostaglandin-stimulating bone-resorbing factor in vitro by human transitional-cell carcinoma cells.

Secretion by tumor cells of circulating bone-resorbing factors may frequently underlie the hypercalcemia that occurs in patients with malignancy. Efforts to identify the responsible mediators have been hampered by a lack of available human tumor cell systems suitable for study of the pathogenesis of the humoral hypercalcemia syndrome. We have established a transitional-cell carcinoma (TCC) line in vitro from a patient with humoral hypercalcemia. These cells are tumorigenic and cause hypercalcemia in athymic nude mice. Culture medium conditioned by TCC cells contains potent bone-resorbing activity in vitro, the physical and biological properties of which are similar to those of bone-resorbing activity present in the original patient's urine. The bone-resorbing activity of the TCC factor is accompanied by increased prostaglandin release from bone and is blocked by indomethacin and calcitonin. The TCC-derived bone-resorbing activity coelutes with prostaglandin-stimulating activity during gel filtration with an approximate molecular weight of 15,000. This activity is nondialyzable, stable to concentrated urea and reducing agents, and destroyed by boiling. The TCC factor does not increase cyclic AMP production in bone or kidney bioassays and does not exhibit transforming growth factor activity. We conclude that a unique macromolecular factor released by TCC cells causes bone resorption by a mechanism dependent upon stimulation of bone cell cyclooxygenase, and that this factor is the probable cause of the hypercalcemia in vivo. The TCC cell line provides a new model for study of the human humoral hypercalcemia syndrome.

Growth-dependent synthesis of c-myc-encoded proteins: early stimulation by serum factors in synchronized mouse 3T3 cells.

Synthesis of the c-myc gene product was measured during the entire cell cycle of subconfluent mouse 3T3 cells with an antibody raised against a human c-myc synthetic peptide. The antiserum recognized two mouse c-myc-encoded proteins with apparent molecular weights in sodium dodecyl sulfate-polyacrylamide gels of 62,000 and 60,000. Cell-derived p62 was compared with the mouse c-myc gene product synthesized in vitro. Immunoprecipitation, electrophoretic analyses, and peptide mapping provided evidence that p62 is encoded by the mouse c-myc gene. The rate of synthesis of the c-myc proteins was tightly coupled to the cellular growth state of nontransformed A31 3T3 cells, but not to that of their benzo(a)pyrene-transformed derivative (BPA31). Furthermore, the synthesis of the c-myc proteins was stimulated by the exposure of confluent, density-arrested A31 cells to platelet-derived growth factor or fibroblast growth factor. Tightly synchronized cell populations were obtained on the addition of serum factors to subconfluent, serum-deprived A31 cells, and c-myc expression could be monitored for more than one complete cell cycle. One hour after stimulation the steady-state level of the 2.2 kilobase c-myc transcript increased 30-fold relative to that of quiescent cells and decreased thereafter to the level observed during exponential growth. The rate of synthesis of c-myc-encoded proteins was determined by immunoprecipitation after a 2-h labeling period. After an initial sevenfold increase detectable 2 h after serum addition, the rate of synthesis remained constant throughout the rest of the cell cycle. No further changes associated with the late prereplicative period, S phase, G2, or mitosis could be demonstrated. Pulse-chase and long-term labeling experiments revealed different half-lives for the two c-myc-encoded proteins. The half-lives of the c-myc proteins, however, were independent of the cellular growth state. The sustained expression observed throughout the cell cycle suggests that the growth-related function of c-myc may be required during the G0-G1 transition and in all phases of the cycle of the growing cell.

Multiple growth-associated nuclear proteins immunoprecipitated by antisera raised against human c-myc peptide antigens.

Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with, particularly for p65, nucleolar enhancement that left an unstained annulus surrounding the nucleolus.

Expression of neurotrophin-4 mRNA during oogenesis in Xenopus laevis.

Neurotrophin-4 (NT-4), a recently discovered novel member of the family of neurotrophic factors structurally related to nerve growth factor (NGF), is abundantly expressed in the Xenopus laevis ovary. In this study we have localized NT-4 mRNA expressing cells in the Xenopus ovary by in situ hybridization and have used this technique together with Northern blot analyses to quantify NT-4 mRNA expression during oogenesis in Xenopus. In situ hybridization of sections through the Xenopus ovary using an alpha-[35S]-dATP labeled Xenopus NT-4 mRNA specific probe showed an intense labeling over the cytoplasm of oocytes with a diameter of 50-200 microns corresponding to stage I according to Dumont (1972). Labeling was also seen over the cytoplasm of stages II to IV although with a lower intensity than over stage I oocytes. No labeling was seen over more mature oocytes of stages V and VI. NT-4 mRNA could not be detected in the early embryo from the onset of cleavage division to the neurula stage suggesting that the NT-4 gene is not expressed during Xenopus early embryogenesis. The confinement of NT-4 mRNA in the Xenopus ovary to immature oocytes suggests that NT-4 mRNA expression is strictly regulated during oogenesis and that the NT-4 protein could play a role as a maturation factor for immature oocytes.

Characterization of human NOV in biological fluids: an enzyme immunoassay for the quantification of human NOV in sera from patients with diseases of the adrenal gland and of the nervous system.

Immunochromatography has shown that human NOV (NOVH), a member of the CCN (CTGF/CYR61/NOV) family, forms a physiological complex with fibulin-1 in blood. We developed an enzyme immunoassay specific for NOVH and showed for the first time that the concentration of NOVH differs in each of these biological fluids. The normal concentration of NOVH circulating in the blood is 350-400 ng/ml, but this concentration varies with age. By using sera from patients with adrenal gland diseases we found that in vivo ACTH or glucocorticoids are not responsible for the high concentration of NOVH in this endocrine gland. However, the NOVH concentration was significantly modified in malignant adrenocortical tumors, but not in benign adrenocortical tumors. The concentration of NOVH was significantly decreased in patients suffering from astrocytomas or multiple sclerosis, two diseases of the nervous system. Thus, NOVH is a potentially useful marker for the diagnosis of these diseases.

Binding of alanine-substituted peptides to the MHC class I protein, Kd.

Peptides eluted from the native MHC class I molecule, Kd, are generally nonamers that display a strong preference for Tyr in position 2. We investigated the molecular basis for this 'consensus motif' by synthesizing a virally derived peptide, NP 147-155, that is known to be presented by Kd on living cells, and peptide variants of NP 147-155 in which the amino acids in the different positions were sequentially replaced by Ala. All of the peptides bound to purified Kd molecules in vitro with high affinity, except for the peptide in which Tyr2 was replaced by Ala, for which the affinity for Kd decreased at least 100-fold. These results confirm the interpretation of the in vivo studies; namely, that Tyr2 is a critical anchor residue for binding to Kd.

Mitogenic activity of high molecular weight forms of insulin-like growth factor-II in amniotic fluid.

Amniotic fluid (AF) collected from ewes and goats at mid gestation displayed mitogenic activity in mouse fibroblasts. Upon fractionation of this material by size exclusion chromatography, the mitogenic activity was resolved into two peaks, whose activity was inhibited by an anti-IGF type 1 receptor blocking antibody. One of the peaks contained IGF-I and IGF-II (mature form), whereas the other contained high M(r) precursor forms of IGF-II. The presence in this latter fraction of IGF-binding proteins (IGFBP) suggests that the AF IGFBPs do not efficiently inhibit the mitogenic activity of the high M(r) forms of IGF-II. In agreement with this conclusion, exogenous IGFBP-1 failed to affect this activity. Analysis of IGF-II in sheep AF showed that the AF concentrations of both forms of IGF-II increased dramatically from mid pregnancy until 106-120 days of gestation, and fell thereafter. The amniotic IGFBPs followed a similar evolution. High M(r) forms of IGF-II were also found in human AF, with a pattern of electrophoretic migration different from that of sheep. We suggest that the precursor forms of IGF-II may play an important role in foetal development.

[Spinal tuberculosis in children: contribution of imaging to diagnostic and therapeutic management]. / Tuberculose vertébrale de l'enfant: place de l'imagerie dans la démarche diagnostique et thérapeutique.

BACKGROUND: Spinal tuberculosis is an increasingly common condition, particularly in children. CASE REPORT: A 2-year-old boy presented with spinal tuberculosis with psoas abscesses. Radiological imaging played a major role in the diagnostic process. DISCUSSION: In children, spinal tuberculosis is a serious condition due to its rapid spread. Clinical symptoms are often insidious and histological and bacteriological studies may fail to disclose the diagnosis. Radiological investigations are essential in the diagnostic approach.

Xenopus mono(adenosine diphosphate ribosyl) transferase: purification, assay, and properties.

Various tissues of Xenopus laevis possess mono(ADP-ribosyl) transferase activity. The Xenopus liver enzyme was purified approximately 40,000-fold by sequential chromatography on phenyl Sepharose, DEAE-Sephadex, NAD+-Agarose, and Con A-Sepharose. The enzyme is relatively heat-stable, has no cation requirements, transfers monomers of ADP-ribose from NAD+ to arginine-rich histones H3 and H4, and is not dependent upon DNA for activity. Its Mr was estimated to be 30 kilodaltons (KD). The amino acid acceptor of the ADP-ribose moiety is probably arginine, and the bond formed is resistant to the hydrolytic action of neutral hydroxylamine. The transferase activity was neither inhibited nor precipitated by antisera directed against the A fragment of cholera or diphtheria toxins. Theophylline, thymidine, nicotinamide, and some of its analogues blocked the enzymatic activity, while nicotinic acid was ineffective. When a DNA-free extract of HeLa cells was incubated with the transferase system, several proteins were found to be ADP-ribosylated. A band at position 90 KD showed high specific radioactivity. A zymographic method for assaying transferase activity in crude tissue extracts was developed. In the initial step, the extracts were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Using this method, a histone-dependent mono(ADP-ribosyl) transferase corresponding to an Mr of about 30 KD was demonstrated in every tissue examined. Treatment with reducing agents practically abolished transferase activity in contrast to the enhancement of choleragen A fragment activity. A variant of the transferase at about 80 KD was detected. It differed from the 30 KD enzyme in that histones inhibited its activity while agmatine had no influence.

Replication inhibition by nucleoside analogues of a recombinant Autographa californica multicapsid nuclear polyhedrosis virus harboring the herpes thymidine kinase gene driven by the IE-1(0) promoter: a new way to select recombinant baculoviruses.

The expression of the thymidine-thymidylate kinase (HSV1-TK), (ATP: thymidine 5'-phosphotransferase; EC 2.7.1.21) of herpes simplex virus type 1 endows the host cell with a conditional lethal phenotype which depends on the presence of nucleoside analogues metabolized by this enzyme into toxic inhibitors of DNA replication. To generate a recombinant baculovirus that could be selected against by nucleoside analogs, the HSV1-tk coding sequence was placed under the control of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) immediate early promoterm IE-1(0), and this construction was introduced via homologous recombination into the polyhedrin locus of AcMNPV. Two recombinant baculoviruses harboring this gene construct at the polyhedrin locus were isolated and tested for their ability to replicate in the presence of various concentrations of the nucleoside analog 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (Ganciclovir). Neither Sf9 lepidopteran cell viability nor replication of wild type or beta-Galactosidase-expressing recombinant AcMNPVs were affected by concentrations of Ganciclovir up to 100 microM. In contrast, replication of the recombinant AcMNPV virus harboring the HSV1-tk gene was inhibited by Ganciclovir in a dose-dependent manner. The inhibition was detectable at 2 microM and complete at 100 microM. This property was exploited in model isolations aimed at purifying new recombinant viruses having lost this counter-selectable gene marker as a result of homologous recombination at the polyhedrin locus after cotransfection of the viral DNA with a replacement vector. After being propagated in the presence of Ganciclovir, the progeny of such co-transfections contained over 85% recombinant viruses, demonstrating that counter-selection of parental HSV1-tk-containing viruses by Ganciclovir constitutes a novel approach for recombinant baculovirus isolation.

Early stimulation of phospholipid methylation in Xenopus oocytes by progesterone.

Progesterone induced a transient increase in the incorporation of [3H]methyl groups into phospholipids of Xenopus oocytes followed by a rise in 45Ca2+ uptake. Phospholipid methylation reached a maximum as early as 15 s after progesterone treatment and returned to basal level within 2 min. Steroids inactive in promoting oocyte maturation were less effective in affecting phospholipid methylation. Methyltransferase inhibitors, 3-deaza-SIBA, SIBA, and Sinefungin, inhibited progesterone-activated stimulation of phospholipid methylation, calcium uptake and meiotic maturation. Phospholipid methylation is the earliest detectable biochemical event occurring in oocytes after exposure to progesterone followed by calcium influx and leading to germinal vesicle dissolution.

Tumor promoter PMA stimulates the synthesis and secretion of mouse pro-urokinase in MSV-transformed 3T3 cells: this is mediated by an increase in urokinase mRNA content.

In mouse MSV-3T3 cells the synthesis of the urokinase form of plasminogen activator was increased 10-fold after addition of the tumor promoter phorbol-12-myristate-13-acetate (PMA). PMA also stimulated the secretion of the protein into the culture medium, mostly in the form of enzymatically inactive pro-urokinase. When assayed by injecting RNA into Xenopus laevis oocytes, the concentration of functional urokinase mRNA was found to be 6- to 10-fold higher in the PMA-treated cells; a similar increase in urokinase mRNA content was measured by hybridisation with a mouse urokinase cDNA probe. Thus, the induction of plasminogen activator by PMA in MSV-3T3 cells is accounted for by an increased content of urokinase mRNA.

Mono(adenosine diphosphate ribosyl) transferase in Xenopus tissues. Direct demonstration by a zymographic localization in sodium dodecyl sulfate--polyacrylamide gels.

A semiquantitative method to measure mono(adenosine diphosphate ribosyl) transferase activity [mADPRT] in tissue extracts is described. After electrophoretic separation in sodium dodecyl sulfate (SDS)--polyacrylamide gels, renatured enzymatic activity is demonstrated in situ by incubation of the slab gels with radiolabeled NAD+ and histones. Precipitation of the radiolabeled product in the gel allows localization of the enzyme by autoradiography. This method is suitable for two-dimensional gel electrophoresis, whereby proteins are electrofocused in the presence of 9 M urea and subsequently subjected to electrophoresis in SDS. A single major band showing mADPRT activity of Mr approximately 30 Kda was observed in all crude extracts of Xenopus tissues examined. Accumulation of acid-insoluble radiolabeled products was dependent on added histones and was specifically inhibited by agmatine. The ADPRT activity of cholera toxin A fragment could also be demonstrated by this technique. Reducing agents stimulated the activity of cholera toxin A fragment while depressing that of Xenopus mADPRT.

Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems.

Rab6 protein (rab6p) belongs to a family of ras-like GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. We have constructed a recombinant baculovirus in order to express rab6p in insect cells. We report here the characterization of four forms of this protein which are found in cytosolic and membrane fractions of infected Sf9 cells. The two major forms are a cytosolic 24 kD protein which represents the unprocessed precursor form of rab6p and a membrane-bound isoprenylated 23 kD protein which represents the processed form. Two other minor forms were also detected: a cytosolic isoprenylated 23 kD protein which may represent a pool in equilibrium with the 23 kD membrane-bound form and a 24 kD non-isoprenylated membrane-bound form which may represent an intermediate in the processing of rab6p.

Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart.

Peptide binding to MHC class I proteins measured with a novel fluorescent technique.

An N-dansylated peptide derived from the Plasmodium berghei circumsporozoite protein (PbCS 253-260) bound in an allele-specific manner to a single-chain Kd molecule (SC-Kd), and its binding resulted in significant fluorescent enhancement. The binding kinetics of unlabelled peptides could be determined by pre-incubating dansylated PbCS with a concentrated suspension of SC-Kd, and then diluting this mixture in the presence of unmodified peptide. The time-dependence of the ensuing fluorescence decrease could be fitted to a single-exponential, which gave an association rate constant of 77 M-1s-1 for unlabelled PbCS.

A single-chain murine class I major transplantation antigen.

Single-chain mouse Kd molecules (SC-Kd) were engineered by connecting residue 276 of Kd heavy chain to the first residue of beta 2-microglobulin through spacers of various lengths, and expressed intracellularly in monkey COS-1 cells. Labeled SC-Kd molecules were found to react with several monoclonal antibodies which recognize native Kd molecules. SC-Kd-15 (with a spacer of 15 residues) was studied in more details. It could be purified and shown to regain a native-like structure after treatment with denaturing agents. Purified SC-Kd-15 could bind certain peptides in a manner qualitatively similar to the Kd.

Fas-mediated liver damage in MRL hemopoietic chimeras undergoing lpr-mediated graft-versus-host disease.

Fas is an apoptosis-signaling receptor that triggers cell death upon binding to its ligand (FasL). Autoimmune-prone MRL/lpr mice, characterized by a spontaneous mutation of Fas, exhibit a defect in the activation-induced cell death of mature T cells through a Fas-mediated pathway. As a consequence of this defect, activated T cells accumulating in this strain overexpress the FasL and can therefore mediate in vitro Fas-dependent cytotoxicity. To determine whether hepatic injury could be the result of an interaction between T lymphocytes bearing FasL and Fas-expressing liver cells, the livers of lethally irradiated MRL+/+ recipients reconstituted with MRL/lpr lymphoid cells were studied. After transfer of MRL/lpr spleen cells, livers were infiltrated by polyclonal CD8+ T lymphocytes of lpr origin with a peak on day 21 postgrafting. These donor-derived intrahepatic lymphocytes overexpressed the FasL and exerted in vitro Fas-mediated cytotoxicity against Fas+ thymocytes, which was specifically inhibited by soluble recombinant Fas in a dose-dependent manner. These intrahepatic lymphocytes induced apoptosis in vitro, irrespective of MHC restriction, in Fas-expressing primary cultured hepatocytes. Histologic examination of the liver revealed severe endothelialitis as well as periportal and intralobular infiltrations of activated lymphocytes with apoptotic hepatocytes in their vicinity. Simultaneously, liver damage was ascertained by elevated serum transaminase levels. These observations support the notion that an Ag-independent mechanism involving FasL may play a role in certain liver pathologies.