Partial duplication of the short arm of chromosome 9 (p13→p22) in a child with typical 9p trisomy phenotype

Summary A 3-year-old girl with duplication 9 (p22p13) is reported. The presence of a classical 9p trisomy phenotype in this patient suggests that this region (or part of it) is responsible for the major, typical clinical stigmata of this partial autosomal trisomy syndrome.     

Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding

During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.     

Hot-spot mutations in p110α of phosphatidylinositol 3-kinase (PI3K): Differential interactions with the regulatory subunit p85 and with RAS

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110α have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110α. The present studies add support to the proposal that helical and kinase domain mutations in p110α trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding, but depends on the interaction with p85.     

Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

Parkinson’s disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein α-synuclein (α-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and α-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between α-syn and Fe(II) (5.8 × 10³ M⁻¹) has been measured, but studies on the binding between α-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between α-syn and Fe(II) and the redox property of the resultant α-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O₂) to the putative α-syn-Fe(III) complex, with H₂O₂ as a co-product. The reduction potential was estimated to be 0.025 V vs. Ag/AgCl, which represents a shift by −0.550 V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between α-syn and Fe(III), 1.2 × 10¹³ M⁻¹, to be deduced. Despite the relatively high binding affinity, α-syn-Fe(III) generated from the oxidation of α-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)₃ and/or ferrihydrite gel. The roles of α-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed α-syn aggregation, and iron transfer processes.     

Effect of electro-acupuncture on ovarian expression of α (1)- and β (2)-adrenoceptors,and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries

Background  

Estradiol valerate (EV)-induced polycystic ovaries (PCO) in rats is associated with an increase in ovarian sympathetic outflow. Low-frequency (2 Hz) electro-acupuncture (EA) has been shown to modulate sympathetic markers as well as ovarian blood flow as a reflex response via the ovarian sympathetic nerves, in rats with EV-induced PCO.     

Multiphasic activation of smooth muscle adenylate cyclase by pretreatment with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) suggests multiple enzyme populations

The activation of uterine smooth muscle adenylate cyclase was studied by pretreating the particulate form of the enzyme with the GTP analog guanyl-5′-yl imidodiphosphate (Gpp(NH)p). Pretreatment with Gpp(NH)p left the enzyme in an irreversibly activated state which survived subsequent washing in guanyl nucleotide-free buffer. Activation under these conditions was multiphasic with rapid and slow components. At 23 °C slow activation proceeded at about $\text{1}\text{10}\text{th}$ the rate of rapid activation. The onset of the slow phase took longer at lower temperatures. Routine adenylate cyclase assay conditions (conversion of [³²P]ATP to cyclic [³²P]AMP) carried out without pretreatment probably characterized the rapidly activated component. The simplest kinetic model suggests not only the generally accepted two-step association reaction, but also implies the existence of more than one enzyme form, each of which is characterized by a separate activation rate. The complex kinetics of activation might be explained by a heterogeneous mixture of unassociated and preassociated nucleotide binding and catalytic subunits.     

Interaction of poly(ADP-ribose)polymerase with DNA polymerase α

Homogeneously purified poly(ADP-ribose) polymerase (PARP) specifically stimulated the activity of immunoaffinity-purified calf or human DNA polymerase by about 6 to 60-fold. Apparently, poly(ADP-ribosyl)ation of DNA polymerase was not necessary for the stimulation. The effects of PARP on DNA polymerase were biphasic: at very low concentrations of DNA, it rather inhibited its activity, whereas, at higher DNA concentrations, PARP greatly stimulated it. The autopoly(ADP-ribosyl)ation of PARP suppressed both its stimulatory and inhibitory effects. By immunoprecipitation with an anti-DNA polymerase antibody, it was clearly shown that PARP may be physically associated with DNA polymerase . Stimulation of DNA polymerase may be attributed to the physical association between the two, rather than to the DNA-binding capacity of PARP, since the PARP fragment containing only the DNA binding domain showed little stimulatory activity. The existence of PARP-DNA polymerase complexes were also detected in crude extracts of calf thymus.     

Dandy-Walker malformations in a case of partial trisomy 9p (p12.1→pter) due to maternal translocation t(9;12)(p12.1;p13.3)

We describe a five-year-old proband presented with Dandy-Walker malformations, right microopthalmia, hamstring contractures, undescended testis with absence of testis in right scrotum in addition to typical trisomy 9p clinical features. Routine cytogenetic studies with GTG - banding showed 46,XY,der(12)t(9;12) (p12;q13.3),mat karyotype (trisomy 9p). Chromosomal analysis of the father was normal and phenotypically normal mother had 46,XX,t(9;12)(p12;q13) karyotype. Fluorescence in situ hybridization analysis with single copy probes bA5OIA2 (9p11.2), bA562M8 (12p12.1) and centromere probes (9) showed break point at 9p12.1 region. The gene dosage effect of Chromosome 9p along with environmental factors might be associated with Dandy- Walker malformations in the patient.     

Description of the larva of Kempnyia colossica (Navás) (Plecoptera: Perlidae) with biological notes

In this paper, the larva of Kempnyia colossica (Navás 1934) is described based on material collected in Parque Estadual Intervales, São Paulo State, and biological notes are presented.     

Charcot-Marie-Tooth disease 1A (CMT1A) associated with a maternal duplication of chromosome 17p11.2→12

We report here the second case of Charcot-Marie-Tooth disease 1A (CMT1A) with a cytogenetically visible de novo direct duplication of 17p11.117p12. A male child who was initially referred for developmental delay and dysmorphism was subsequently shown to have significantly reduced motor nerve conduction velocities characteristic of CMT1A. This patient was not informative for the DNA markers mapping to the CMT1A region; however, with DNA markers pA10–41 and EW503 that map proximally and distally with respect to the disease locus, a dosage difference was observed between the two alleles. Comparison with parental genotypes indicated a de novo maternal duplication. Pulsed field gel analysis using probe VAW409R3a indicated that a 500-kb SacII junction fragment usually associated with CMT1A was absent in this patient. These findings confirm that the disease phenotype is probably caused by a gene dosage effect.     

Interaction of human β-defensin 2 (HBD2) with glycosaminoglycans

Human β-defensin 2 (HBD2) is a member of the defensin family of antimicrobial peptides that plays important roles in the innate and adaptive immune system of both vertebrates and invertebrates. In addition to their direct bactericidal action, defensins are also involved in chemotaxis and Toll-like receptor activation. In analogy to chemokine/glycosaminoglycan (GAG) interactions, GAG-defensin complexes are likely to play an important role in chemotaxis and in presenting defensins to their receptors. Using a gel mobility shift assay, we found that HBD2 bound to a range of GAGs including heparin/heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate. We used NMR spectroscopy of (15)N-labeled HBD2 to map the binding sites for two GAG model compounds, a heparin/HS pentasaccharide (fondaparinux sodium; FX) and enzymatically prepared DS hexasaccharide (DSdp6). We identified a number of basic amino acids that form a common ligand binding site, which indicated that these interactions are predominantly electrostatic. The dissociation constant of the [DSdp6-HBD2] complex was determined by NMR spectroscopy to be 5 ± 5 μM. Binding of FX could not be quantified because of slow exchange on the NMR chemical shift time scale. FX was found to induce HBD2 dimerization as evidenced by the analysis of diffusion coefficients, (15)N relaxation, and nESI-MS measurements. The formation of FX-bridged HBD2 dimers exhibited features of a cooperative binding mechanism. In contrast, the complex with DSdp6 was found to be mostly monomeric.     

Mechanism for sympathetic modulation of activity of cutaneous receptors (with Aδ fibers)

The afferent flow in A fibers from cutaneous receptors was studied in acute experiments with cats. A decline in the number of fibers activated during adequate receptor stimulation was observed at 5 and 35 sec after the excitation of sympathetic efferents. Similar changes in the afferent flow were recorded in identical time intervals after the preliminary stretching and cooling of the skin. The results obtained indicate that the quantitative characteristics of afferent flow during the first 30 sec after stimulation of the sympathetic chain are primarily due to the change in the mechanical state of the tissues surrounding the receptors.N. I. Lobachevskii Institute of Mathematics and Cybernetics, Gor'kii University. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 517–521, July–August, 1985.     

Reactions of Re(III) with α-diketones: Oxidative addition

The reactions of [ReCl₃(CH₃CN)(PPh₃)₂] with benzil PhC(O)C(O)Ph, and with a natural 1,2-naphthoquinone derivative, β-lapachone (Lap), result in oxidative addition with the formation of Re(V) complexes with stilbenediolate, [ReCl₃(PhC(O)C(O)Ph)(PPh₃)] (1) and with a reduced semiquinonic form of lapachone, [Re^IVCl₃(Lap⁻)(PPh₃)] (2). The structures of both compounds were established by X-ray crystallography.     

Fusarium verticillioides (Saccardo) Nirenberg Associated with Hardlock of Cotton

The development of new immune potentiators for human vaccines is an important and expanding field of research. In the present study, the ability of the capsular polysaccharide from Neisseria meningitidis serogroup A (CPS-A), a mannose-containing carbohydrate, to enhance the antibody production against a co-administered model vaccine antigen, is examined. A protein-meningococcal serogroup C capsular polysaccharide (CPS-C) conjugate was selected as the model antigen for this study. After subcutaneous immunization of Balb/C mice, the conjugate mixed with CPS-A induced higher anti-CPS-C IgG and IgG_2a antibody levels and higher anti-meningococcal serogroup C bactericidal titers than the conjugate alone or mixed with CPS-C. The immuno-stimulatory properties exhibited by CPS-A and the fact that vaccines based on purified CPS-A has been safely used during decades to fight the serogroup A meningococcal disease, support the proposal to use CPS-A as immune potentiator for human vaccination studies.     

Center for barr body condensation. A case of Turner's syndrome with 45,X/46,X,dic(X) (Xqter→ p22::p22→qter)

Summary An 11-year-old girl with karyotype 45,X/46,X,dic(X) (Xqterp22::p22qter) is presented. The abnormal X is always found to be the inactive and late replicating X, and according to previous investigations by Therman et al. (1974) part of the cells are seen to have bipartite Barr bodies.     

Dynamics of the phosphoinositide 3-kinase p110δ interaction with p85α and membranes reveals aspects of regulation distinct from p110α

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.