Group B streptococcus GAPDH is released upon cell lysis, associates with bacterial surface, and induces apoptosis in murine macrophages.

Glyceraldehyde 3-phosphate dehydrogenases (GAPDH) are cytoplasmic glycolytic enzymes that, despite lacking identifiable secretion signals, have been detected at the surface of several prokaryotic and eukaryotic organisms where they exhibit non-glycolytic functions including adhesion to host components. Group B Streptococcus (GBS) is a human commensal bacterium that has the capacity to cause life-threatening meningitis and septicemia in newborns. Electron microscopy and fluorescence-activated cell sorter (FACS) analysis demonstrated the surface localization of GAPDH in GBS. By addressing the question of GAPDH export to the cell surface of GBS strain NEM316 and isogenic mutant derivatives of our collection, we found that impaired GAPDH presence in the surface and supernatant of GBS was associated with a lower level of bacterial lysis. We also found that following GBS lysis, GAPDH can associate to the surface of many living bacteria. Finally, we provide evidence for a novel function of the secreted GAPDH as an inducer of apoptosis of murine macrophages.

Rhesus macaque θ-defensin isoforms: expression, antimicrobial activities, and demonstration of a prominent role in neutrophil granule microbicidal activities.

Mammalian defensins are cationic, antimicrobial peptides that play a central role in innate immunity. The peptides are composed of three structural subfamilies: α-, ß-, and θ-defensins. θ-defensins are macrocyclic octadecapeptides expressed only in Old World monkeys and orangutans and are produced by the pair-wise, head-to-tail splicing of nonapeptides derived from their respective precursors. The existence of three active θ-defensin genes predicts that six different RTDs (1-6) are produced in this species. In this study, we isolated and quantified RTDs 1-6 from the neutrophils of 10 rhesus monkeys. RTD-1 was the most abundant θ-defensin, constituting ~50% of the RTD content; total RTD content varied by as much as threefold between animals. All peptides tested were microbicidal at ∼1 µM concentrations. The contribution of θ-defensins to macaque neutrophil antimicrobial activity was assessed by analyzing the microbicidal properties of neutrophil granule extracts after neutralizing θ-defensin content with a specific antibody. θ-defensin neutralization markedly reduced microbicidal activities of the corresponding extracts. Macaque neutrophil granule extracts had significantly greater microbicidal activity than those of human neutrophils, which lack θ-defensins. Supplementation of human granule extracts with RTD-1 markedly increased the microbicidal activity of these preparations, further demonstrating a prominent microbicidal role for θ-defensins.

Induction of proto-oncogene fos transcription through the adenylate cyclase pathway: characterization of a cAMP-responsive element.

Transcription of proto-oncogene fos is induced by elevated levels of intracellular cAMP. We report that human c-fos promoter recombinants transfected into rat pheochromocytoma cells (PC12) and human choriocarcinoma cells (JEG-3) are induced by stimulation of adenylate cyclase and that this induction is diminished considerably in the mutant PC12 cell line A126-1B2, which is deficient in cAMP-dependent protein kinase II. An element centered at position -60 of the c-fos promoter, which encompasses a consensus cAMP response element (CRE), is sufficient to confer cAMP responsiveness to a herpes thymidine kinase/CAT fusion gene. The specific binding of a nuclear protein to the c-fos CRE can be competed by the somatostatin and alpha-chorionic gonadotropin (alpha-CG) promoter regions that contain CREs. Gel mobility shift assays with double-stranded oligonucleotides containing either the wild-type or mutated c-fos CRE sequence have demonstrated that binding occurs only to the wild-type CRE. The nuclear factor binding to the c-fos CRE is likely to be transcription factor CREB (CRE nuclear binding protein), because an affinity-purified 43-kD CREB isolated from PC12 cells binds efficiently in a DNA footprinting assay. Thus, regulation of the c-fos gene transcription appears to involve a mechanism common to many genes that respond to cAMP as a second message leading to cell growth and differentiation.

The regulation of SV40 early gene expression in embryonal carcinoma stem cells--faithful transcriptional regulation in vitro.

We have derived from F9 murine embryonal carcinoma stem cells, and from differentiated derivatives induced with retinoic acid and cAMP, whole cell extracts which efficiently and accurately transcribe a variety of supercoiled DNA templates in vitro. These extracts and control elements from viral genomes have been used to study changes in transcriptional activity which accompany differentiation. The SV40 enhancer is inefficiently utilized in stem cells but is activated upon differentiation to parietal endoderm and the in vitro systems mimick this regulation. Mixing experiments demonstrate that the differentiated cell phenotype is dominant, suggesting that stem cells contain limiting amounts of factors required for enhancer activity. Our results may explain the developmental regulation of cellular genes with enhancer motifs in their control sequences.

Binding properties of monoclonal antibody to the cytoplasmic domain of transferrin receptor.

Hybridomas secreting monoclonal antibodies to transferrin receptor (TFR) were isolated. One of these antibodies, U-1, recognized the cytoplasmic domain of TFR and the others, N-2 and W-3, recognized its cell surface domains. Only antibody W-3 competed with transferrin (TF) for binding to TFR. Antibody U-1 bound to purified TFR but not to 35S- or 125I-TFR in cell extracts. 125I-Antibody U-1 bound to TFR alone in cell extracts when TFR was bound to antibody N-2-Sepharose 4B, but even in the presense of cell extracts it did not bind to TFR bound to antibody W-3-Sepharose 4B. Antibody W-3 co-precipitated TFR and a protein of about 30 kDa from cell extracts, and also reacted with the 30 kDa protein in cell extracts in the absence of TFR. Based on these results, the existence of two different states of the cytoplasmic domain of TFR is discussed.

Estradiol-binding molecules in the hepatocytes of the female water frog, Rana esculenta, and plasma estradiol and vitellogenin levels during the reproductive cycle.

Estradiol-binding molecules have been found both in cytosol and nuclear extract of hepatocytes of the female green frog Rana esculenta. These molecules show the properties of an estradiol receptor (Re): high and specific affinity for estrogens (2.10-7.10 x 10(-10) M), stability of binding at 0 and 20 degrees, localization in the nucleus, and cytosolic versus nuclear binding shift in estradiol-treated frogs. In hepatocytes, both filled and unfilled Re is detectable in all stages of the R. esculenta annual cycle. It increases during the recovery phase (September to January) when vitellogenetic processes are active in the ovary. These changes are positively correlated with vitellogenin. Only nuclear filled Re, moreover, is positively correlated to level of circulating estradiol. This suggests a direct connection: plasma estradiol level versus nuclear filled Re versus plasma vitellogenin level.

No evidence for bradykinin hydrolysis in human erythrocyte suspensions: 1H NMR studies.

In view of their permeability to small peptides, it has been postulated that human erythrocytes may play a role in terminating the action of some circulating peptide hormones. Work using classical paper chromatographic techniques for detecting free amino acids indicated that the octapeptide, des-(Arg9)-bradykinin, enters these cells and its amino-terminal arginine residue is released by cytosolic aminopeptidase-P. We have used 1H NMR to monitor directly the release of arginine from bradykinin. The hydrolytic reaction rate in hemolysates, with an initial peptide concentration of 13.0 mmol l-1 was 6.5 mmol (1 packed red cell)-1 h-1. But no reaction was evident after a 4.5-h incubation with intact cells, thus contradicting the earlier suggestion that erythrocytes are involved in the primary inactivation of this hormone. This is consistent with our previous findings that the pentapeptide leu-enkephalin fails to enter human erythrocytes but that its lower-order degradation products may do so.

A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells.

Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60 degrees C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.

Accurate transcription of mouse metallothionein-I gene in a fractionated nuclear extract from a rat hepatoma.

Nuclear extract from Morris hepatoma 3924A was fractionated by DEAE-Sephadex chromatography. The fraction eluting with 300 mM (NH4)2SO4 (DE-C) was used for transcribing cloned mouse metallothionein-I (MT-I) gene in a run-off assay. This fraction contained the majority of RNA polymerase II as well as the transcription factor(s). Accuracy of MT-I DNA transcription was confirmed by S1 nuclease mapping. Low concentrations (1 microgram/ml) of alpha-amanitin inhibited the reaction, indicating that RNA polymerase II directed the transcription. Unfractionated nuclear extracts from the hepatoma or a rat mammary adenocarcinoma as well as whole cell extract obtained from the mammary tumor also transcribed MT-I gene. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-C greater than whole cell extract derived from rat mammary adenocarcinoma cells greater than nuclear extract derived from rat hepatoma or rat mammary adenocarcinoma cells. These studies have demonstrated that a fractionated nuclear extract obtained from a tissue supports efficient and accurate RNA polymerase II-mediated transcription of MT-I DNA.

Effect of reduced food intake on blood levels of a hypertensive factor in spontaneously hypertensive rats.

We have previously demonstrated the existence of an active component(s) of extracts prepared from rat erythrocytes that stimulates the in vitro uptake of calcium in aortic rings and induces a sustained elevation in the blood pressure of normotensive rats. The apparent concentration of the calcium stimulatory component and the responsiveness of aortic rings to stimulation of calcium uptake by the compound is exaggerated in adult spontaneously hypertensive rats (SHR) compared with normotensive controls. Although no effect was observed on tissue responsiveness to the compound, a decrease in the concentration of the compound was observed in the blood of adult SHR subjected to dietary manipulation designed to blunt the elevation of blood pressure. These data suggest that blood concentration of the active compound may be influenced by external stimuli such as dietary manipulation.

Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation.

The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.

Cyclic AMP-dependent protein kinase in Molt 4b lymphoblasts: identification by photoaffinity labeling and activation in intact cells by vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI).

Molt 4b lymphoblasts have previously been shown to possess a single class of pharmacologically specific, high affinity receptors for vasoactive intestinal polypeptide (VIP). This study further explores the molecular basis for modulation of human lymphocyte function by VIP. Dose-dependent stimulation of adenylate cyclase was observed in Molt lymphoblasts over the range of 0.1 nM to 1 microM VIP. VIP-mediated by guanine nucleotide. Accumulation of intracellular cAMP was observed in the presence of either VIP or the diterpene, forskolin. The effects of these two agonists were synergistic. Two neuropeptides that share sequence homology with VIP were also studied; both peptide histidine isoleucine (PHI) and human pancreatic growth hormone releasing factor (1-44 GHRF) competed for 125I-VIP binding to Molt cells. PHI stimulated intracellular cAMP accumulation and demonstrated synergism with forskolin, whereas GHRF had no effect on cAMP. Photoaffinity labeling of 100,000 X G soluble proteins with 8-N3-[32P]cAMP followed by SDS gel electrophoresis demonstrated the presence of cAMP-dependent protein kinases I and II. Cyclic AMP-dependent protein kinase II predominated in the soluble fraction and was the only isozyme observed in particulate fractions. Protein phosphorylation was studied in Molt 4b cells preincubated with [32P]PO43- followed by addition of media alone, 1 microM peptide, or 10 microM forskolin. Cells were lysed and subjected to two-dimensional electrophoresis. Increased phosphorylation of a specific 41,000 Mr protein was observed after addition of forskolin, VIP, or PHI. A much lower concentration of VIP (1 nM) also caused a significant net increase in phosphorylation, which was of a lower magnitude. In contrast, no net effect on protein phosphorylation was seen with GHRF. These data demonstrate the presence of a functional VIP receptor that is linked to the G protein-adenylate cyclase complex. The demonstration of cAMP-dependent protein kinase and of VIP- and PHI-mediated protein phosphorylation in Molt 4b lymphoblasts provides evidence on a molecular level for neuropeptide modulation of human lymphocyte function.

Monoclonal antibody AHN-1 inhibits phagocytosis by human neutrophils.

The granulocyte-specific monoclonal antibody, AHN-1, immunoprecipitates two major surface-iodinated proteins of 105,000 and 145,000 to 150,000 daltons from normal human neutrophils. In this study, the effect of AHN-1 on a number of neutrophil functions was evaluated in vitro. Both complement- and antibody-mediated phagocytosis were inhibited when human neutrophils were pretreated with AHN-1 and opsonized bacteria were used as targets. The inhibition of phagocytosis was specific, in that lysosomal enzyme release and chemotaxis were not altered by treatment with AHN-1. AHN-1 did inhibit superoxide production by neutrophils in response to particulate stimuli, but not in response to the soluble stimulus, 12-O-tetradecanoylphorbol-13-acetate. The data indicate that one or both of these surface proteins may be important in the process of phagocytosis. AHN-1 should be useful in isolating and further characterizing the nature of these molecules.

A radioimmunoassay with monoclonal antibodies for the detection of antigenic cell-free Fc receptor.

Making use of 125I-labelled monoclonal rat antibodies against mouse Fc receptor we have developed a solid-phase radioimmunoassay for cell-free antigenic mouse Fc receptor (FcR). An 80% acetone solution was used for fixation of relatively large amounts of soluble proteins on PVC microtiter plates. As a result of this treatment FcR practically lost its activity to bind the Fc portion of an IgG molecule but retained its antigenicity, the binding of specific anti-FcR (aFcR) antibody being increased following acetone fixation. Concentrations of cell-free FcR in NP-40 extracts of FcR-expressing cells were calculated from the linear part of a standard curve and expressed in units of antigenic activity, 1 unit being the amount of antigenic FcR capable of binding 1 microgram of 125I-aFcR. The method may be used for detecting cell-free FcR as a minor constituent in a mixture of proteins.

A simple method for the preparation of extracts from animal cells which catalyze efficient in vitro protein synthesis.

A rapid, simple procedure is described for the preparation of cell-free extracts of both uninfected and virus-infected pig kidney and baby hamster kidney cells which are very active in the in vitro translation of not only endogenous mRNA, but also of exogenous mRNA added to extracts that had been depleted of endogenous mRNA by treatment with micrococcal nuclease. This procedure appears to be adaptable with only minor variations to many eukaryotic cell lines and should greatly facilitate in vitro molecular studies of protein synthesis and gene regulation at the level of translation.

Detection by ELISA of shed cell-surface molecules in serum.

Monoclonal antibodies OKT9 and OKT9A, which recognize distinct epitopes on the transferrin receptor, were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for this molecule. A 115,000 dalton molecule was detected in serum which probably represents a proteolytic cleavage fragment of the 186,000 dalton (dimer of two 93,000 chains) found on the cell surface of replicating cells. Serum levels of the shed transferrin receptor in human sera may be studied by means of this assay.

Alloreactive cloned T cell lines. V. Differential kinetics of IL 2, CSF, and BCSF release by a cloned T amplifier cell and its variant.

A noncytolytic, Thy-1+, MIs-responsive cloned T amplifier cell, designated L2, derived from secondary C57BL/6 anti-DBA/2 mixed leukocyte culture was found to produce IL 2 (Interleukin 2), granulocyte/macrophage colony-stimulating factor (CSF), and a polyclonal B cell-stimulating factor (BCSF) after alloantigenic stimulation. IL 2 activity was present transiently, with maximal levels observed 12 to 24 hr after exposure to alloantigen. Only trace amounts were present 96 hr after initiation of culture. CSF and BCSF activities were present at high levels by 24 hr and remained elevated throughout an 8-day culture period. Further evidence that the lymphokine(s) having IL 2 activity is different from those having CSF and BCSF activities was provided by L2V, a variant of L2 that did not produce IL 2. The time course of CSF and BCSF production by L2V after stimulation with alloantigen was similar to that found with L2, although IL 2 activity was not detected at any time. Since the release of lymphokines from both L2 and L2V cells could be induced by Con A in the absence of stimulating alloantigen, it is likely that the biologically active factors were produced by the cloned T cell.

Bacterial degradation of 3,4,5-trimethoxyphenylacetic and 3-ketoglutaric acids.

When grown at the expense of 3,4,5-trimethoxyphenylacetic acid, a species of Arthrobacter readily oxidized 3,4-dihydroxy-5-methoxyphenylacetic acid, but other structurally related aromatic acids were oxidized only slowly. Cell extracts contained a dioxygenase for 3,4-dihydroxy-5-methoxyphenylacetate, and the corresponding trihydroxy acid, which was not attacked by the enzyme, inhibited oxidation of this ring-fission substrate. Cell suspensions did not release carbon dioxide from 3,4-[methoxyl-14C]dihydroxy-5-methoxyphenylacetate but accumulated 1 mol of methanol per mol of 3,4,5-trimethoxyphenylacetate oxidized. A cell extract converted the ring-fission substrate into stoichiometric amounts of pyruvate and acetoacetate, formed from 3-ketoglutarate by the action of an induced decarboxylase. 3-Ketoglutaric acid served as sole source of carbon for many soil isolates.