Differentiated mouse kidney tubuloids as a novel in vitro model to study collecting duct physiology.

Kidney tubuloids are cell models that are derived from human or mouse renal epithelial cells and show high similarities with their in vivo counterparts. Tubuloids grow polarized in 3D, allow for long-term expansion, and represent multiple segments of the nephron, as shown by their gene expression pattern. In addition, human tubuloids form tight, functional barriers and have been succesfully used for drug testing. Our knowledge of mouse tubuloids, on the other hand, is only minimal. In this study, we further characterized mouse tubuloids and differentiated them towards the collecting duct, which led to a significant upregulation of collecting duct-specific mRNAs of genes and protein expression, including the water channel AQP2 and the sodium channel ENaC. Differentiation resulted in polarized expression of collecting duct water channels AQP2 and AQP3. Also, a physiological response to desmopressin and forskolin stimulation by translocation of AQP2 to the apical membrane was demonstrated. Furthermore, amiloride-sensitive ENaC-mediated sodium uptake was shown in differentiated tubuloids using radioactive tracer sodium. This study demonstrates that mouse tubuloids can be differentiated towards the collecting duct and exhibit collecting duct-specific function. This illustrates the potential use of mouse kidney tubuloids as novel in vitro models to study (patho)physiology of kidney diseases.

Prenatal diagnosis of facial clefting as part of the oculo-auriculo-vertebral spectrum.

We report a rare facial cleft (type 2 according to the Tessier classification) as the first presenting echographic sign of the oculo-auriculo-vertebral spectrum (OAVS) (Goldenhar syndrome). Associated malformations included a left lateral cleft with macrostomia, left ear hypoplasia, left preauricular tag, single umbilical artery, hyposegmentation of the left lung and imperforatio ani.

Atmospheric pressure changes and outdoor temperature changes in relation to spontaneous pneumothorax.

STUDY AIMS: To examine the influence of atmospheric pressure (AP) and temperature changes on the incidence of idiopathic spontaneous pneumothorax (SP). METHODS: From December 1991 through November 1993, 115 consecutive SP cases were selected. Patients were included after being in Amsterdam at least 1 full day before contracting the SP. Differences in air temperature and AP (provided hourly by the national weather bureau) for the days of the SP occurrence and the days previous to it were recorded to measure influences of air temperature and AP. The correlation between days with lightning and SP and clustering of SP was evaluated. RESULTS: SP occurred on 14.7% of the days in the 2-year period. There was no relationship between SP and a rise or fall in AP (Poisson regression). There was an average temperature rise of 0.57 degrees C from the day prior to the day of the SP, compared with a 0.08 degrees C fall on the days without SP. This difference is statistically significant and was consistent over the four seasons and both years. Seventy-three percent of the SP cases were clustered. A relationship between SP and thunderstorms was found. CONCLUSIONS: AP differences do not seem to influence the chance of developing SP. SP occurs in clusters, and more often 1 to 2 days after thunderstorms. Whether the identified temperature rise prior to the SP is a causative factor is unlikely; coexisting weather phenomena might explain this unexpected finding and should be studied in the future.

Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.

Selective biochemical modification of functional residues in recombinant human macrophage colony-stimulating factor beta (rhM-CSF beta): identification by mass spectrometry.

A rapid method combining classical chemical modification with mass spectrometry was developed to identify amino acids in the recombinant human macrophage colony-stimulating factor (rhM-CSF) protein of potential import to the ligand-receptor interaction. Diethyl pyrocarbonate modification of rhM-CSF beta (under nondenaturing conditions) results in a time- and concentration-dependent loss in receptor binding and biological activity. Peptide mapping of the reaction products by mass spectrometry showed that, with low DEP:M-CSF ratios (< 50:1), there was selective modification of histidine residues, whereas at higher ratios (> 50:1), Tyr and Lys residues were also modified. The loss in rhM-CSF beta activity was directly correlated with the extent of carbethoxylation of His9 and His15, as determined by matrix-assisted laser desorption/ionization mass spectrometric molecular weight determinations (MALDIMS). For these residues mono-modification was observed. By contrast, C-terminal histidine residues His176 and His210 showed bis-modifications, the extent of which had no correlation to losses in biological activity. These data suggest the importance of residues in the A-helix (His9 and His15) to ligand-receptor binding.

Immunoaffinity purification of epitope-tagged human beta 2-adrenergic receptor to homogeneity.

To obtain large quantities of pure human beta 2-adrenergic receptor (beta 2-AR) needed for structural studies, an efficient method for beta 2-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged beta 2-AR was expressed in Sf9 cells with a specific activity of 5-20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-beta-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60-70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of > 30%. The purified receptor was concentrated to > 1 mg/ml without loss of ligand binding activity.

Functional wild-type and carboxyl-terminal-tagged rat substance P receptors expressed in baculovirus-infected insect Sf9 cells.

The rat substance P (SP) receptor (SPR) was expressed in insect Sf9 cells by infection with recombinant baculovirus. The receptor bound SP with high affinity (KD = 360 pM) and had a rank order of affinity of SP > neurokinin A > neurokinin B. Ligand activation of the receptor resulted in an increase in both inositol lipid hydrolysis and intracellular Ca2+ concentration ([Ca2+]i). However, high-level expression of the receptor, in the absence of ligand, was correlated with increased basal turnover of inositol lipids and an elevated rate of Ca2+ influx. These results demonstrate that the Sf9 cells provide a suitable environment for the high-level expression of a functionally active SPR. Two carboxy-terminal epitope-tagged receptors (SPR-KT3 = SPR-TPPPEPET, COOH; SPR-Glu = SPR-EEEEYMPME, COOH) were also expressed. The affinity of the KT3-tagged receptor for ligand was similar to that of the wild-type receptor (KD = 405 pM), and that of the Glu-tagged receptor was slightly lower (KD = 1,082 pM). The high-affinity SP binding site of all three receptors was sensitive to guanosine 5'-O-(3-thiotriphosphate) pretreatment. The maximal signal-transducing ability of the epitope-tagged receptors was comparable to that of the wild-type receptor ([Ca2+]i rise as a percentage of wild-type: SPR-KT3, 80-100%; SPR-Glu, 88-100%). These data show that heterologous expression in the baculovirus system results in high expression of functional wild-type and tagged receptors.

Decreased macrophage colony-stimulating factor mRNA expression from activated cord versus adult mononuclear cells: altered posttranscriptional stability.

We have previously shown that protein production and mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin-3 are decreased in stimulated mononuclear cells (MNCs) from human umbilical cord compared with adult peripheral blood. These deficiencies may contribute to the increased susceptibility of neonates to infection. Macrophage colony-stimulating factor (M-CSF) regulates the proliferation, differentiation, and functional activation of monocytes. In the present study, we compared the regulation of M-CSF gene expression and protein production from stimulated cord and adult MNCs. Upon adhesion to tissue culture flasks, both cord and adult MNCs constitutively expressed M-CSF mRNA. In response to both adhesion and recombinant human GM-CSF (rhGM-CSF) stimulation for 120 hours, radioimmunoassays and bioassays showed that cord MNCs produced twofold to threefold less M-CSF protein compared with adult MNCs. Northern blot analysis also showed a fourfold decrease in M-CSF mRNA expression in both unstimulated and GM-CSF-induced cord versus adult MNCs. M-CSF mRNA expression in both cord and adult MNCs peaked between 16 and 24 hours and decreased to normal levels by 48 hours. We next determined the relative rates of transcription of the M-CSF gene by nuclear run-on assays in both cord and adult MNCs. The basal level signal of the M-CSF gene was similar between cord and adult MNCs. The transcriptional rate after stimulation with rhGM-CSF appeared to increase to a similar extent in both cord and adult MNCs (130% +/- 10% v 150% +/- 15%, C v A, n = 3, mean +/- SD). The comparative stability of M-CSF mRNA from cord versus adult MNCs was next determined by actinomycin D decay studies. The half-life of M-CSF mRNA from stimulated adult MNCs was 70 +/- 7.0 minutes (n = 4) compared with 47 +/- 2.8 minutes (n = 3) from stimulated cord MNCs (mean +/- SD, P < .05). To further determine the involvement of labile protein factors in posttranscriptional regulation, cord and adult MNCs were incubated with cycloheximide (CHX; 10 micrograms/mL). There was a significant increase in the induction of M-CSF mRNA by CHX treatment in both cord and adult MNCs. The increase of M-CSF mRNA induction by CHX was 2.5 times higher in cord MNCs compared with that in adult MNCs. These results suggest that there are one or more labile proteins that regulate M-CSF transcript stability in both cord and adult MNCs.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of exposure reducing measures on parameters of genetic risk in a population occupationally exposed to coal fly ash.

In a previous study we found increased SCE frequencies in peripheral blood lymphocytes (PBLs) of workers occupationally exposed in a coal fly ash processing industry, as compared to a non-exposed control population. Shortly after this study, measures were taken in this plant to reduce fly ash levels. The objective of the present study, conducted 2 years later in the same plants, was to evaluate the effect of these measures with respect to genotoxic risk. A group of 18 male workers of the coal fly ash processing industry agreed to participate in the study. The control population consisted of 18 male workers from a flour processing industry, who were matched for age and smoking behavior. In contrast to our previous study, no increased SCE frequencies were found in PBLs of workers potentially exposed to coal fly ash when compared to the control group (mean SCEs: 6.4 +/- 1.2 and 7.0 +/- 0.9, respectively). In addition, no differences were observed between the exposed and control groups for frequencies of gene mutations at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus in PBLs, for micronucleus frequencies using the cytokinesis block method, or for urinary mutagen excretion measured with Salmonella typhimurium tester strains TA98 and TA97 with and without metabolic activation. In smokers, however, SCE frequencies in PBLs were significantly increased in comparison to non-smokers (7.1 +/- 1.1 vs. 6.1 +/- 0.5; P < 0.005), as was 24-h urinary mutagen excretion measured with strain TA98 with S9 mix (2373 +/- 1870 vs. 156 +/- 211; P < 0.001) and with TA98 with S9 mix and beta-glucuronidase/arylsulfatase (2361 +/- 1958 vs. 538 +/- 396; P < 0.005). In addition, hprt variant frequencies in PBLs were higher in smokers than in non-smokers (15.0 +/- 23.5 x 10(-6)6 vs. 2.6 +/- 2.8 x 10(-6); P < 0.05). No differences were observed for micronucleus induction between smokers and non-smokers. It is concluded that the protective measures taken in the coal fly ash processing plant appear to have been sufficient, since an effect of exposure to coal fly ash on parameters of genetic risk was not found any longer.

Cytokines in infectious disease: the battle between host and pathogen.

The immune system is poised like a fulcrum to respond quickly to challenge by infectious agents, but can produce excess inflammatory signals or excess suppressive signals when out of balance. During the past year, significant progress has been made in our understanding of how certain pathogens promote immune suppression and shift the balance from the host in their favor. Understanding the mechanisms that underlie excessive inflammatory responses or the suppressive effects of the micro-organism will aid in the development of new therapies.

The substance P receptor, which couples to Gq/11, is a substrate of beta-adrenergic receptor kinase 1 and 2.

The agonist-occupied forms of several G-protein-coupled receptors that modulate the activity of adenylycyclase via Gs (e.g. beta 2-adrenergic) or Gi (e.g. alpha 2-adrenergic and cardiac muscarinic) are phosphorylated by beta-adrenergic receptor kinases (beta ARK 1 and beta ARK 2). beta ARK-catalyzed phosphorylation of these receptors appears to correlate with their agonist-induced desensitization. The possibility that beta ARK isozymes may also be involved in the desensitization of other G-protein-coupled receptors such as those mediating phosphoinositide (PI) hydrolysis was tested by determining the phosphorylation of the substance P receptor (SPR), which is coupled to PI hydrolysis in numerous tissues. Rat SPR was expressed in Sf9 cells, partially purified, and reconstituted in phospholipid vesicles. The reconstituted SPR bound the SPR agonist substance P, 125I-labeled with Bolton-Hunter reagent, with low affinity. However, addition of purified Gq/11 to the reconstituted SPR resulted in the conversion of all the receptors to a high affinity state, suggesting that SPR couples to Gq/11. Phosphorylation of the reconstituted SPR with purified beta ARK 1 or 2 in the absence and presence of substance P (SP) was then studied. In the presence of 100 microM SP, both kinases promoted phosphorylation of the receptor to a stoichiometry of 9 +/- 2 mol of phosphate/mol of receptor. However, no phosphorylation of the receptor could be detected in the absence of agonist. Agonist-induced phosphorylation of the receptor was blocked by coincubation with the SPR antagonist spantide. These results show that beta ARK isozymes may regulate the function of both adenylylcyclase as well as PI-coupled receptors, and suggest a role for beta ARK isozymes in SPR signal transduction.

Assignment of the inter- and intramolecular disulfide linkages in recombinant human macrophage colony stimulating factor using fast atom bombardment mass spectrometry.

The disulfide bridges in recombinant human macrophage colony stimulating factor (rhM-CSF), a 49-kDa homodimeric protein, were assigned. The 18 cysteines in the dimer form three intermolecular and two sets of three intramolecular disulfide bonds. The intermolecular disulfide bridges hold the dimer together and form symmetric bonds in which Cys31 and Cys157/Cys159 from one monomer unit are linked to the corresponding cysteines of the second monomer. The intramolecular disulfide bonds are located between Cys7-Cys90, Cys48-Cys139, and Cys102-Cys146, respectively. The resistance of native M-CSF to proteolytic cleavage was overcome by an initial chemical cleavage reaction using BrCN. The close proximity of four cysteines (Cys139, Cys146, Cys157, and Cys159) results in a tight core complex that makes the protein undigestable for most proteases. Digestion using endoprotease Asp-N resulted in cleavage at Asp156 near the C-terminal end of this region, thereby opening the complex structure.

Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry.

The primary structure of mouse interleukin-3 (IL-3) expressed by recombinant baculovirus-infected silkworm (Bombyx mori) larvae was analyzed by subjecting isolated IL-3 derived peptides to liquid secondary ion mass spectrometry. Two species of IL-3 were isolated from the silkworm hemolymph by reverse-phase high-pressure liquid chromatography. The major component has M(r)20-22 x 10(3) as determined by SDS-PAGE. Liquid secondary ion mass spectrometric analysis was carried out on the reduced tryptic and endopeptidase lysyl-C peptides of glycosylated and deglycosylated IL-3. These studies provided evidence that (1) Asn-16 is heterogeneously glycosylated with four different oligosaccharides, (2) Asn-86 is either nonglycosylated or has attached to it one oligosaccharide, (3) the N-glycosylation sites Asn-44 and Asn-51 are not glycosylated, and (4) there is no O-glycosylation. Liquid secondary ion mass spectrometric analysis of the unreduced tryptic peptides provided evidence for disulfide linkages between Cys-140 and Cys-79 or Cys-80 and between Cys-17 and Cys-79 or Cys-80. In comparison to the major component, a minor IL-3 species (M(r) 17-19 x 10(3) by SDS-PAGE) isolated from the hemolymph showed no difference with respect to the glycosylation pattern or the disulfide linkages, but it was cleaved between Ala-127 and Ser-128, and only a disulfide linkage between Cys-140 and Cys-79 or Cys-80 held the molecule together.(ABSTRACT TRUNCATED AT 250 WORDS)

AIC2A is a component of the purified high affinity mouse IL-3 receptor: temperature-dependent modulation of AIC2A structure.

IL-3, a potent hemopoietic growth factor, interacts with distinct classes of receptor, one of high affinity and the other of low affinity. The gene for a 115 kDa, low affinity IL-3 binding protein (AIC2A) was recently cloned. Ligand affinity purification was used to show that the AIC2A gene product participates in the formation of a high affinity IL-3 receptor (IL-3R). Cells were incubated with biotin-IL-3 at 4 degrees C and IL-3 bound to the low affinity site was removed by washing, cells were detergent extracted, and then streptavidin - agarose was used to purify proteins bound to biotin-IL-3. A 115 kDa phosphotyrosine (Ptyr)-containing protein was specifically purified and its identity as AIC2A was shown in Western assays using polyclonal anti-AIC2A antibodies. A brief temperature shift of the intact, biotin-IL-3-treated cells from 4 to 37 degrees C, prior to receptor purification, results in structural and compositional changes in the IL-3R, including: (i) a 10-20 kDa increase in the apparent Mr of both the AIC2A and the Ptyr antigens, and (ii) the association of a serine/threonine kinase. These observations indicate that in its native environment, the low affinity IL-3 binding protein, AIC2A, participates to form the high affinity IL-3R and is a substrate for a tyrosine kinase. Moreover, a ligand-induced, temperature-regulatable structural change in the IL-3R may be of importance in the transduction of information through the receptor, as suggested by the enhanced association of the IL-3R with a serine/threonine kinase.

Identification of a novel protein capable of interacting with the IL-3 receptor.

IL-3 has numerous functions in hematopoiesis yet its receptor has not been fully characterized. We have developed two mAb, 4G8 and 2F2, that markedly inhibited IL-3-dependent proliferation whereas only marginally affecting IL-2 or IL-4-induced proliferation. On Western blots, both antibodies identified the same protein, which varied in size from 115 to 145 kDa in six cell lines tested. The 4G8/2F2 Ag was detected at moderate density, on a wide variety of cells including IL-3-dependent cell lines and T lymphocytes. Radioligand binding studies revealed that 4G8, but not 2F2, could inhibit the binding of 125I-IL-3 to the high affinity IL-3R. These data suggest that the mAb 4G8 and 2F2 recognize different epitopes on the same Ag, and suggest furthermore that the inhibition of IL-3-dependent proliferation mediated by 2F2, in particular, does not occur via inhibition of ligand binding. Neither antibody showed an enhanced level of fluorescent staining of Cos 7 cells transfected with the low affinity IL-3R cDNA. In addition, 4G8 did not inhibit IL-3 binding to L cells transfected with the cloned IL-3R or IL-4R despite the fact that 4G8 was expressed on these cells. These data suggest that the 4G8/2F2 Ag is a unique cell surface protein that can interact with the endogenous functional IL-3R.

Characterization of three related murine interleukin-3 surface receptor proteins.

Iodinated interleukin-3 (IL-3) can be covalently cross-linked to three specific surface glycoproteins with net molecular masses of 170, 140, and 65-70 kDa under conditions in which ligand internalization and degradation do not occur. These three proteins plus two additional non-ligand-binding proteins of 90 and 55 kDa can be purified by IL-3 affinity chromatography. Comparative two-dimensional analysis of the tryptic digests of these five proteins indicates that the ligand-binding proteins are highly related at the peptide level. Incubation of cells with 125I-IL-3 at 37 degrees C results in rapid time- and energy-dependent internalization and degradation of ligand. Under these conditions only the 140- and 65-70-kDa binding proteins, which can recycle to the surface after internalization, can be identified. The lability of the 170-kDa protein indicates that it may not recycle. Thus, an energy-dependent mechanism is responsible for internalization and may be necessary for any potential interconversion of the higher 170- or 140-kDa proteins to the lower 140- and/or 65-70-kDa binding proteins.

Multiple in vivo effects of interleukin-3 and interleukin-6 on murine megakaryocytopoiesis.

The in vivo effects of interleukin-3 (IL-3), interleukin-6 (IL-6), and a combination of IL-3 plus IL-6 on murine megakaryocytopoiesis and thrombopoiesis were examined. Human recombinant IL-6 was administered subcutaneously as 14 equal injections of 5,000 units each during a 102-hour period. Murine recombinant IL-3 was given as 8 injections of 80,000 units each during the first 54 hours. Megakaryopoiesis and thrombopoiesis were evaluated 120 hours after initial administration of the cytokines. Platelet levels increased by 20% following IL-3 alone, 35% following IL-6 alone and 61% after administration of both IL-3 and IL-6. Platelet production, as measured by 75Se-selenomethionine incorporation, increased by approximately 120% in animals that had received IL-6 or IL-3 plus IL-6. Megakaryocyte ploidy analysis by two-color flow cytometry showed a shift in the modal ploidy class from 16N to 32N and a significant increase in the frequency of 64N cells only in IL-6 treated animals. Both bone marrow and splenic megakaryocyte colony-forming cells were significantly increased following either IL-3 or IL-6. Bone marrow megakaryocyte size increased 18%, 43%, and 38%, respectively, after administration of IL-3, IL-6, or the combination of IL-3 plus IL-6. Leukocyte counts and hematocrits were unaffected by either cytokine. Additional groups of mice received the same injection schedule as above and the serial effects on peripheral blood cell levels were assessed for 30 days. Platelet levels, which had been elevated by IL-3 or IL-6, fell to control values within 4 days following the last injection. Animals given IL-6 or IL-3 plus IL-6 were subsequently thrombocytopenic relative to controls on days 7 through 9 following cessation of treatment. Temporary 'cycling' of platelet levels was observed for 3 weeks following treatment with IL-6 or the combination of IL-3 plus IL-6. We conclude that IL-6 and to a lesser extent IL-3 stimulate platelet production in vivo and that their combined effects on platelet levels are approximately additive. Following discontinuation of IL-3 or IL-6, the effects are rapidly reversed, presumably by negative feedback mechanisms, resulting in a period of 'rebound thrombocytopenia' in mice that had received IL-6.

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3.

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and 252Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17,000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites. Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.