Human-Specific ARHGAP11B Acts in Mitochondria to Expand Neocortical Progenitors by Glutaminolysis.

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP).

FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing.

Background: Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors.

Results: To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC".

Molecular parallelism in fast-twitch muscle proteins in echolocating mammals.

Detecting associations between genomic changes and phenotypic differences is fundamental to understanding how phenotypes evolved. By systematically screening for parallel amino acid substitutions, we detected known as well as novel cases (Strc, Tecta, and Cabp2) of parallelism between echolocating bats and toothed whales in proteins that could contribute to high-frequency hearing adaptations. Our screen also showed that echolocating mammals exhibit an unusually high number of parallel substitutions in fast-twitch muscle fiber proteins.

Stability of proICA512/IA-2 and its targeting to insulin secretory granules require β4-sheet-mediated dimerization of its ectodomain in the endoplasmic reticulum.

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that β2- or β4-strands in the mature ectodomain (ME ICA512) form dimers in vitro.

Purification of tubulin from porcine brain.

Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availability of purified αβ-tubulin. In this chapter, we describe the process of purification of heterodimeric αβ-tubulin from porcine brain.

Biogenesis of secretory granules.

Secretory granules of neuroendocrine cells store and release peptide hormones and neuropeptides in response to various stimuli. Generation of granules from the Golgi complex involves the aggregation of cargo proteins and their sorting from non-regulated secretory molecules. Recent findings on knockout mice lacking individual granule constituents have challenged the hypothesis that an 'essential' protein for the assembly of these organelles exists, while studies on polypyrimidine tract-binding protein and ICA512/IA-2 have provided insight into the mechanisms for adjusting granule production in relation to stimulation and secretory activity.

The small chemical vacuolin-1 inhibits Ca(2+)-dependent lysosomal exocytosis but not cell resealing.

Resealing after wounding, the process of repair following plasma membrane damage, requires exocytosis. Vacuolins are molecules that induce rapid formation of large, swollen structures derived from endosomes and lysosomes by homotypic fusion combined with uncontrolled fusion of the inner and limiting membranes of these organelles. Vacuolin-1, the most potent compound, blocks the Ca(2+)-dependent exocytosis of lysosomes induced by ionomycin or plasma membrane wounding, without affecting the process of resealing.

Polypyrimidine tract-binding protein promotes insulin secretory granule biogenesis.

Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown.

Regulated exocytosis: a novel, widely expressed system.

Electrophysiological studies in some secretory and non-secretory cells have identified an extensive form of calcium-induced exocytosis that is rapid (hundreds of milliseconds), insensitive to tetanus toxin and distinct from regulated secretion. We have now identified a marker of the process, desmoyokin-AHNAK, in a clonal derivative of the neuronal cell line, PC12. In resting cells, desmoyokin-AHNAK is localized within the lumen of specific vesicles, but appears on the cell surface during stimulation.

Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1.

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction.

Neurosecretory cells without neurosecretion: evidence of an independently regulated trait of the cell phenotype.

Neurosecretion competence is a fundamental property that enables differentiated neurones and professional neurosecretory cells to store neurotransmitters and hormones in specialized organelles, the synaptic-like vesicles and dense granules, and to release them by regulated exocytosis. In our laboratory, the study of rat phaeochromocytoma (PC12) clones that fail to express the above organelles or any other components involved in neurosecretion, whilst maintaining most of the general markers of the parental population, has served to demonstrate that this trait is controlled independently from the rest of the phenotype. The present review focuses on recent advances in elucidating the molecular mechanisms governing neurosecretion competence.

Neurosecretion competence, an independently regulated trait of the neurosecretory cell phenotype.

Neurosecretion competence is intended as the ability of neurosecretory cells to express dense and clear vesicles discharged by regulated exocytosis (neurotransmitter release). Such a property, which so far has never been studied independently, is investigated here by a heterotypic cell fusion approach, using a clone of rat pheochromocytoma PC12 cells totally incompetent for neurosecretion that still largely maintains its typical molecular and cellular phenotype. When fused with wild-type partners of various species (rat, human) and specialization (PC12, neuroblastoma SH-SY5Y, HeLa), the defective cells reacquire their competence as revealed by the expression of their secretion-specific proteins.

MSJ-1, a new member of the DNAJ family of proteins, is a male germ cell-specific gene product.

A cDNA encoding for a new member of the DnaJ protein family has been isolated by screening a mouse spermatogenic cell expression library. The full-length cDNA obtained by extension of the original clone with RT-PCR has been characterized with respect to its DNA sequence organization and expression. The predicted open reading frame encodes a protein of 242 amino acid residues whose sequence is similar to that of bacterial DnaJ proteins in the amino-terminal portion since it contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ protein-protein interactions.

Recruitment of circulating allergen-specific T lymphocytes to the lung on allergen challenge in asthma.

Background: In allergic subjects with asthma, the migration of CD4+ T cells to the lungs in the hours after allergen exposure may contribute to allergic inflammation in the target organ.

Objective: We studied allergen-specific T cells from the peripheral blood and lungs of allergic subjects with asthma at baseline and after allergen challenge.

Methods: In each patient, blood samples were taken 10 minutes before and 24 hours after the inhalation of a major sensitizing allergen.

Antigen-driven C-C chemokine-mediated HIV-1 suppression by CD4(+) T cells from exposed uninfected individuals expressing the wild-type CCR-5 allele.

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1-specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32-base pair deletion in the C-C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1.

Depletion of circulating allergen-specific TH2 T lymphocytes after allergen exposure in asthma.

Background: In allergic asthma, CD4+ T lymphocytes are a fundamental component of local chronic inflammation. Their cytokine profile is oriented toward a TH2 phenotype, characterized by production of IL-4, IL-5, IL-10, and IL-13. Egress of T cells from blood to airways after allergen challenge has been described.

Overall lack of regulated secretion in a PC12 variant cell clone.

A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D.

sp42, the boar sperm tyrosine kinase, is a male germ cell-specific product with a highly conserved tissue expression extending to other mammalian species.

sp42, a tyrosine kinase of 42 kDa originally found in ejaculated boar spermatozoa, is so far the only tyrosine protein kinase to have been purified from mature male germ cells. We have developed and characterized rabbit polyclonal antibodies specifically directed against the boar sperm enzyme, which has been here purified to homogeneity. Anti-sp42 serum and sp42 affinity-purified antibodies work very well in western blot, immunoprecipitation and immunocytochemistry, and do not inhibit sp42 catalytic activity.

Autoantibodies to CD4 in HIV type 1-exposed seronegative individuals.

The aim of this study was to investigate the presence and the fine specificity of anti-CD4 autoantibodies in seronegative subjects sexually exposed to HIV-1. Anti-CD4 autoantibodies were previously detected in a fraction of HIV-1-seropositive individuals. Whole sera, purified IgG fractions, and supernatants of EBV-transformed lymphoblastoid cell lines were analyzed by means of ELISA, Western blot, and by competition assays using monoclonal antibodies with known fine specificities.

The repertoire of T-lymphocytes recovered by bronchoalveolar lavage from healthy nonsmokers.

We reasoned that persistent exposure to a limited set of airborne antigens could drive the preferential expansion of single T-cell clones in the lower respiratory tract of normal individuals. To explore this issue, the normal human alpha/beta T-cell receptor repertoire was studied in lung lymphocytes obtained by bronchoalveolar lavage (BAL) from the humen of the lower respiratory tract. BAL T-cells obtained from five healthy volunteers were first analysed using polymerase chain reaction to amplify all known V alpha and V beta genes of the T-cell receptor.

Oligoclonality of lung T lymphocytes following exposure to allergen in asthma.

We were interested in studying the lung allergen-specific T cell repertoire in different conditions of allergen exposure in subjects with atopic asthma. Twenty-one allergic individuals were studied: 17 subjects suffering mainly from asthma and 4 from rhinitis. They all performed spirometry and methacholine challenge.