Plasma clearance of radiolabelled IGF-1 in the late gestation ovine fetus

We investigated the distribution of radiolabelled IGF-1 in the late gestation ovine fetus by exclusion gel chromatography following intravenous injection of 125I rh (recombinant human) met-IGF-1 into the chronically instrumented fetal lamb (120-130 days, n = 7). One minute after injection of 125I rh met-IGF-1 into the fetal femoral vein, 20.9 +/- 3.1% of the counts circulated in the 150K binding protein region, 55.0 +/- 3.7% in the 50K binding protein region and 18.7 +/- 0.6% in the free or 7K region. The chromatographic profiles obtained in the fetus were in general similar to those previously seen in the adult sheep. After an initial equilibration phase the half life of IGF-1 associated with the 150K binding fractions were 412.1 +/- 103.6 min. Two phases of clearance were observed for IGF-1 in association with the 50K binding fractions, an initial phase with a half life of 30.6 +/- 4.5 min followed by a second phase with a half life of 202.3 +/- 10.3 min. The 7K or 'free' form of IGF-1 had an initial half life of 12.6 +/- 5.1 min. Chromatography of samples of fetal tracheal fluid, fetal urine, amniotic fluid, maternal uterine venous plasma and maternal systemic plasma showed no movement of intact IGF-1 out of the fetal circulation into the fetal fluids or into the maternal circulation. However, when simultaneous samples were obtained from the fetal femoral artery and umbilical vein, higher radioactivity was consistently observed in the fetal femoral artery raising the possibility of placental uptake of IGF-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

Electrical consequences of spine dimensions in a model of a cortical spiny stellate cell completely reconstructed from serial thin sections

We built a passive compartmental model of a cortical spiny stellate cell from the barrel cortex of the mouse that had been reconstructed in its entirety from electron microscopic analysis of serial thin sections (White and Rock, 1980). Morphological data included dimensions of soma and all five dendrites, neck lengths and head diameters of all 380 spines (a uniform neck diameter of 0.1 m was assumed), locations of all symmetrical and asymmetrical (axo-spinous) synapses, and locations of all 43 thalamocortical (TC) synapses (as identified from the consequences of a prior thalamic lesion). In the model, unitary excitatory synaptic inputs had a peak conductance change of 0.5 nS at 0.2 msec; conclusions were robust over a wide range of assumed passive-membrane parameters. When recorded at the soma, all unitary EPSPs, which were initiated at the spine heads, were relatively iso-efficient; each produced about 1 mV somatic depolarization regardless of spine location or geometry. However, in the spine heads there was a twentyfold variation in EPSP amplitudes, largely reflecting the variation in spine neck lengths. Synchronous activation of the TC synapses produced a somatic depolarization probably sufficient to fire the neuron; doubling or halving the TC spine neck diameters had only minimal effect on the amplitude of the composite TC-EPSP. As have others, we also conclude that from a somato-centric viewpoint, changes in spine geometry would have relatively little direct influence on amplitudes of EPSPs recorded at the soma, especially for a distributed, synchronously activated input such as the TC pathway. However, consideration of the detailed morphology of an entire neuron indicates that, from a dendro-centric point of view, changes in spine dimension can have a very significant electrical impact on local processing near the sites of input.     

Assay of interferon and viral antibodies in the cerebrospinal fluid in clinical neurology and psychiatry

Cerebrospinal fluids (CSF) of 245 neurological and 194 psychiatric patients were tested for viral antibodies and interferon. Complement dependent neutralizing antibodies to Herpesvirus hominis 1 were found in the CSF of patients with encephalitis (50.6%), meningitis (35.4%), lesions of peripheral nerves (36.9%), sclerosis multiplex (41.2%), schizophrenia (31.9%), senile dementia (51.4%), mental retardation (11.1%), ethylism (43.5%). Neutralizing antibodies to tick-borne encephalitis virus were found in the CSF of 38% patients with encephalitis, in 14% meningitis, 11% lesions of peripheral nerves and also in 5.6--11.8% of psychiatric patients. In encephalitis, meningitis and in lesions of peripheral nerves were found in the CSF frequently plaque neutralizing antobidies to the tick-borne orbivirus Lipovník, complement-fixing antibodies to lymphocytic choriomeningitis virus and hemagglutination inhibiting antibodies to measles virus. In multiple sclerosis were detected CSF antibodies to measles virus (44%), Herpesvirus hominis 1 (41.2%) and Lipovník virus (52.6%). In neurological patients were observed CSF antibodies simultaneously to two or three viruses in 16.7 to 40.6%, while in psychiatric patients in zero to 4.6%. CSF interferon was found in psychiatric patients with an equal or even higher incidence (33.7 to 57.1%) than in the neurological patients (29.6--38.6%, in multiple sclerosis only 16.7%). Non-interferon virus inhibitors were excluded. The evaluation of the ratio of serum and CSF titers of viral antibodies and of interferon indicated local synthesis of both in the central nervous system -- with the exception of antibodies to Herpesvirus hominis 1 in CSF of some patients with very high titres in serum and probable lesions of the blood brain barrier.     

The effect of the frequency of subcutaneous insulin-like growth factor-1 administration on weight gain in growth hormone deficient mice.

To ascertain the frequency of subcutaneous IGF-1 administration necessary to promote growth we examined the weight gain of male homozygous lit/lit mice in response to either sc. IGF-1 or bovine GH administration. Lit/lit mice showed a dose dependent response to treatment with GH. Bovine GH induced a response in body weight gain within 3 days of the start of treatment. Following a single subcutaneous injection of IGF-1, plasma IGF-1 levels were elevated for 4-6 hours. Three treatment schedules for IGF-1 were used (once daily, twice daily and four times daily), each employing the same total daily dose of IGF-1 (30 micrograms). With IGF-1 treatment, a significant effect on body weight gain was obtained when administered four times daily. The growth rate with IGF-1 treatment 6 hourly was similar to that observed following treatment with bGH (10 micrograms sc daily). Twelve hourly IGF-1 administration only had a significant effect on body weight gain when weight was measured in the evening. Lit/lit mice treated once daily with 30 micrograms IGF-1 had no weight gain response and became severely hypoglycaemic. Frequent subcutaneous IGF-1 administration is one approach to growth enhancement in GH deficiency; higher doses administered less frequently do not promote growth and may cause hypoglycaemia.     

Prioritization of Copy Number Variation Loci Associated with Autism from AutDB–An Integrative Multi-Study Genetic Database

Copy number variants (CNVs) are thought to play an important role in the predisposition to autism spectrum disorder (ASD). However, their relatively low frequency and widespread genomic distribution complicates their accurate characterization and utilization for clinical genetics purposes. Here we present a comprehensive analysis of multi-study, genome-wide CNV data from AutDB (http://mindspec.org/autdb.html), a genetic database that accommodates detailed annotations of published scientific reports of CNVs identified in ASD individuals. Overall, we evaluated 4,926 CNVs in 2,373 ASD subjects from 48 scientific reports, encompassing ∼2.12×10⁹ bp of genomic data. Remarkable variation was seen in CNV size, with duplications being significantly larger than deletions, (P  =  3×10⁻¹⁰⁵; Wilcoxon rank sum test). Examination of the CNV burden across the genome revealed 11 loci with a significant excess of CNVs among ASD subjects (P<7×10⁻⁷). Altogether, these loci covered 15,610 kb of the genome and contained 166 genes. Remarkable variation was seen both in locus size (20 - 4950 kb), and gene content, with seven multigenic (≥3 genes) and four monogenic loci. CNV data from control populations was used to further refine the boundaries of these ASD susceptibility loci. Interestingly, our analysis indicates that 15q11.2-13.3, a genomic region prone to chromosomal rearrangements of various sizes, contains three distinct ASD susceptibility CNV loci that vary in their genomic boundaries, CNV types, inheritance patterns, and overlap with CNVs from control populations. In summary, our analysis of AutDB CNV data provides valuable insights into the genomic characteristics of ASD susceptibility CNV loci and could therefore be utilized in various clinical settings and facilitate future genetic research of this disorder.     

ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling,Determining Human Brain Size

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.