Further insight into the neurobehavioral pattern of children carrying the 2p16.3 heterozygous deletion involving NRXN1: Report of five new cases

Increasing evidence links heterozygosity for NRXN1 gene deletions to a clinically wide spectrum of neurodevelopmental, psychiatric, and neurological disorders. However, to date, the neurocognitive and social communication features of children carrying this genomic rearrangement have not been assessed in detail. The cognitive and behavioral profiles of five children carrying a heterozygous NRXN1 deletion were investigated through systematic assessment of the cognitive and developmental levels, adaptive profile and presence of behavioral symptoms and autistic features. Furthermore, four transmitting parents were assessed by means of cognitive, psychopathological and parental stress tests. A below‐average cognitive level was documented in all children, and defective adaptive levels were observed in four of them. Three of the five children were diagnosed as having autism spectrum disorder in comorbidity with intellectual disability/global developmental delay, with a major impairment in social communication skills. The remaining two children presented with isolated intellectual disability and an unclassifiable neurodevelopmental disorder, respectively. This study provide data contributing to a more accurate characterization of the neurobehavioral phenotype of individuals carrying heterozygous NRXN1 deletions. This analysis indicates that these structural rearrangements are associated with a variable expression of neuropsychiatric symptoms, and cast some doubts about the incomplete penetrance of the disorder.     

Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1

In human neutrophils, IL-8 induces chemotaxis, the respiratory burst, and granule release, and enhances cellular adhesion, a beta(2) integrin-dependent event. IL-8 stimulates neutrophil adhesion to purified fibrinogen in a Mac-1-dependent manner. Mitogen-activated protein kinase (MAPK) activation was detected in human neutrophil lysates after treatment with IL-8 and PMA, but not the activating mAb CBR LFA 1/2. IL-8-stimulated neutrophil adhesion to fibrinogen was blocked 50% by the MAPK/extracellular signal-related kinase-activating enzyme inhibitor PD098059. Adhesion was blocked approximately 75% by inhibition of the phosphatidylinositol-3 kinase (PI3K) pathway with LY294002, supporting that activation of both MAPK and PI3K may play a role in IL-8-dependent inside-out signals that activate Mac-1. Activation of MAPK was inhibited in IL-8-stimulated cells in the presence of PI3K inhibitors LY294002 or wortmannin, supporting a model in which PI3K is upstream of MAPK. IL-8-stimulated neutrophil adhesion was inhibited 50% by bisindolylmaleimide-I, implicating protein kinase C (PKC) in the intracellular signaling from the IL-8R to Mac-1. A 74-kDa molecular mass species was detected by an activation-specific Ab to PKC when cells were stimulated with PMA or IL-8, but not a beta(2)-activating Ab. Inhibition of either MAPK or PKC resulted in partial inhibition of IL-8-stimulated polymorphonuclear neutrophil adhesion, and treatment with both inhibitors simultaneously completely abolished IL-8-stimulated adhesion to ligand. Inhibition of PI3K blocked MAPK activation, but not PKC activation, suggesting a branch point that precedes PI3K activation. These data suggest that both MAPK and PKC are activated in response to IL-8 stimulation, and that these may represent independent pathways for beta(2) integrin activation in neutrophils.     

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP – kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase – MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.     

Heteroplasmy in bovine fetuses produced by intra- and inter-subspecific somatic cell nuclear transfer: neutral segregation of nuclear donor mitochondrial DNA in various tissues and evidence for recipient cow mitochondria in fetal blood

Varying degrees of mitochondrial DNA (mtDNA) heteroplasmy have been observed in nuclear transfer embryos, fetuses, and offspring, but the mechanisms leading to this condition are unknown. We have generated a clone of 12 bovine somatic cell nuclear transfer fetuses, using nuclear donor cells, recipient oocytes, and recipient heifers with defined mtDNA genotypes, to study nuclear-mitochondrial interactions and the origins of mtDNA heteroplasmy. Embryos were reconstructed from granulosa cells with Bos taurus mtDNA type A and recipient oocytes collected from three different maternal lineages with B. taurus mtDNA type B, B. taurus mtDNA type C, or B. indicus mtDNA. Sequence differences in the control region (CR) of B. taurus mtDNAs ranged from 6 to 11 nucleotides and differences between B. taurus and B. indicus CRs from 45 to 50 nucleotides. Fetuses were recovered from recipient heifers with B. taurus mtDNA type B on Day 80 after nuclear transfer (eight B. taurus A/B, two B. taurus A/C, and two B. taurus A/B. indicus). Agarose gel analysis of the CR by polymerase chain reaction-based restriction fragment length polymorphism failed to detect nuclear donor mtDNA in 11 investigated tissues of 10 viable fetuses and in DNA samples of two fetuses in resorption (one B. taurus A/B and one B. taurus A/C). A more sensitive analysis of 1801 plasmid clones with CR inserts derived from tissues of a B. taurus A/B. indicus fetus detected no or very low levels of heteroplasmy (0.5-0.7%). However, the analyses detected considerable amounts ( approximately 2.5% and 5%) of recipient heifer mtDNA in blood samples from two fetuses. Our data do not suggest a replicative advantage of somatic nuclear donor cell mtDNA in bovine transmitochondrial clones produced with oocytes from domestic forms of the same or a different aurochs (B. primigenius) subspecies. Detection of mtDNA from the recipient animal in the circulation of two fetuses points to leakage of the placental barrier, mimicking heteroplasmy.     

Dissociation of Bimolecular ��IIb��3-Fibrinogen Complex under a Constant Tensile Force

The regulated ability of integrin αIIbβ3 to bind fibrinogen plays a crucial role in platelet aggregation, adhesion, and hemostasis. Employing an optical-trap-based electronic force clamp, we studied the thermodynamics and kinetics of αIIbβ3-fibrinogen bond formation and dissociation under constant unbinding forces, mimicking the forces of physiologic blood shear on a thrombus. The distribution of bond lifetimes was bimodal, indicating that the αIIbβ3-fibrinogen complex exists in two bound states with different mechanical stability. The αIIbβ3 antagonist, abciximab, inhibited binding without affecting the unbinding kinetics, whereas Mn²⁺ biased the αIIbβ3-fibrinogen complex to the strong bound state with reduced off-rate. The average bond lifetimes decreased exponentially with increasing pulling force from ∼5 pN to 50 pN, suggesting that in this force range the αIIbβ3-fibrinogen interactions are classical slip bonds. We found no evidence for catch bonds, which is consistent with the known lack of shear-enhanced platelet adhesion on fibrinogen-coated surfaces. Taken together, these data provide important quantitative and qualitative characteristics of αIIbβ3-fibrinogen binding and unbinding that underlie the dynamics of platelet adhesion and aggregation in blood flow.     

The fine structure of the swine sweat gland. II. The coiled ducts

The duct of the swine sweat gland crosses the dermis and epidermis in sequence. The cells of the dermic segment seem to be related with cellular secretion and absorption. In the epidermic segment of the duct the whole morphology of the cells resembles the cellular morphology of the epidermic cells.     

Locomotion induced by non-contingent intracranial stimulation: comparison to psychomotor stimulant

Non-contingent experimenter-applied stimulation (nEAS) to the ventral mesencephalon, unlike contingent intracranial self-stimulation (ICSS), elicits high rates of general locomotion. This locomotion may be due to the nature of the presentation of stimulation, in that nEAS is non-contingent, while ICSS depends on a specific and focused response (e.g., bar pressing). Psychomotor stimulants also elicit high amounts of general locomotion, with the locomotion attributed to increased dopamine release. Interestingly, dopamine release decreases or is absent with repeated ICSS, but not nEAS. This suggests that the locomotion elicited by nEAS may be the result of DA release similar to that observed with psychomotor stimulants. To determine the relationship between locomotion induced by nEAS and psychomotor stimulants, locomotion elicited by nEAS was directly compared to that produced by cocaine, a psychomotor stimulant and indirect DA agonist. Six groups of rats were examined: (1) DA+ group: rats were implanted with a stimulating electrode in the ventral mesencephalon and activation of DA neurons was verified during surgery by monitoring DA release in the striatum; (2) DA- group: rats were also implanted with stimulating electrodes, but the location in the ventral mesencephalon did not elicit DA release; (3) 10-mg/kg cocaine group: rats were exposed to a low dose (10 mg/kg) of cocaine; (4) 40-mg/kg cocaine group: rats were exposed to a high dose (40 mg/kg) of cocaine; (5) saline group: rats were injected with saline; and (6) naive group: rats received no treatment. The topography of behavior was assessed in all rats during four periods: a pre-treatment baseline, treatment, early post-treatment, and a late post-treatment end point. The results suggest that locomotion elicited by nEAS was stereotypic, dependent upon DA release and similar, but not identical, to psychomotor stimulant-induced locomotion.     

Trp290Cys mutation in exon IIIa of the fibroblast growth factor receptor 2 (FGFR2) gene is associated with Pfeiffer syndrome

Pfeiffer syndrome is a skeletal disorder characterized by craniosynostosis associated with foot and hand anomalies. Mutations in the genes encoding fibroblast growth factor receptors 1 and 2 (FGFR1 and FGFR2) have recently been implicated in the aetiology of such a syndrome, as well as of other craniosynostotic conditions. We now report a novel missense mutation, a G to C transversion at position 1049 (exon IIIa) of FGFR2, detected in a patient with severe Pfeiffer clinical features. The mutation results in the substitution of a cysteine for tryptophan-290 in the third immunoglobulin-like domain and affects both spliceoforms of FGFR2. Mutations causing replacement of tryptophan-290 have also been reported previously in Crouzon syndrome, a similar but clinically distinct craniosynostotic disorder. This finding confirms the involvement of mutations of FGFR2 exon IIIa in Pfeiffer syndrome, and emphasizes both the extensive heterogeneity of the FGFR2 mutations that result in the Pfeiffer phenotype and the perturbations caused by unpaired cysteine residues in receptor dimerization and transduction of the FGFs signal. Received: 15 August 1996 / Revised: 19 October 1996     

Cryopreservation of dog platelets with dimethyl sulfoxide: therapeutic effectiveness of cryopreserved platelets in the treatment of thrombocytopenic dogs, and the effect of platelet storage at -80 degrees C

Dog platelets were frozen with 6% dimethyl sulfoxide at 2-3 degrees C per minute in a -80 degrees C mechanical freezer. The frozen platelets were stored at -80 degrees C for as long as 39 months. After storage at -80 degrees C for less than 1 year, platelet in vitro freeze-thaw-wash recovery values were 70%, and in vivo survival values 1 to 2 hr after transfusion were 40% those of fresh platelets. After 2 years or longer storage, in vitro freeze-thaw-wash recovery values were 60%, and in vivo survival values 1 to 2 hr after transfusion were 20% those of fresh platelets. These results indicate that significant deterioration of the dog platelets occurred between the first and second year of storage at -80 degrees C. Platelets that were stored frozen at -80 degrees C for less than 1 year and washed before transfusion into lethally irradiated thrombocytopenic dogs were hemostatically effective.     

Rat MAbs to the product of the c-erbB-2 proto-oncogene for diagnosis and therapy in breast cancer

The product of the c-erbB-2 protooncogene (p185) is a member of the EGF receptor family of transmembrane tyrosine kinases. Amplification of this gene and overexpression of the product has been observed in adenocarcinomas and has been correlated with poor prognosis in patients with breast and ovarian cancer. The very low levels of expression of p185 by normal adult tissues makes the receptor an almost tumor-specific target. We have prepared rat monoclonal antibodies against five distinct epitopes on the external domain of the c-erbB-2 product overexpressed by the breast cancer line BT474. The antibodies bind to the protein core of p185 and stain specifically the membranes in frozen sections of tumors overexpressing the c-erbB-2 product. Three of the antibodies, ICR12 (epitope A), ICR54, and ICR55 (epitope E), also stain the cell membrane in formalin-fixed, paraffin-embedded sections and bind to p 185 in Western blots. An investigation of the stability of the antigen-antibody complexes indicates that the majority are not readily internalized by SKOV3 cells growing in vitro. Antibodies ICR12 (IgG2a) and ICR55 (IgG2a), which are directed against separate epitopes on the c-erbB-2 p185, are both of high affinity and immunoreactivity (>75%) and localize specifically and stably to xenografted breast and ovarian carcinomas growing in athymic mice when labeled with¹²⁵I (up to 13% injected dose/g, ICR12 and ICR55) or a range of other radionuclides (up to 20% id/g, ICR12). We conclude that these antibodies may be useful as therapeutic vehicles for targeting radionuclides (imaging and therapy) or enzymes for activating prodrugs (ADEPT).