Mouse Horizontal Cells do not Express Connexin26 or Connexin36

Gap junctions between neurons function as electrical synapses, and are present in all layers of mammalian and teleost retina. These synapses are largest and most prominent between horizontal cells where they function to increase the receptive field of a single neuron beyond the width of its dendrites. Receptive field size and the extent of gap junctional coupling between horizontal cells is regulated by ambient light levels and may mediate light/dark adaptation. Furthermore, teleost horizontal cell gap junction hemichannels may facilitate a mechanism of feedback inhibition between horizontal cells and cone photoreceptors. As a prelude to using mouse genetic models to study horizontal cell gap junctions and hemichannels, we sought to determine the connexin complement of mouse horizontal cells. Cx36, Cx37, Cx43, Cx45 and Cx57 mRNA could be detected in mouse retina by RT-PCR. Microscopy was used to further examine the distribution of Cx26 and Cx36. Cx26 immunofluorescence and a β-gal reporter under regulatory control of the Cx36 promoter did not colocalize with a horizontal cell marker, indicating that these genes are not expressed by horizontal cells. The identity of the connexin(s) forming electrical synapses between mouse horizontal cells and the connexin that may form hemichannels in the horizontal cell telodendria remains unknown.     

Macrophages and T cells do not express Mlsa determinants

In order to test the tissue distribution of Mlsa determinants, we have generated highly purified stimulator cell populations. First, Mlsa expression in bone marrow derived macrophages (M phi) of Mlsa genotype was tested in primary MLR and on Mlsa-specific T cell hybridomas (THy). Second, a similar experimental approach was used to analyze thioglycolate, peptone or Con A elicited peritoneal M phi. In all cases, these M phi cell populations were able to generate an excellent alloresponse, whereas no functional Mlsa determinants could be detected. Third, to further investigate whether the expression of Mlsa is lymphocyte specific, but dependent on expression of class II molecules, we have transfected I-Ek alpha and beta cDNA into a panel of thymomas of Mlsa genotype. Although we achieved a high level of surface I-Ek expression in all of these T cell tumors, none of them was able to trigger the Mlsa-specific THy. These results strongly suggest that Mlsa expression is limited to B cells. It is likely that Mlsa is a tissue-specific self-peptide that associates with class II molecules.     

Platelets do not express the oxidized or reduced forms of tissue factor

Background

Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.

Methods

Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.

Results

Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.

Conclusion and significance

Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.     

Natural killer cells do not lyse resting or mitogen-stimulated B cells

It has recently been reported that isolated resting natural killer cells lyse autologous resting and mitogen-stimulated B cells. In this report, we have been unable to corroborate these observations and provide indirect evidence that lytic susceptibility is attributable to exposure of the target cells to xenogeneic antigens present in fetal calf serum (FCS). Moreover, we show that interleukin-2-activated killer cells potently lyse normal peripheral blood mononuclear cells which are exposed to FCS.     

Xenopus laevis larval thymocytes do not express surface immunoglobulin

Xenopus laevis larval thymocytes and splenocytes were examined for the presence of Ig determinants by an indirect immunofluorescence technique, using rabbit antiserum to deglycosylated Xenopus immunoglobulins. Thymocytes had no detectable surface membrane Ig, while Ig determinants were identified on the surface of a large percentage of the lymphocytes from the spleen. The positive fluorescent staining that one obtains on the surface of thymocytes using antisera to intact Ig's is due to antibody molecules directed to the carbohydrate determinants of the Ig's which cross-react with thymocytes' surface carbohydrate determinants.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Mouse Dach2 mutants do not exhibit gross defects in eye development or brain function

Drosophila dachshund is a critical regulator of eye, brain, and limb formation. Vertebrate homologs, Dach1 and Dach2, are expressed in the developing retina, brain, and limbs, suggesting functional conservation of the dachshund/Dach gene family. Dach1 mutants die postnatally, but exhibit grossly normal development. Here we report the generation of Dach2 mutant mice. Although deletion of Dach2 exon 1 results in abrogation of RNA expression, Dach2 mutants are viable and fertile. Histochemical analysis reveals grossly normal Dach2 mutant eye development. In addition, a battery of neurological assays failed to yield significant differences in behavior between Dach2 mutants and controls. We discuss these findings in the light of published observations of DACH2 mutations in the human population. Finally, to test the functional conservation hypothesis, we generated Dach2; Dach1 double mutant mice. Dach double mutants die after birth, similar to Dach1 homozygotes. However, unlike Drosophila dachshund mutants that lack eyes and exhibit leg truncations, the eyes and limbs of Dach double mutants are present, suggesting differences between Dach and dachshund gene function during embryonic eye and limb formation.     

Plasmacytoid dendritic cells do not migrate in intestinal or hepatic lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRalphabeta or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.     

Kinetics of Ii synthesis, processing, and turnover in n-butyrate-treated Burkitt's lymphoma cell lines which express or do not express class II antigens and in hairy leukemic cells

We have sought to understand the role of the electrophoretically invariant chain (Ii) in class II antigen functions, particularly in certain transformed cells in which we have previously demonstrated hyperexpression of Ii. Molecular structures and relative kinetics of Ii synthesis, processing and turnover were compared in paired, Ia+ and Ia- Burkitt's lymphoma (BL) cell lines and in hairy cell leukemia (HCL) cells. Cells were metabolically labeled with [35S] methionine for 15 min (with or without a cold methionine chase to 3 hr) or were continuously labeled for 3 hr. One- and two-dimensional gel electrophoresis resolved immunoprecipitates formed with a) a heteroantiserum to purified class II antigen (demonstrating alpha and beta chains and Ii associated with that complex), b) a heteroantiserum to hairy cell leukemia (HCL) membranes (demonstrating principally the dominant, basic form of Ii molecules, class I antigens, and some additional proteins), and c) a monoclonal antibody to human Ii. Treatment of Ia+ Jijoye and its daughter, Ia- P3HR-1, BL cells with 4 mM butyrate for 48 hr enhanced the synthesis of the dominant, basic form of Ii but did not affect apparent turnover rates of that pool of Ii chains in either cell line. In Ia+ Jijoye cells but not in Ia- P3HR-1 cells Ii was terminally processed to more acidic, sialic acid-derivatized forms. Butyrate treatment did not alter the relative turnover rate of terminally processed Ii in Jijoye cells. The level of the dominant, basic form of Ii in HCL cells equaled that in butyrate-treated Jijoye cells, and relative turnover rates of this terminally unprocessed Ii pool were similar in HCL and Jijoye cells. However, HCL Ia-associated Ii was not terminally processed, as was Ia-associated Ii in Jijoye cells. The expression of Ia auxiliary proteins, p41 and p25, was also enhanced in Jijoye cells by butyrate treatment and was prominent in HCL cells. From these experiments, we may hypothesize the following. In lymphoblastoid cells, two pathways for Ii turnover could exist. One is through association with Ia complexes and progressive terminal processing of carbohydrate side chains and a second is not associated with Ia or, apparently, with such processing. Because Ii is not found to be terminally processed in the absence of class II antigen, Ia might be considered to direct the processing of a subset of Ii towards some function (rather than vice versa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Maturation ameloblasts of the porcine tooth germ do not express amelogenin

 Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. Accepted: 14 January 1999     

Human neutrophils do not express purinergic P2X7 receptors

It has been reported that in human neutrophils, external ATP activates plasma membrane purinergic P2X₇ receptors (P2X₇R) to elicit Ca²⁺ entry, production of reactive oxygen species (ROS), processing and release of pro-inflammatory cytokines, shedding of adhesion molecules and uptake of large molecules. However, the expression of P2X₇R at the plasma membrane of neutrophils has also been questioned since these putative responses are not always reproduced. In this work, we used electrophysiological recordings to measure functional responses associated with the activation of membrane receptors, spectrofluorometric measurements of ROS production and ethidium bromide uptake to asses coupling of P2X₇R activation to downstream effectors, immune-labelling of P2X₇R using a fluorescein isothiocyanate-conjugated antibody to detect the receptors at the plasma membrane, RT-PCR to determine mRNA expression of P2X₇R and Western blot to determine protein expression in neutrophils and HL-60 cells. None of these assays reported the presence of P2X₇R in the plasma membrane of neutrophils and non-differentiated or differentiated HL-60 cells—a model cell for human neutrophils. We concluded that P2X₇R are not present at plasma membrane of human neutrophils and that the putative physiological responses triggered by external ATP should be reconsidered.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Growth inhibition by connexin26 expression in cultured rodent tumor cells

The Connexin (Cx) gene family acts as a tumor suppressor. However, it is unclear whether the tumor-suppressing activity acquired by Cx gene transfection is mainly due to the recovery of the gap junction-mediated intercellular communication (GJIC) or to other unknown mechanisms. In order to elucidate the mechanism of the Cx-induced tumor-suppressing activity, we transfected Cx26 cDNA into a rodent mammary tumor cell-line (BICR-M1Rk) in which Cx43 had been normally expressed and a typical pattern of GJIC had been observed. The exogenous Cx26 was mainly localized on the nuclear envelope, whereas most of the endogenous Cx43 resided at the plasma membrane of the transfected BICR-M1Rk. Consistent with the localization of Cx26, GJIC was not increased upon the transfection of Cx26 when it was assessed by a scrape-loading dye transfer technique. A conventional [3H]-thymidine incorporation study showed that the growth rate of the Cx26-transfected cells was significantly decreased (70%), compared to that of the control BICR-M1Rk. Therefore, our results clearly demonstrate that the exogenously expressed Cx26 in the BICR-M1Rk cancer cell-line exerts an anti-proliferate activity in a GJIC-independent manner.