A lipoprotein receptor cluster IV mutant preferentially binds amyloid-ß and regulates its clearance from the mouse brain.

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ~70% of amyloid ß-peptide (Aß) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aß peripheral binding and higher levels of free Aß in plasma. Experimental studies have shown that free circulating Aß re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aß from entering the brain. Treatment of Alzheimer APPsw(+/0) mice with WT-LRPIV has been shown to reduce brain Aß pathology. In addition to Aß, LRPIV binds multiple ligands. To enhance LRPIV binding for Aß relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aß40 and Aß42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3-1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aß40 and Aß42 25-27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw(+/0) mice with LRPIV-D3674G (40 µg/kg/day) reduced Aß40 and Αß42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60-80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aß clearance therapy.

Na+ modulates anion permeation and block of P2X7 receptors from mouse parotid glands.

We previously reported that mouse parotid acinar cells display anion conductance (I(ATPCl)) when stimulated by external ATP in Na+-free extracellular solutions. It has been suggested that the P2X7 receptor channel (P2X7R) might underlie I(ATPCl). In this work we show that I (ATPCl) can be activated by ATP, ADP, AMP-PNP, ATPgammaS and CTP. This is consistent with the nucleotide sensitivity of P2X7R. Accordingly, acinar cells isolated from P2X7R( -/- ) mice lacked I(ATPCl). Experiments with P2X7R heterologously expressed resulted in ATP-activated currents (I(ATP-P2X7)) partially carried by anions. In Na(+)-free solutions, I (ATP-P2X7) had an apparent anion permeability sequence of SCN(-) > I(-) congruent with NO3(-) > Br(-) > Cl(-) > acetate, comparable to that reported for I(ATPCl) under the same conditions. However, in the presence of physiologically relevant concentrations of external Na+, the Cl(-) permeability of I(ATP-P2X7) was negligible, although permeation of Br(-) or SCN(-) was clearly resolved. Relative anion permeabilities were not modified by addition of 1 mM: carbenoxolone, a blocker of Pannexin-1. Moreover, cibacron blue 3GA, which blocks the Na(+) current activated by ATP in acinar cells but not I(ATPCl), blocked I(ATP-P2X7) in a dose-dependent manner when Na+ was present but failed to do so in tetraethylammonium containing solutions. Thus, our data indicate that P2X7R is fundamental for I(ATPCl) generation in acinar cells and that external Na+ modulates ion permeability and conductivity, as well as drug affinity, in P2X7R.

Age and gender related differences in human parotid gland gene expression.

OBJECTIVE: The present study evaluated differences in gene expression associated with age and gender in the human parotid gland. DESIGN: Parotid gland tissue was analysed using the Affymetrix GeneChip HGU133plus2.0 array. RESULTS: Differential gene expression, defined as a statistically significant difference with a 1.5-fold or greater change, was detected in 787 gene probe sets; 467 (approximately 59%) showed higher expression in females. Several genes associated with saliva secretion were differentially expressed in male and female parotid glands including vesicle-associated membrane protein 3 VAMP3, synaptosomal-associated protein SNAP23, RAS oncogene family member RAB1A and the syntaxin binding protein STXBP1. Evaluation of gene expression in the youngest and the oldest female subjects revealed that the expression of 228 probe sets were altered during aging; 155 genes were up-regulated in the aged female parotid gland. However, of the genes that were altered during aging, 22 of the 30 probes (73%) classified as being associated with immune responses were down-regulated in the aged parotid gland. A panel of differentially expressed, age- and gender-related genes was selected for validation by quantitative, real-time RT-PCR. Comparable differences in gene expression were detected by both Affymetrix array and quantitative, real-time RT-PCR methods. CONCLUSIONS: Our data suggest that salivary gland function may be adversely affected in the aged population due, at least in part, to the altered regulation of several categories of genes. Moreover, the gender specific differences in gene expression identified in the present study correlate with the previously observed sexual dimorphism in salivary gland function.

A variant of the Ca2+-activated Cl channel Best3 is expressed in mouse exocrine glands.

Fluid secretion by exocrine glands requires the activation of an apical Ca2+-dependent Cl channel, the molecular identity of which is unknown. We found that mouse exocrine glands expressed an alternately spliced variant of Best3, a member of the Bestrophin (Vmd2) Ca2+-activated Cl channel gene family, whereas the heart expressed full-length Best3. The spliced transcript lacked exons 2, 3 and 6 (Best3-Delta2,3,6) and is predicted to generate an in-frame protein missing the entire cytoplasmic N terminus, the initial two transmembrane domains and part of the first intracellular loop. In addition to exocrine glands, the Best3-Delta2,3,6 splice variant transcript was detected in lung, testis and kidney. The parotid gland and heart expressed proteins of the predicted size for Best3-Delta2,3,6 and full-length Best3, respectively, that targeted to the plasma membrane in HEK293 cells. HEK293 cells expressing Best3 displayed Ca2+-dependent Cl(-) currents that were sensitive to the Cl channel blocker DIDS. In contrast, no Ca2+-dependent Cl(-) currents were detected in cells expressing Best3-Delta2,3,6. Cotransfection of Best3-Delta2,3,6 with Best3 or Best2 (also expressed in salivary gland acinar cells) had no significant effects on the currents generated by either of these Ca2+-dependent Cl channels. Our results demonstrate that exocrine glands express a unique splice variant of Best3. Nevertheless, Best3-Delta2,3,6 does not produce Ca2+-dependent Cl(-) currents, nor does it regulate the activity of Best2 or the full-length Best3 channel.

Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells.

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion. The resting membrane potential of human acinar cells was near -51 mV, while the intracellular [Cl-] was approximately 62 mM, about fourfold higher than expected if Cl ions were passively distributed. Active Cl- uptake mechanisms included a bumetanide-sensitive Na+ -K+ -2Cl- cotransporter and paired DIDS-sensitive Cl-/HCO3- and EIPA-sensitive Na+/H+ exchangers that correlated with expression of NKCC1, AE2, and NHE1 transcripts, respectively. Intracellular Ca2+ stimulated a niflumic acid-sensitive Cl- current with properties similar to the Ca2+ -gated Cl channel BEST2. In addition, intracellular Ca2+ stimulated a paxilline-sensitive and voltage-dependent, large-conductance K channel and a clotrimazole-sensitive, intermediate-conductance K channel, consistent with the detection of transcripts for KCNMA1 and KCNN4, respectively. Our results demonstrate that the ion transport mechanisms in human parotid glands are equivalent to those in the mouse, confirming that animal models provide valuable systems for testing therapies to prevent salivary gland dysfunction.

Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands.

We have recently shown that the IK1 and maxi-K channels in parotid salivary gland acinar cells are encoded by the K(Ca)3.1 and K(Ca)1.1 genes, respectively, and in vivo stimulated parotid secretion is severely reduced in double-null mice. The current study tested whether submandibular acinar cell function also relies on these channels. We found that the K(+) currents in submandibular acinar cells have the biophysical and pharmacological footprints of IK1 and maxi-K channels and their molecular identities were confirmed by the loss of these currents in K(Ca)3.1- and K(Ca)1.1-null mice. Unexpectedly, the pilocarpine-stimulated in vivo fluid secretion from submandibular glands was essentially normal in double-null mice. This result and the possibility of side-effects of pilocarpine on the nervous system, led us to develop an ex vivo fluid secretion assay. Fluid secretion from the ex vivo assay was substantially (about 75%) reduced in animals with both K(+) channel genes ablated - strongly suggesting systemic complications with the in vivo assay. Additional experiments focusing on the membrane potential in isolated submandibular acinar cells revealed mechanistic details underlying fluid secretion in K(+) channel-deficient mice. The membrane potential of submandibular acinar cells from wild-type mice remained strongly hyperpolarized (-55 +/- 2 mV) relative to the Cl(-) equilibrium potential (-24 mV) during muscarinic stimulation. Similar hyperpolarizations were observed in K(Ca)3.1- and K(Ca)1.1-null mice (-51 +/- 3 and -48 +/- 3 mV, respectively), consistent with the normal fluid secretion produced ex vivo. In contrast, acinar cells from double K(Ca)3.1/K(Ca)1.1-null mice were only slightly hyperpolarized (-35 +/- 2 mV) also consistent with the ex vivo (but not in vivo) results. Finally, we found that the modest hyperpolarization of cells from the double-null mice was maintained by the electrogenic Na(+),K(+)-ATPase.

Molecular identification and physiological roles of parotid acinar cell maxi-K channels.

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca(2+)-activated Cl(-) and K(+) channels. Parotid acinar cells express two types of Ca(2+)-activated K(+) channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the K(Ca)3.1 gene, and the K(Ca)1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the K(Ca)1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in K(Ca)1.1-null animals. We examined fluid secretion from animals with both K(+) channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K(+) channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.

The Chlamydomonas reinhardtii gtr gene encoding the tetrapyrrole biosynthetic enzyme glutamyl-trna reductase: structure of the gene and properties of the expressed enzyme.

Plants, algae, cyanobacteria and many other bacteria synthesize the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate by means of a tRNAGlu-mediated pathway. The enzyme glutamyl-tRNA reductase (GTR) catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. Chlamydomonas reinhardtii mRNA encoding gtr was sequenced from a cDNA and genomic libraries. The 3179-bp gtr cDNA contains a 1566-bp open reading frame that encodes a 522-amino acid polypeptide. After removal of the predicted transit peptide, the mature 480-residue GTR has a calculated molecular weight of 52,502. The deduced C. reinhardtii mature GTR amino acid sequence has more than 55% identity to a GTR sequence of Arabidopsis thaliana, and significant similarity to GTR proteins of other plants and prokaryotes. Southern blot analysis of C. reinhardtii genomic DNA indicates that C. reinhardtii has only one gtr gene. Genomic DNA sequencing revealed the presence of a small intron near the putative transit peptide cleavage site. Expression constructs for the full-length initial gtr translation product, the mature protein after transit peptide removal, and the coding sequence of the second exon were cloned into expression vector that also introduced a C-terminal His6 tag. All of these constructs were expressed in E. coli, and both the mature protein and the exon 2 translation product complemented a hemA mutation. The expressed proteins were purified by Ni-affinity column chromatography to yield active GTR. Purified mature GTR was not inhibited by heme, but heme inhibition was restored upon addition of C. reinhardtii soluble proteins.

Cellular levels of glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase do not control chlorophyll synthesis in Chlamydomonas reinhardtii.

5-Aminolevulinic acid (ALA) is the first committed universal precursor in the tetrapyrrole biosynthesis pathway. In plants, algae, and most bacteria, ALA is generated from glutamate. First, glutamyl-tRNA synthetase activates glutamate by ligating it to tRNA(Glu). Activated glutamate is then converted to glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR). Finally, GSA is rearranged to ALA by GSA aminotransferase (GSAT). In the unicellular green alga Chlamydomonas reinhardtii, GTR and GSAT were found in the chloroplasts and were not detected in the mitochondria by immunoblotting. The levels of both proteins (assayed by immunoblotting) and their mRNAs (assayed by RNA blotting) were approximately equally abundant in cells growing in continuous dark or continuous light (fluorescent tubes, 80 micromol photons s(-1) m(-2)), consistent with the ability of the cells to form chlorophyll under both conditions. In cells synchronized to a 12-h-light/12-h-dark cycle, chlorophyll accumulated only during the light phase. However, GTR and GSAT were present at all phases of the cycle. The GTR mRNA level increased in the light and peaked about 2-fold at 2 h into the light phase, and GTR protein levels also increased and peaked 2-fold at 4 to 6 h into the light phase. In contrast, although the GSAT mRNA level increased severalfold at 2 h into the light phase, the level of GSAT protein remained approximately constant in the light and dark phases. Under all growth conditions, the cells contained significantly more GSAT than GTR on a molar basis. Our results indicate that the rate of chlorophyll synthesis in C. reinhardtii is not directly controlled by the expression levels of the mRNAs for GTR or GSAT, or by the cellular abundance of these enzyme proteins.

Glutamyl-tRNA reductase of Chlorobium vibrioforme is a dissociable homodimer that contains one tightly bound heme per subunit.

delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His(6) tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.

Characterization of the 820-nm transmission signal paralleling the chlorophyll a fluorescence rise (OJIP) in pea leaves.

Monitoring transmission changes at 820 nm, a measure of the redox states of plastocyanin (PC) and P700, is a good complementary technique for chlorophyll (chl) a fluorescence induction measurements. A thorough characterization of the properties of the 820-nm transmission kinetics during the first second after a dark-to-light transition is provided here for pea (Pisum sativum L.) leaves. The data indicate that plastocyanin in a dark-adapted leaf is in the reduced state. Three photosystem I (PSI)-related components, PC, P700 and ferredoxin, can contribute to the 820-nm transmission signal. The contribution of ferredoxin, however, is only approximately 5%, thus, it can be neglected for further analysis. Here, we show that by monitoring the sequential oxidation of PC and P700 during a far-red pulse and analysing the re-reduction kinetics it is possible to assign the three re-reduction components to PC (τ = 7-14 s) and P700 (τ = 35-55 ms and 1.2-1.6 s). Our data indicate that the faster re-reduction phase (τ =35-55 ms) may represent a recombination reaction between P700+ and the acceptor side of PSI. This information made it possible to show that the ratio between the potential contributions of PC : P700 is 50 : 50 in pea and Camellia leaves and 40 : 60 in sugar beet leaves.

The guard cell chloroplast: a perspective for the twenty-first century.

The guard cell chloroplast is the site of perception of blue light and of photosynthetically active radiation, and of at least one of the mechanisms sensing CO2 in the guard cell. The guard cell chloroplast has been the focus of intense controversy over its capacity for light sensing and photosynthetic carbon fixation, and the osmoregulatory mechanisms mediating stomatal movements. It is argued here that a primary reason behind these long-lived controversies is the remarkable plasticity of the guard cell, which has resulted in responses being generalized as basic properties when opposite responses appear to be the norm under different environmental or experimental conditions. Examples of guard cell plasticity are described, including variation of chlorophyll fluorescence transients over a daily course, acclimation of the guard cell responses to blue light and CO2 , the shift from potassium to sucrose in daily courses of osmoregulation and the transduction of red light into different osmoregulatory pathways. Recent findings on the properties of the guard cell chloroplast are also presented, including the role of the chloroplastic carotenoid, zeaxanthin, in blue light photoreception, the blue-green reversibility of stomatal movements, and the involvement of phytochrome in the stomatal response to light in the orchid, Paphiopedilum.