Protein kinase C targeting of luminal (T-47D), luminal/HER2-positive (BT474), and triple negative (HCC1806) breast cancer cells in-vitro with AEB071 (Sotrastaurin) is efficient but mediated by subtype specific molecular effects.

PURPOSE: Protein kinase C (PKC) plays a pivotal role in malignant cell proliferation, apoptosis, invasiveness and migration. However, its exploitation as therapeutic target in breast cancer has been merely explored. Here were evaluated the AEB071 (Sotrastaurin™) treatment efficiency of breast cancer cell lines derived from estrogen receptor positive (T-47D), estrogen/HER2 receptor positive (BT474), and triple negative (HCC1806) breast cancer cells under 2D (monolayer) and 3D (multicellular tumor spheroids) culture conditions. Additionally, spheroid cocultures of BC and N1 fibroblasts were analyzed. METHODS: We quantitatively assessed the proliferation capacity of breast cancer cells and fibroblasts as a function of AEB071 treatment using flow cytometry. The activities of PKC isoforms, substrates, and key molecules of the PKC signaling known to be involved in the regulation of tumor cell proliferation and cellular survival were additionally evaluated. Moreover, a multigene expression analysis (PanCancer Pathways assay) using the nanoString™ technology was applied. RESULTS: All breast cancer cell lines subjected to this study were sensitive to AEB071 treatment, whereby cell proliferation in 2D culture was considerably (BT474) or moderately (HCC1806) retarded in G0/G1 or in G2/M phase (T-47D) of the cell cycle. Regardless of the breast cancer subtype the efficiency of AEB071 treatment was significantly lower in the presence of N1 fibroblast cells. Subtype specific driver molecules, namely IL19, c-myb, and NGFR were mostly affected by the AEB071 treatment. CONCLUSION: A combined targeting of PKC and a subtype specific driver molecule might complement specified breast cancer treatment.

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-ß.

Extracellular plaques of amyloid-ß and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-ß fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-ß. Interestingly, many adverse responses to amyloid-ß, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-ß are strongly associated with Alzheimer's disease, are more toxic than amyloid-ß, residues 1-42 (Aß(1-42)) and Aß(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aß may trigger Alzheimer's disease. Aß(3(pE)-42) co-oligomerizes with excess Aß(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aß(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aß(3(pE)-42) plus 95% Aß(1-42) (5% pE-Aß) seed new cytotoxic LNOs through multiple serial dilutions into Aß(1-42) monomers in the absence of additional Aß(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aß(3(pE)-42), and enhanced Aß(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aß(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aß(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aß(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.

Glutaminyl cyclase-mediated toxicity of pyroglutamate-beta amyloid induces striatal neurodegeneration.

BACKGROUND: Posttranslational modifications of beta amyloid (Aß) have been shown to affect its biophysical and neurophysiological properties. One of these modifications is N-terminal pyroglutamate (pE) formation. Enzymatic glutaminyl cyclase (QC) activity catalyzes cyclization of truncated Aß(3-x), generating pE3-Aß. Compared to unmodified Aß, pE3-Aß is more hydrophobic and neurotoxic. In addition, it accelerates aggregation of other Aß species. To directly investigate pE3-Aß formation and toxicity in vivo, transgenic (tg) ETNA (E at the truncated N-terminus of Aß) mice expressing truncated human Aß(3-42) were generated and comprehensively characterized. To further investigate the role of QC in pE3-Aß formation in vivo, ETNA mice were intercrossed with tg mice overexpressing human QC (hQC) to generate double tg ETNA-hQC mice. RESULTS: Expression of truncated Aß(3-42) was detected mainly in the lateral striatum of ETNA mice, leading to progressive accumulation of pE3-Aß. This ultimately resulted in astrocytosis, loss of DARPP-32 immunoreactivity, and neuronal loss at the sites of pE3-Aß formation. Neuropathology in ETNA mice was associated with behavioral alterations. In particular, hyperactivity and impaired acoustic sensorimotor gating were detected. Double tg ETNA-hQC mice showed similar Aß levels and expression sites, while pE3-Aß were significantly increased, entailing increased astrocytosis and neuronal loss. CONCLUSIONS: ETNA and ETNA-hQC mice represent novel mouse models for QC-mediated toxicity of truncated and pE-modified Aß. Due to their significant striatal neurodegeneration these mice can also be used for analysis of striatal regulation of basal locomotor activity and sensorimotor gating, and possibly for DARPP-32-dependent neurophysiology and neuropathology. The spatio-temporal correlation of pE3-Aß and neuropathology strongly argues for an important role of this Aß species in neurodegenerative processes in these models.

Selective hippocampal neurodegeneration in transgenic mice expressing small amounts of truncated Aß is induced by pyroglutamate-Aß formation.

Posttranslational amyloid-ß (Aß) modification is considered to play an important role in Alzheimer's disease (AD) etiology. An N-terminally modified Aß species, pyroglutamate-amyloid-ß (pE3-Aß), has been described as a major constituent of Aß deposits specific to human AD but absent in normal aging. Formed via cyclization of truncated Aß species by glutaminyl cyclase (QC; QPCT) and/or its isoenzyme (isoQC; QPCTL), pE3-Aß aggregates rapidly and is known to seed additional Aß aggregation. To directly investigate pE3-Aß toxicity in vivo, we generated and characterized transgenic TBA2.1 and TBA2.2 mice, which express truncated mutant human Aß. Along with a rapidly developing behavioral phenotype, these mice showed progressively accumulating Aß and pE3-Aß deposits in brain regions of neuronal loss, impaired long-term potentiation, microglial activation, and astrocytosis. Illustrating a threshold for pE3-Aß neurotoxicity, this phenotype was not found in heterozygous animals but in homozygous TBA2.1 or double-heterozygous TBA2.1/2.2 animals only. A significant amount of pE3-Aß formation was shown to be QC-dependent, because crossbreeding of TBA2.1 with QC knock-out, but not isoQC knock-out, mice significantly reduced pE3-Aß levels. Hence, lowering the rate of QC-dependent posttranslational pE3-Aß formation can, in turn, lower the amount of neurotoxic Aß species in AD.

Significant impact of circulating tumour DNA mutations on survival in metastatic breast cancer patients.

Mutational analysis of circulating tumour (ct) DNA holds promise as an effective tool to predict the course of metastatic breast cancer (MBC). In the present study we used targeted next generation sequencing of ctDNA to evaluate the impact of cancer driven mutations on the prognosis of MBC. The study included 59 oestrogen receptor-positive (ER+), HER2-negative MBC patients. Sequencing analysis was performed in ESR1, PIK3CA, ERBB2, PTEN, TP53, KRAS, HRAS, NRAS, and AR. At baseline, patients started receiving either chemotherapy (34%; n = 20) or cyclin-dependent kinase 4/6 inhibitor therapy in combination with endocrine therapy (CDK4/6i+ET; 66%; n = 39). Overall, 64.4% (n = 38) of the patients carried at least one pathogenic or likely-pathogenic mutation. Number of ctDNA mutations was significantly linked with worse progression free survival (PFS; p = 0.003) and overall survival (OS; p = 0.007). Furthermore, ctDNA load, defined by the number of mutant ctDNA molecules per mL plasma, significantly correlated with PFS (p < 0.001) and OS (p = 0.001). Furthermore, mutational status of ESR1 and TP53 significantly predicted PFS (p = 0.024 and p = 0.035, respectively) and OS (p < 0.001 and p = 0.035, respectively). These results emphasizes the clinical value of ctDNA mutational analysis in the management of advanced breast cancer.

Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice.

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

Inhibition of glutaminyl cyclase prevents pGlu-Abeta formation after intracortical/hippocampal microinjection in vivo/in situ.

Modified amyloid beta (Abeta) peptides represent major constituents of the amyloid deposits in Alzheimer's disease and Down's syndrome. In particular, N-terminal pyroglutamate (pGlu) following truncation renders Abeta more stable, increases hydrophobicity and the aggregation velocity. Recent evidence based on in vitro studies suggests that the cyclization of glutamic acid, leading to pGlu-Abeta, is catalyzed by the enzyme glutaminyl cyclase (QC) following limited proteolysis of Abeta at the N-terminus. Here, we studied the pGlu-formation by rat QC in vitro as well as after microinjection of Abeta(1-40) and Abeta(3-40) into the rat cortex in vivo/in situ with and without pharmacological QC inhibition. Significant pGlu-Abeta formation was observed following injection of Abeta(3-40) after 24 h, indicating a catalyzed process. The generation of pGlu-Abeta from Abeta(3-40) was significantly inhibited by intracortical microinjection of a QC inhibitor. The study provides first evidence that generation of pGlu-Abeta is a QC-catalyzed process in vivo. The approach per se offers a strategy for a rapid evaluation of compounds targeting a reduction of pGlu formation at the N-terminus of amyloid peptides.

Extracellular Citrate Affects Critical Elements of Cancer Cell Metabolism and Supports Cancer Development In Vivo.

Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy.Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513-23. ©2018 AACR.

Shift in prevalence of HPV types in cervical cytology specimens in the era of HPV vaccination.

The aim of the present population-based cohort study was to analyze the association between the prevalence of 32 types of human papilloma virus (HPV) in 615 female patients with abnormal cervical cytopathology findings. In total, 32 HPV types were screened by DNA array technology. HPV infection was detected in 470 women (76.42%), 419 of whom (89.15%) were infected with ≥1 high-risk (HR)-HPV type. HPV16, which is recognized as the main HR-HPV type responsible for the development of cervical cancer, was observed in 32.98% of HPV+ participants, followed by HPV42 (18.09%), HPV31 (17.66%), HPV51 (13.83%), HPV56 (10.00%), HPV53 (8.72%) and HPV66 (8.72%). The prevalence of HR-HPV types, which may be suppressed directly (in the case of HPV16 and 18), or possibly via cross-protection (in the case of HPV31) following vaccination, was considerably lower in participants ≤22 years of age (HPV16, 28.57%; HPV18, 2.04%; HPV31, 6.12%), compared with participants 23-29 years of age (HPV16, 45.71%; HPV18, 7.86%; HPV31, 22.86%), who were less likely to be vaccinated. Consequently, the present study hypothesizes that there may be a continuous shift in the prevalence of HPV types as a result of vaccination. Furthermore, the percentage of non-vaccine HR-HPV types was higher than expected, considering that eight HPV types formerly classified as 'low-risk' or 'probably high-risk' are in fact HR-HPV types. Therefore, it may be important to monitor non-vaccine HPV types in future studies, and an investigation concerning several HR-HPV types as risk factors for the development of cervical cancer is required.

Cell type specific expression of secretory TFF peptides: colocalization with mucins and synthesis in the brain.

The "TFF domain" is an ancient cysteine-rich shuffled module forming the basic unit for the family of secretory TFF peptides (formerly P-domain peptides and trefoil factors). It is also an integral component of mosaic proteins associated with mucous surfaces. Three mammalian TFF peptides are known (i.e., TFF1-TFF3); however, in Xenopus laevis the pattern is more complex (xP1, xP4.1, xP4.2, and xP2). TFF peptides are typical secretory products of a variety of mucin-producing epithelial cells (e.g., the conjunctiva, the salivary glands, the gastrointestinal tract, the respiratory tract, and the uterus). Each TFF peptide shows an unique expression pattern and different mucin-producing cells are characterized by their specific TFF peptide/secretory mucin combinations. TFF peptides have a pivotal role in maintaining the surface integrity of mucous epithelia in vivo. They are typical constituents of mucus gels, they modulate rapid mucosal repair ("restitution") by their motogenic and their cell scattering activity, they have antiapoptotic effects, and they probably modulate inflammatory processes. Pathological expression of TFF peptides occurs as a result of chronic inflammatory diseases or certain tumors. TFF peptides are also found in the central nervous system, at least in mammals. In particular, TFF3 is synthesized from oxytocinergic neurons of the hypothalamus and is released from the posterior pituitary into the bloodstream.

A luminous efficiency function, V*(lambda), for daylight adaptation.

We propose a new luminosity function, V*(lambda), that improves upon the original CIE 1924 V(lambda) function and its modification by D. B. Judd (1951) and J. J. Vos (1978), while being consistent with a linear combination of the A. Stockman & L. T. Sharpe (2000) long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) cone fundamentals. It is based on experimentally determined 25 Hz, 2 degrees diameter, heterochromatic (minimum) flicker photometric data obtained from 40 observers (35 males, 5 females) of known genotype, 22 with the serine variant L(ser180), 16 with the alanine L(ala180) variant, and 2 with both variants of the L-cone photopigment. The matches, from 425 to 675 nm in 5-nm steps, were made on a 3 log troland xenon white (correlated color temperature of 5586 K but tritanopically metameric with CIE D65 standard daylight for the Stockman and Sharpe L- and M-cone fundamentals in quantal units) adapting field of 16 degrees angular subtense, relative to a 560-nm standard. Both the reference standard and test lights were kept near flicker threshold so that, in the region of the targets, the total retinal illuminance averaged 3.19 log trolands. The advantages of the new function are as follows: it forms a consistent set with the new proposed CIE cone fundamentals (which are the Stockman & Sharpe 2000 cone fundamentals); it is based solely on flicker photometry, which is the standard method for defining luminance; it corresponds to a central 2 degrees viewing field, for which the basic laws of brightness matching are valid for flicker photometry; its composition of the serine/alanine L-cone pigment polymorphism (58:42) closely matches the reported incidence in the normal population (56:44; Stockman & Sharpe, 1999); and it specifies luminance for a reproducible, standard daylight condition. V*(lambda) is defined as 1.55L(lambda) + M(lambda), where L(lambda) and M(lambda) are the Stockman & Sharpe L- & M-cone (quantal) fundamentals. It is extrapolated to wavelengths shorter than 425 nm and longer than 675 nm using the Stockman & Sharpe cone fundamentals.

A novel missense mutation in the mouse growth hormone gene causes semidominant dwarfism, hyperghrelinemia, and obesity.

The SMA1-mouse is a novel ethyl-nitroso-urea (ENU)-induced mouse mutant that carries an a-->g missense mutation in exon 5 of the GH gene, which translates to a D167G amino acid exchange in the mature protein. Mice carrying the mutation are characterized by dwarfism, predominantly due to the reduction (sma1/+) or absence (sma1/sma1) of the GH-mediated peripubertal growth spurt, with sma1/+ mice displaying a less pronounced phenotype. All genotypes are viable and fertile, and the mode of inheritance is in accordance with a semidominant Mendelian trait. Adult SMA1 mice accumulate excessive amounts of sc and visceral fat in the presence of elevated plasma ghrelin levels, possibly reflecting altered energy partitioning. Our results suggest impaired storage and/or secretion of pituitary GH in mutants, resulting in reduced pituitary GH and reduced GH-stimulated IGF-1 expression. Generation and identification of the SMA1 mouse exemplifies the power of the combination of random mouse mutagenesis with a highly detailed phenotype-analysis as a successful strategy for the detection and analysis of novel gene-function relationships.

The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions.

Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE(1) -CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis.

Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology.

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.

IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.

IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-alpha, IL-1beta, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation (P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration (P < 0.00001) as well as mRNA expression of TNF-alpha, IL-8, and human beta-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn's disease and correlated highly with the IL-8 expression in these lesions (r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and IEC migration.

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry.

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.

The molecular basis of dichromatic color vision in males with multiple red and green visual pigment genes.

We investigated the genotypic variation in 50 red-green color vision deficient males (27 deuteranopes and 23 protanopes) of middle European ancestry who possess multiple genes in the X-linked photopigment gene array. We have previously shown that only the first two genes of the array are expressed and contribute to the color vision phenotype. Therefore, the hypothesis is that the first two genes possessed by multigene-dichromats encode pigments of identical or nearly identical spectral sensitivity: one gene normal (R or G) and the other a hybrid (G/R or R/G). The spectral sensitivities of the encoded pigments were inferred from published in vitro and in vivo data. The color vision phenotype was assessed by standard anomaloscopy. Most genotypes (92%) included hybrid genes whose sequence and position and whose encoded pigment correlated exactly with the phenotype. However, one and possibly two of the protanopes had gene arrays consistent with protanomaly rather than protanopia, since two spectrally different pigments may be encoded by their arrays. Two of the deuteranopes had only R- and G-photopigment genes, without any detectable G/R-hybrid genes or any as-of-yet identified point mutation or coding/promoter sequence deletions. Further, an unexpectedly high number of multigene-deuteranopes (11%) had the C203R mutation in their most upstream G-pigment gene, suggesting a founder effect of middle European origin for this mutation. About half of the protanopes possessed an upstream R/G-hybrid gene with different exon 2 coding sequences than their downstream G-pigment gene(s), which is inconsistent with published data implying that a single amino acid substitution in exon 2 can confer red-green color discrimination capacity on multigene-protans by altering the optical density of the cones.

Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase.

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.