Saturable Manner Research Articles

The central cavity in trimeric glutamate transporters restricts ligand diffusion

A prominent aqueous cavity is formed by the junction of three identical subunits in the excitatory amino acid transporter (EAAT) family. To investigate the effect of this structure on the interaction of ligands with the transporter, we recorded currents in voltage-clamped Xenopus oocytes expressing EAATs and used concentration jumps to measure binding and unbinding rates of a high-affinity aspartate analog that competitively blocks transport (β-2-fluorenyl-aspartylamide; 2-FAA). The binding rates of the blocker were approximately one order of magnitude slower than l-Glu and were not significantly different for EAAT1, EAAT2, or EAAT3, but 2-FAA exhibited higher affinity for the neuronal transporter EAAT3 as a result of a slower dissociation rate. Unexpectedly, the rate of recovery from block was increased by l-Glu in a saturable and concentration-dependent manner, ruling out a first-order mechanism and suggesting that following unbinding, there is a significant probability of ligand rebinding to the same or neighboring subunits within a trimer. Consistent with such a mechanism, coexpression of wild-type subunits with mutant (R447C) subunits that do not bind glutamate or 2-FAA also increased the unblocking rate. The data suggest that electrostatic and steric factors result in an effective dissociation rate that is approximately sevenfold slower than the microscopic subunit unbinding rate. The quaternary structure, which has been conserved through evolution, is expected to increase the transporters' capture efficiency by increasing the probability that following unbinding, a ligand will rebind as opposed to being lost to diffusion.

Interaction of Actin with Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) Receptor in Liposomes Is Ca2+- and Phospholipid-dependent

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.

Decorin Interacts with Connective Tissue Growth Factor (CTGF)/CCN2 by LRR12 Inhibiting Its Biological Activity

Fibrotic disorders are the end point of many chronic diseases in different tissues, where an accumulation of the extracellular matrix occurs, mainly because of the action of the connective tissue growth factor (CTGF/CCN2). Little is known about how this growth factor activity is regulated. We found that decorin null myoblasts are more sensitive to CTGF than wild type myoblasts, as evaluated by the accumulation of fibronectin or collagen III. Decorin added exogenously negatively regulated CTGF pro-fibrotic activity and the induction of actin stress fibers. Using co-immunoprecipitation and in vitro interaction assays, decorin and CTGF were shown to interact in a saturable manner with a K(d) of 4.4 nM. This interaction requires the core protein of decorin. Experiments using the deletion mutant decorin indicated that the leucine-rich repeats (LRR) 10-12 are important for the interaction with CTGF and the negative regulation of the cytokine activity, moreover, a peptide derived from the LRR12 was able to inhibit CTGF-decorin complex formation and CTGF activity. Finally, we showed that CTGF specifically induced the synthesis of decorin, suggesting a mechanism of autoregulation. These results suggest that decorin interacts with CTGF and regulates its biological activity.

Occupancy of dopamine D₂ and D₃ and serotonin 5-HT₁A receptors by the novel antipsychotic drug candidate, cariprazine (RGH-188), in monkey brain measured using positron emission tomography.

Cariprazine is a novel antipsychotic drug candidate that exhibits high selectivity and affinity to dopamine D(3) and D(2) receptors and moderate affinity to serotonin 5-HT(1A) receptors. Targeting receptors other than D(2) may provide a therapeutic benefit for both positive and negative symptoms associated with schizophrenia. Positron emission tomography (PET) can be used as a tool in drug development to assess the in vivo distribution and pharmacological properties of a drug. The objective of this study was to determine dopamine D(2)/D(3) and serotonin 5-HT(1A) receptor occupancy in monkey brain after the administration of cariprazine. We examined three monkeys using the following PET radioligands: [(11)C]MNPA (an agonist at D(2) and D(3) receptors), [(11)C]raclopride (an antagonist at D(2) and D(3) receptors), and [(11)C]WAY-100635 (an antagonist at 5-HT(1A) receptors). During each experimental day, the first PET measurement was a baseline study, the second after a low dose of cariprazine, and the third after the administration of a high dose. We found that cariprazine occupied D(2)/D(3) receptors in a dose-dependent and saturable manner, with the lowest dose occupying ~5% of receptors and the highest dose showing more than 90% occupancy. 5-HT(1A) receptor occupancy was considerably lower compared with D(2)/D(3) occupancy at the same doses, with a maximal value of ~30% for the raphe nuclei. We conclude that cariprazine binds preferentially to dopamine D(2)/D(3) rather than to serotonin 5-HT(1A) receptors in monkey brain. These findings can be used to guide the selection of cariprazine dosing in humans.

The stable prostacyclin-analogue, iloprost, unlike prostanoids and leukotrienes, potently stimulates cyclic adenosine monophosphate synthesis of primary astroglial cell cultures

The effect of different eicosanoids on adenosine-3', 5'-cyclic-monophosphate (cAMP) accumulation in primary astroglial cell cultures prepared from newborn rat brain was studied. The stable prostacyclin-analogue, iloprost, effectively stimulated cAMP synthesis in a concentration-dependent, saturable manner, the EC50 being about 3 x 10(-8) M. Prostaglandin (PG) E2 was less potent, without reaching plateau even at 10(-5) M. Prostaglandins D2 and F2 alpha, and the stable thromboxane A2-analogue, U 46619, as well as leukotrienes (LT) B4, C4, D4 and E4 were not effective and did not attenuate basal or isoprenaline (10(-8) M)-stimulated astroglial cAMP formation. This is the first indication for the existence of a prostacyclin receptor coupled positively to the adenylate cyclase in astrocytes. Other eicosanoids are unlikely to be involved in receptor-mediated regulation of astroglial cAMP levels.

Clarithromycin Accumulation by Phagocytes and Its Effect on Killing of Aggregatibacter actinomycetemcomitans

Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [(3)H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs. Mature human PMNs, HL-60 cells differentiated into granulocytes, and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a K(m) of ≈250 μg/ml and a V(max) of 473 ng/min/10(6) cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half-times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.

Mhp107 Is a Member of the Multifunctional Adhesin Family of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1(Mhp107), F2(Mhp107), and F3(Mhp107), spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1(Mhp107) was found to bind porcine heparin (K(D) ∼90 nM) in a dose-dependent and saturable manner, whereas F3(Mhp107) bound fibronectin (K(D) ∼180 nM) at physiologically relevant concentrations. F1(Mhp107) also bound porcine plasminogen (K(D) = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3(Mhp107) mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1(Mhp107)-F3(Mhp107)) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniae adhesin family of surface proteins and contributes to both adherence to the host and pathogenesis.

Identification and characterization of a heme periplasmic-binding protein in Haemophilus ducreyi

Haemophilus ducreyi, a gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Heme acquisition in H. ducreyi proceeds via a receptor mediated process in which the initial event involves binding of hemoglobin and heme to their cognate outer membrane proteins, HgbA and TdhA, respectively. Following this specific interaction, the fate of the periplasmic deposited heme is unclear. Using protein expression profiling of the H. ducreyi periplasmic proteome, a periplasmic-binding protein, termed hHbp, was identified whose expression was enhanced under heme-limited conditions. The gene encoding this protein was situated in a locus displaying genetic characteristics of an ABC transporter. The purified protein bound heme in a dose-dependent and saturable manner and this binding was specifically competitively inhibited by heme. The hhbp gene functionally complemented an Escherichia coli heme uptake mutant. Expression of the heme periplasmic-binding protein was detected in a limited survey of H. ducreyi and H. influenzae clinical strains. These results indicate that the passage of heme into the cytoplasm of H. ducreyi involves a heme dedicated ABC transporter.

Essential Role for Protein Kinase Cζ in Oleic Acid-Induced Glucagon-Like Peptide-1 Secretion in Vivo in the Rat

Luminal monounsaturated long-chain fatty acids [e.g. oleic acid (OA)] increase secretion of the incretin, glucagon-like peptide-1 (GLP-1) from the ileocolonic L cell. However, it is not known whether OA ingestion causes a sufficient increase in distal luminal concentrations to directly enhance GLP-1 secretion. Furthermore, we have demonstrated that protein kinase Cζ (PKCζ) is required for OA-induced GLP-1 secretion in vitro; however, the physiological relevance of this finding remains unknown. Therefore, we have determined luminal OA concentrations in OA-fed rats and examined the effects of direct OA stimulation on GLP-1 secretion using a novel model of intestinal-specific PKCζ knockdown. Murine GLUTag L cells express numerous fatty acid transport proteins and take up OA in a saturable manner. Oral administration of OA increased the ileal chyme content of OA by 140-fold over 60-120 min (P < 0.05-0.01), peaking at 105 ± 50 μmol/g. To evaluate the direct effects of OA on GLP-1 secretion, 125 mm OA was rectally infused into the colon and terminal ileum of rats. Plasma bioactive GLP-1 increased from 20 ± 6 to 102 ± 21 pg/ml at 60 min (P < 0.01). However, pretreatment with ileocolonic adenoviral PKCζ small interfering RNA resulted in a 68 ± 8% reduction in the GLP-1 response to rectal OA (P < 0.001). The results of these studies indicate that OA levels in the rat terminal gut after oral ingestion are sufficient to induce GLP-1 secretion and that PKCζ is necessary for the effects of OA on GLP-1 secretion in vivo. PKCζ may therefore serve as a novel therapeutic target to enhance GLP-1 levels in patients with type 2 diabetes.

Dose-dependent binding of AZD3783 to brain 5-HT1B receptors in non-human primates and human subjects: a positron emission tomography study with [11C]AZ10419369

The serotonin 5-HT(1B) receptor is a potential target for the pharmacologic treatment of depression. Positron emission tomography (PET) determination of 5-HT(1B) receptor occupancy with drug candidates targeting this receptor in non-human primate and human subjects may facilitate translation of research from animal models and guide dose selection for clinical studies. AZD3783 is a recently developed, orally bioavailable 5-HT(1B) receptor antagonist with potential antidepressant properties. To determine the relationship between plasma concentration of AZD3783 and occupancy at primate brain 5-HT(1B) receptors using PET and the radioligand [(11)C]AZ10419369. PET studies with [(11)C]AZ10419369 were performed in three non-human primates at baseline and after intravenous injection of AZD3783. Subsequently, PET measurements were undertaken in six human subjects at baseline and after administration of different single oral doses of AZD3783 (1-40 mg). After administration in non-human primates and human subjects, AZD3783 reduced regional [(11)C]AZ10419369 binding in a dose-dependent and saturable manner. The AZD3783 plasma concentration required for 50% receptor occupancy (K (i,plasma)) for monkeys was 25 and 27 nmol/L in occipital cortex and striatum, respectively. Corresponding estimates for human occipital cortex and ventral striatum were 24 and 18 nmol/L, respectively. The potential antidepressant AZD3783 binds in a saturable manner to brain 5-HT(1B) receptors with a similar in vivo affinity for human and monkey receptors. [(11)C]AZ10419369 can be successfully used to determine occupancy at brain 5-HT(1B) receptors in vivo and constitutes a useful tool for dose selection in clinical studies with 5-HT(1B) receptor compounds.

Entry of Bacillus anthracis spores into epithelial cells is mediated by the spore surface protein BclA, integrin α2β1 and complement component C1q

Inhalational anthrax is initiated by pulmonary exposure to Bacillus anthracis spores. Spore entry into lung epithelial cells is observed both in vitro and in vivo and evidence suggests it is important for bacterial dissemination and virulence. However the specific host receptor and spore factor that mediate the entry process were unknown. Here, we report that integrin α2β1 is a major receptor for spore entry. This is supported by results from blocking antibodies, siRNA knock-down, colocalization, and comparison of spore entry into cells that do or do not express α2. BclA, a major spore surface protein, is found to be essential for entry and α2β1-mediated entry is dependent on BclA. However, BclA does not appear to bind directly to α2. Furthermore, spore entry into α2-expressing cells is dramatically reduced in the absence of serum, suggesting that additional factors are involved. Finally, complement component C1q, also an α2β1 ligand, appears to act as a bridging molecule or a cofactor for BclA/α2β1-mediated spore entry and BclA binds to C1q in a dose-dependent and saturable manner. These findings suggest a novel mechanism for pathogen entry into host cells as well as a new function for C1q-integrin interactions. The implications of these findings are discussed.

A Cost‐effective High‐resolution Melting Approach using the EvaGreen Dye for DNA Polymorphism Detection and Genotyping in Plants

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.

Binding of Semenogelin I to Intact Human Spermatozoa Studied by Flow Cytometry and Surface Plasmon Resonance

Approximately 1 in 10 couples is infertile. No definite cause can be found in about 25% of those cases. Studies have indicated that seminal vesicle secretion functions as an optimizer of fertilization. The Zn(2+) binding protein semenogelin I (SgI) represents a major fraction of the proteins present in seminal vesicle fluid, and it serves as a structural component of the coagulum that is formed after ejaculation. Cleavage of SgI by prostate-specific antigen results in liquefaction of the coagulum. Fragmented SgI has antibacterial effects and inhibits spermatozoa mobility. SgI has also been found complexed to eppin on spermatozoa, and this complex has been suggested to be of importance for fertility. Here, we used flow cytometry and surface plasmon resonance to study SgI regarding its association with spermatozoa and the interaction dependency on Zn(2+). The concentration of Zn(2+) in seminal plasma is approximately 100 times higher than in blood plasma, and the metal ion is known to change the structure of SgI. We found that SgI binds to spermatozoa in a concentration-dependent and saturable manner. In solution, SgI bound to spermatozoa in a non-Zn(2+)-dependent way, whereas immobilized SgI interacts with spermatozoa only in the presence of Zn(2+). It indicates that SgI must exhibit a specific structure or free flexibility to be able to interact with that ligand. Our results indicate that the association of SgI to spermatozoa is conformation dependent and specific. These findings could constitute a basis for the development of a male contraceptive.

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

Liposomes modified by carbohydrate ligands can target B cells for the treatment of B-cell lymphomas

Evaluation of: Chen WC, Completo GC, Sigal DS, Crocker PR, Saven A, Paulson JC. In vivo targeting of B-cell lymphoma with glycan ligands of CD22. Blood 115(23), 4778–4786 (2010).A strategy has been developed to deliver selectively chemotherapeutic drugs to B cells by employing doxorubicin-loaded liposomes modified by a ligand for the B-cell-specific cell-surface protein CD22, also known as Siglec-2. The liposomes bound in a rapid and saturable manner to the human Burkitt lymphoma Daudi B-cell line and exhibited significantly higher cytotoxicity in vitro and in vivo compared with similar untargeted liposomes. The CD22-targeted liposome bound to B cells isolated from lymphoma patients and although binding was proportional to CD22 expression on the cell surface, low levels of expression on chronic lymphocytic leukemia cells were sufficient to effect cell neutralization. The glycan-based strategy for delivery of chemotherapeutic agents may provide a new strategy for the treatment of B-cell lymphomas.

An appraisal of serum preptin levels in PCOS

Plasma preptin levels were higher in women with PCOS compared with the control group (mean±SD, 823.2±140.7 vs. 324.9±147.3 pg/mL). This article suggests a potential role of preptin in the pathogenesis of PCOS.

A Processed Multidomain Mycoplasma hyopneumoniae Adhesin Binds Fibronectin, Plasminogen, and Swine Respiratory Cilia

Porcine enzootic pneumonia is a chronic respiratory disease that affects swine. The etiological agent of the disease, Mycoplasma hyopneumoniae, is a bacterium that adheres to cilia of the swine respiratory tract, resulting in loss of cilia and epithelial cell damage. A M. hyopneumoniae protein P116, encoded by mhp108, was investigated as a potential adhesin. Examination of P116 expression using proteomic analyses observed P116 as a full-length protein and also as fragments, ranging from 17 to 70 kDa in size. A variety of pathogenic bacterial species have been shown to bind the extracellular matrix component fibronectin as an adherence mechanism. M. hyopneumoniae cells were found to bind fibronectin in a dose-dependent and saturable manner. Surface plasmon resonance was used to show that a recombinant C-terminal domain of P116 bound fibronectin at physiologically relevant concentrations (K(D) 24 ± 6 nm). Plasmin(ogen)-binding proteins are also expressed by many bacterial pathogens, facilitating extracellular matrix degradation. M. hyopneumoniae cells were found to also bind plasminogen in a dose-dependent and saturable manner; the C-terminal domain of P116 binds to plasminogen (K(D) 44 ± 5 nm). Plasminogen binding was abolished when the C-terminal lysine of P116 was deleted, implicating this residue as part of the plasminogen binding site. P116 fragments adhere to the PK15 porcine kidney epithelial-like cell line and swine respiratory cilia. Collectively these data suggest that P116 is an important adhesin and virulence factor of M. hyopneumoniae.

Lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal phylogenetic diversity in heat shock protein 70 sequences

We previously found that mouse inducible Hsp72 bound more extensively to lymphoblast-like lymphoid neoplastic P388D1 cells than to RAW264.7 monocyte–macrophages. In the present study, we analyzed the characteristics of the binding to P388D1 cells of recombinant HSP70 derived from different species. Recombinant mouse inducible-type Hsp72 bound extensively to P388D1cells in a saturable manner, but not to P815 mastocytoma or EL4 thymoma. Spinach Hsc70-1, highly homologous with mouse Hsp72 in the C-terminal region, also bound to P388D1 cells. In contrast, significant binding was not observed for bacterial DnaK derived from Lactobacillus acidophilus and Escherichia coli which have relatively little homology in this region. Analyses of surface antigens showed that B220, and CD19, but not CD91, LOX-1, and CD40, the HSP70 receptors reported so far, were present on P388D1 cells, suggesting a B-cell lineage for this cell line. A similar discrimination of the diversity between mouse Hsp72 and bacterial DnaK occurred for CD19 + B cells derived from mouse spleen, Peyer's patches, and mesenteric lymph nodes. The binding of HSP70 to P388D1 cells was partially, but significantly, antagonized by fucoidan and maleylated BSA, implying a few types of scavenger receptors to be responsible for the binding of HSP70 to this cell line. Furthermore, photo-affinity labeling revealed several membrane protein candidates larger than 110 kDa to be involved in the recognition of HSP70 molecules. Using a NF-κB-luciferase reporter assay, we found that exogenous HSP70 did not stimulate NF-κB-dependent signal transduction in P388D1 cells. It thus follows that lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal sequences of the HSP70 family. The present results indicate that several types of cells in the innate immune system may distinguish among the phylogenetically specific signals in protein molecules.

Function and morphology correlates of rectal nerve mechanoreceptors innervating the guinea pig internal anal sphincter

Mechanoreceptors to the internal anal sphincter (IAS) contribute to continence and normal defecation, yet relatively little is known about their function or morphology. We investigated the function and structure of mechanoreceptors to the guinea pig IAS. Extracellular recordings from rectal nerve branches to the IAS in vitro, combined with anterograde labeling of recorded nerve trunks, were used to characterize extrinsic afferent nerve endings activated by circumferential distension. Slowly adapting, stretch-sensitive afferents were recorded in rectal nerves to the IAS. Ten of 11 were silent under basal conditions and responded to circumferential stretch in a saturating linear manner. Rectal nerve afferents responded to compression with von Frey hairs with low thresholds (0.3-0.5 mN) and 3.4 ± 0.5 discrete, elongated mechanosensitive fields of innervation aligned parallel to circular muscle bundles (length = 62 ± 16 mm, n = 10). Anterogradely labeled rectal nerve axons typically passed through sparse irregular myenteric ganglia adjacent to the IAS, before ending in extensive varicose arrays within the circular muscle and, to a lesser extent, the longitudinal muscle overlying the IAS. Few (8%) IAS myenteric ganglia contained intraganglionic laminar endings. In eight preparations, mechanotransduction sites were mapped in combination with successful anterograde fills. Mechanotransduction sites were strongly associated with extensive fine varicose arrays within the circular muscle (P < 0.05), and not with any other neural structures. Mechanotransduction sites for low-threshold, slowly adapting mechanoreceptors innervating the IAS are likely to correspond to extensive fine varicose arrays within the circular muscle.

Recombinant expression in human cells of active integrin α 1β 1-blocking RTS-disintegrin jerdostatin

Jerdostatin, an RTS short disintegrin cloned from Protobothrops jerdonii and recombinantly produced in Escherichia coli, is a potent and specific antagonist of the α 1β 1 integrin. Jerdostatin selectively blocked the adhesion of α 1β 1-K562 cell to collagens I and IV in vitro and angiogenesis in vivo. Here we report the recombinant production of jerdostatin in a mammalian cell system, a prerequisite for developing a conditional transgenic mouse to investigate the effect of systemic expression of jerdostatin on tumor development. For proper export of jerdostatin, a secretion leader sequence was engineered at the protein’s N-terminus. A FLAG epitope was also included at the N-terminus of the mature disintegrin to facilitate its isolation and characterization of recombinant jerdostatin (rJerd). This pRc-CMV/FLAG-rJerd construct was transiently expressed in HEK-293 cells and was efficiently secreted into the culture medium. rJerd bound to recombinant soluble α 1β 1 integrin in a saturable and cation-independent manner. Soluble rJerd also inhibited the binding of α 1β 1 integrin to the CB3 fragment of collagen IV in a dose-dependent manner (IC 50 570 nM). Mammalian cell-expressed jerdostatin disrupted the adhesion of RuGli cells to collagen IV. Our results highlight pRc-CMV/FLAG-rJerd as a suitable construct for expressing soluble active α 1β 1-blocking jerdostatin in a mammalian cell system.