Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIα subunit would reveal those tissues and signaling events that require anchored PKA. RIIα knockout mice appear normal and healthy. In adult skeletal muscle, RIα protein levels increased to partially compensate for the loss of RIIα. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIα knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA–AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIα subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIα subunit. The potentiation of the L-type Ca2+ channel in RIIα knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIα is capable of physiologically relevant anchoring interactions.

Ca2+/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca2+ concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca2+ concentration, produced by patterned synaptic activity or intradendritic Ca2+ injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca2+/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca2+ concentration and is expressed as a long-lasting increase of postsynaptic GABAA receptor sensitivity. Herein we show that inhibitors of the Ca2+/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca2+ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.

Overexpression of a Kinase-deficient Transforming Growth Factor-β Type II Receptor in Mouse Mammary Stroma Results in Increased Epithelial Branching

Members of the transforming growth factor-β (TGF-β) superfamily signal through heteromeric type I and type II serine/threonine kinase receptors. Transgenic mice that overexpress a dominant-negative mutation of the TGF-β type II receptor (DNIIR) under the control of a metallothionein-derived promoter (MT-DNIIR) were used to determine the role of endogenous TGF-βs in the developing mammary gland. The expression of the dominant-negative receptor was induced with zinc and was primarily localized to the stroma underlying the ductal epithelium in the mammary glands of virgin transgenic mice from two separate mouse lines. In MT-DNIIR virgin females treated with zinc, there was an increase in lateral branching of the ductal epithelium. We tested the hypothesis that expression of the dominant-negative receptor may alter expression of genes that are expressed in the stroma and regulated by TGF-βs, potentially resulting in the increased lateral branching seen in the MT-DNIIR mammary glands. The expression of hepatocyte growth factor mRNA was increased in mammary glands from transgenic animals relative to the wild-type controls, suggesting that this factor may play a role in TGF-β-mediated regulation of lateral branching. Loss of responsiveness to TGF-βs in the mammary stroma resulted in increased branching in mammary epithelium, suggesting that TGF-βs play an important role in the stromal–epithelial interactions required for branching morphogenesis.

Ca2+/calmodulin-dependent protein kinase II phosphorylation of the presynaptic protein synapsin I is persistently increased during long-term potentiation

Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic and/or postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multiple-electrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-d-aspartate receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Iβ or a membrane-bound cGK II/Iβ chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4+ human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4+ T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1–20. DR3.Ab0 mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses to M. tuberculosis, and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab0 mice immunized with a second major M. tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0 with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity to M. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls1

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca2+. The complex formed in the presence of Ca2+ was more stable than that without Ca2+. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca2+ and B. Removal of the Ca2+ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca2+ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca2+ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca2+ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca2+, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca2+ but also borate and Ca2+ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.

UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.

Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory.

Feasibility of developing training programs designed to improve deficient driver factors. Volume II: identifying accident avoidance behaviors: a guide for accident investigators. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Phtx-ii A Basic Myotoxic Phospholipase A2 From Porthidium Hyoprora Snake Venom, Pharmacological Characterization And Amino Acid Sequence By Mass Spectrometry.

A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.

Elevated plasma membrane and sarcoplasmic reticulum Ca2+ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats

We have observed previously that Ca2+ pump-mediated Ca2+ efflux is elevated in cultured aortic smooth muscle cells from spontaneously hypertensive rats compared to those from Wistar-Kyoto rat controls. The objective of this work was to determine if these strains differ in mRNA levels for the PMCA1 isoform of the plasma membrane Ca2+-ATPase and the SERCA2 isoform of the sarcoplasmic reticulum Ca2+-ATPase. mRNA levels were compared in cultured aortic smooth muscle cells from 10-week-old male rats. PMCA1 and SERCA2 mRNA levels were elevated in SHR compared to WKY. Angiotensin II increased the level of PMCA1 and SERCA2 mRNA in both strains. These studies provide further evidence for alterered Ca2+ homeostasis in hypertension at the level of Ca2+ transporting ATPases in the spontaneously hypertensive rat model. These data are also consistent with the hypothesis that the expression of these two Ca2+ pumps may be linked. (C) 1997 Academic Press

Intracellular free Ca2+ and basal Mn2+ influx in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

Numerous studies investigating the possible role of altered Ca2+ homeostasis in hypertension have compared resting and agonist-stimulated intracellular free Ca2+ ([Ca2+](i)) in cultured aortic smooth muscle cells from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. However, such studies have not given consistent results. Differences in the method used to load cells with the Ca2+-sensitive indicator fura-2 have been investigated here as a possible source of variability between studies. We also describe the adaptation of a fluorescence technique for the assessment of basal Ca2+ permeability in SHR and WKY through the measurement of Mn2+ influx. The results are consistent with the hypothesis that basal Ca2+ influx is elevated in cultured aortic smooth muscle cells from SHR compared to those from WKY. However, this was not reflected as a significant difference between the two strains in basal or angiotensin II (200 nmol/L)stimulated [Ca2+](i). Furthermore, this result was not dependent on the protocol used to load cells with fura-2. Hence, measurement of bulk [Ca2+](i) does not appear to be the most sensitive parameter for altered Ca2+ homeostasis in SHR. Other compartments of the cell may better reflect altered Ca2+ fluxes in hypertension and are discussed in this work.