IGFBP-3 Regulates Mitochondrial Hyperfusion and Metabolic Activity in Ocular Surface Epithelia during Hyperosmolar Stress.

In the eye, hyperosmolarity of the precorneal tear film triggers inflammation and the development of dry eye disease (DED), a highly prevalent condition that causes depression and disability in severe forms. A member of the insulin-like growth factor (IGF) family, the IGF binding protein-3 (IGFBP-3), is a pleiotropic protein with known roles in growth downregulation and survival. IGFBP-3 exerts these effects by blocking IGF-1 activation of the type 1 IGF-receptor (IGF-1R). Here, we examined a new IGF-independent role for IGFBP-3 in the regulation of mitochondrial and metabolic activity in ocular surface epithelial cells subject to hyperosmolar stress and in a mouse model of DED. We found that hyperosmolar stress decreased IGFBP-3 expression in vitro and in vivo. Treatment with exogenous IGFBP-3 induced an early, transient shift in IGF-1R to mitochondria, followed by IGFBP-3 nuclear accumulation. IGFBP-3 nuclear accumulation increased protein translation, blocked the hyperosmolar-mediated decrease in oxidative phosphorylation through the induction of mitochondrial hyperfusion, and restored corneal health in vivo. These data indicate that IGFBP-3 acts a stress response protein in ocular surface epithelia subject to hyperosmolar stress. These findings may lead to the development of first-in-class therapeutics to treat eye diseases with underlying mitochondrial dysfunction.

High Dose Gamma Radiation Selectively Reduces GABAA-slow Inhibition.

Studies on the effects of gamma radiation on brain tissue have produced markedly differing results, ranging from little effect to major pathology, following irradiation. The present study used control-matched animals to compare effects on a well characterized brain region following gamma irradiation. Male Sprague-Dawley rats were exposed to 60 Gy of whole brain gamma radiation and, after 24-hours, 48-hours, and one-week periods, hippocampal brain slices were isolated and measured for anatomical and physiological differences. There were no major changes observed in tissue appearance or evoked synaptic responses at any post-irradiation time point. However, exposure to 60 Gy of irradiation resulted in a small, but statistically significant (14% change; ANOVA p < 0.005; n = 9) reduction in synaptic inhibition seen at 100 ms, indicating a selective depression of the gamma-aminobutyric acid (GABAA) slow form of inhibition. Population spike (PS) amplitudes also transiently declined by ~ 10% (p < 0.005; n = 9) when comparing the 24-hour group to sham group. Effects on PS amplitude recovered to baseline 48 hour and one week later. There were no obvious negative pathological effects; however, a subtle depression in circuit level inhibition was observed and provides evidence for 'radiomodulation' of brain circuits.

Crystal structure of Azotobacter cytochrome c5 at 2.5 A resolution.

The crystal structure of cytochrome c5 from Azotobacter vinelandii has been solved and refined to an R value of 0.29 at 2.5 A resolution. The structure of the oxidized protein was solved using a monoclinic crystal form. The structure was solved by multiple isomorphous replacements, re-fit to a solvent-leveled multiple isomorphous replacement map, and refined by restrained least squares. The structure reveals monomers associated about the crystallographic 2-fold axis by hydrophobic contacts at the "exposed heme edge". The overall conformation for the monomer is similar to that of Pseudomonas aeruginosa cytochrome c551. However, relative to a common heme conformation, c5 and c551 differ by an average of 6.8 A over 82 alpha-carbon positions and the propionates of c5 are much more exposed to solvent. The shortest heme--heme contact at the "dimer" interface is 6.3 A (Fe to Fe 16.4 A). Alignment of c5 and c551 shows that the two cytochromes, in spite of sequence differences, have remarkably similar charge distributions. A disulfide stacks on a tyrosine between the N- and C-terminal helices.

A multicenter trial on interobserver reproducibility in reporting on 99mTc-DMSA planar scintigraphy: a Belgian survey.

UNLABELLED: Conflicting opinions have been expressed regarding reproducibility in 99mTc-dimercaptosuccinic acid (DMSA) planar renal image interpretation. The purpose of this investigation was to determine the level of interobserver variability among a large group of Belgian nuclear medicine physicians who evaluated a randomly selected series of DMSA planar scintigraphic examinations performed on children and adults. METHODS: All Belgian nuclear medicine centers (n = 82) were invited to participate in a reproducibility study on 99mTc-DMSA scintigraphy. 99mTc-DMSA scans obtained on 10 adults and 40 children were randomly selected from the databases of 2 hospitals. Those participating in this investigation (65 centers = 79%) received a series of computer disks containing 50 99mTc-DMSA studies. To avoid potential problems related to unfamiliar display, the disks were formatted to be interpretable using the participants' own computer systems. Each participant was then free to use his or her usual display (hard copies, contrast enhancement, color scale, gray scale, and so forth). For each kidney, the observers had to choose between the following answers: normal, abnormal, equivocal, and lack of quality. RESULTS: Forty-two responses were obtained from a wide variety of institutions and from observers with different levels of experience in interpreting 99mTc-DMSA scintigraphy. Altogether, the following data were obtained: 60.8% normal, 25.2% abnormal, 7.0% equivocal, and 3.2% lack of quality. The median percentage of agreement (overall reproducibility) for the 42 observers was 92%. When the results of all 42 observers were compared, the median agreements on normality and abnormality were 93.5% and 90.5%, respectively. In a small number (n = 4) of kidneys, reproducibility was poor and ranged from 51% to 70%. Except for 2 outliers, all observers had almost the same level of performance. CONCLUSION: A large number of Belgian nuclear medicine physicians participated in evaluating a large randomly selected sample of 99mTc-DMSA studies, and excellent interobserver agreement was found.

Crystals of cadmium, zinc metallothionein.

Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space group is P41212(P43212) with unit cell dimensions a = b = 31.0 A and c = 120.0 A, and one molecule in the crystallographic asymmetric unit. The crystals are square bipyramids elongated on the tetragonal c-axis and are grown by repetitive seeding. The crystals are suitable for high resolution structure analysis. Assays of dissolved crystals show that the crystals have the same Cd and Zn content and amino acid composition as the native, as-isolated protein.

Two siblings with midline field defects and Hirschsprung disease: variable expression of Toriello-Carey or new syndrome?

We describe 2 sibs with multiple congenital anomalies. The main manifestations include hypoplasia of the corpus callosum and/or cerebellar hypoplasia, Robin sequence, pharyngeal and laryngeal hypoplasia, abnormal ears, excessive neck skin, cardiac defect, and Hirschsprung disease. The presence in 2 sibs born to healthy, consanguineous parents suggests autosomal recessive inheritance. These anomalies must have arisen during blastogenesis; the syndrome resembles most the condition described in 1988 by Toriello and Carey.

Single photon emission computed tomography using perfusion tracers in seizure disorders.

Single photon emission computed tomography (SPECT) using perfusion tracers makes it possible to estimate regional cerebral blood flow (rCBF) and, indirectly, local brain metabolism. It may be possible to detect and follow physiopathological alterations, such as may be seen in seizure disorders. The authors review the principles of and some data on perfusion SPECT in seizure disorders, stress advantages as well as major drawbacks and add their initial experience with Tc-99m hexamethylpropyleneamine oxime (HMPAO) SPECT in febrile convulsions.

Monozygotic twins non-concordant for oligomeganephronic renal hypoplasia: artery-vein placental shunting as a possible pathogenetic mechanism.

The etiology and pathogenesis of oligomeganephronic renal hypoplasia (OMN) are not known. In the present paper a second case of monozygotic twins non-concordant for OMN is described. It is hypothesized that one of the mechanisms which have been proposed to explain structural defects in monozygotic twins, namely placental artery-vein shunting, may have been involved in the pathogenesis of OMN in these patients. In OMN in general vascular abnormalities may have to be considered as a pathogenetic mechanism.

Inter-observer reproducibility of relative 99Tcm-DMSA uptake.

99Tcm-DMSA planar images of 49 randomly selected patients (10 adults, 39 children) were sent to 15 physicians at various centres in Belgium. They were asked to calculate, using their own routine program, the relative uptake (expressed as a percentage) of each kidney. The data were sent on disks formatted so that they could be read by all participants, using their own computer systems. For each scan, the inter-observer variability was expressed using the maximum difference and the standard deviation of left renal uptake. Left renal uptake measured by the 15 observers in the 49 patients was 29.0-72.0% (mean +/- s = 49.8 +/- 6.4%). The maximum differences in left renal uptake ranged between 1.7% and 12.0% (4.5 +/- 2.6%); however, the maximum difference did not exceed 8% in about 90% of the patients. The standard deviations of the individual left renal uptake were between 0.6 and 3.9 (1.3 +/- 0.8). The standard deviations were significantly higher in adults (mean standard deviation = 2.05) than in children (mean standard deviation = 1.12) (P < 0.001); this was probably related to the high background observed in three adults with severe renal impairment. Indeed there was a significant correlation (P < 0.001) between the standard deviation and both the signal-to-noise ratio and the degree of asymmetry between the right and left kidneys. The differences between right and left kidney uptake were systematically lower for some observers, suggesting an influence of the calculation programs.

[Chemical pneumonia in children]. / Chemische pneumonie bij kinderen.

Between 1980 and 1989 the Children's Hospital of Antwerp admitted 954 children with signs of intoxication. In 83 cases (9%) these were due to ingestion of hydrocarbons, 17 of these 83 children (21%) had chemical pneumonia. The most frequent chemicals were turpentine, petrol and lamp oil. The main symptoms were vomiting, skin rash, coughing and fever accompanied by an infectious blood count. Roentgen abnormalities in this group were less frequent than reported in the literature. A chest X-ray immediately after admission does not always provide information about pneumonia because abnormalities may already be present, e.g. due to an acute lung condition; nevertheless it is necessary for further study, if any. An X-ray after 24 hours is indispensable to confirm or exclude chemical pneumonia. As regards treatment, only supportive therapy is possible. Pulmonary function studies after a few weeks may be useful. A survey is presented of the epidemiology, clinical findings, diagnostics, pathophysiology, symptomatic therapy, prevention and prognosis.

Long-term use of cisapride (Prepulsid) in premature neonates.

In order to study the effect of cisapride on gastric stasis and to evaluate the possible risk of cholestasis, 20 premature neonates born in the hospital during one year were orally treated with cisapride 0.15 mg/kg q.i.d., over a mean period of 38 days. The gestational age ranged from 26 to 34 weeks and the mean age at the start of the cisapride treatment was 18 days. All patients were ventilated, 13 had a respiratory distress syndrome (hyaline membrane disease), and 9 had gastro-oesophageal reflux (GOR). All patients were given a semielementary formula by means of a continuous nasogastric infusion. The gastric residue was studied during three days: 24 hour baseline and 48 hours under cisapride treatment. The mean residue decreased (p less than 0.0001) from 50.6% during the last 6 baseline hours to 12.1% during the last 6-hours of the cisapride period. The mean feeding volume increased from 24.2 ml to 34.2 ml (p less than 0.001). A group of four patients had reversible cholestasis against the background of an outbreak of Candida, three before and one during cisapride treatment. Therefore, it could not be demonstrated that cisapride plays a role in the development of cholestasis. Because of the risks of GOR and the drawbacks of delayed enteral feeding, it is concluded that the use of cisapride is justified in premature neonates with gastric stasis.

Correlation between beta- and alpha-adrenergic receptor concentrations in human placenta.

alpha 2- and beta-adrenergic receptors in human placental membranes have been investigated using the radioligands [3H]-RX 821002 and [3H]-dihydroalprenolol, respectively. The specific binding of the alpha 2-adrenoceptor antagonist RX 821002 confirms the presence of an alpha 2-adrenoceptor in the human placenta, which has been characterized previously with [3H]-rauwolscine. The major finding presented here is a correlation between the alpha 2- and beta-adrenergic receptor concentrations (r = 0.765) in the human placenta at term. It is suggested that the alpha 2/beta adrenoceptor balance may play an important role in regulation of the vascular bed of the placenta. Determination of the alpha 2/beta ratio may help towards an understanding of the contractility of the placental vascular muscles.

Cat-scratch disease with reversible encephalopathy.

A case of cat-scratch disease (CSD) complicated by reversible encephalopathy is presented. Neurological complications of CSD are uncommon. Laboratory and radiological examinations were negative. There was complete recovery.

The amino acid sequence of the dihaem cytochrome c4 from the bacterium Azotobacter vinelandii.

An amino acid sequence is proposed for the cytochrome c4 from the bacterium Azotobacter vinelandii strain OP. It is a single polypeptide chain of 190 residues, with two sets of haem-attachment cysteine residues at positions 14/17 and 119/122. Proteins with similar sequences are also present in denitrifying pseudomonads. There is similarity in sequence between the two halves of the cytochrome c4 molecule, and each half also shows similarity to the sequences of certain monohaem cytochromes c isolated from organisms that are not obviously closely related to A. vinelandii. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50125 (17 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment.

Structure of Azotobacter vinelandii 7Fe ferredoxin. Amino acid sequence and electron density maps of residues.

The complete amino acid sequence of the 7Fe ferredoxin from Azotobacter vinelandii (Av Fd) was determined by repetitive Edman degradation of the whole protein and of peptides derived from CNBr cleavage or chymotrypsin digestion. The sequence was confirmed by the 2A electron density maps for the residues calculated with difference Fourier coefficients. The density maps for all residues are included in the paper. Av Fd has several important differences with the clostridial-type ferredoxins: (i) Av Fd is 106 residues (versus 55-60 for other bacterial ferredoxins); (ii) Av Fd has 9 cysteines, one of which (residue 24) is not homologous with the bacterial ferredoxins; (iii) Av Fd has 2 extra residues between 2 cysteines (residues 11 and 16) homologous to cysteines in the bacterial ferredoxins; and (iv) Av Fd has the unique sequence -Cys-Val-Glu-Val-Cys- (residues 16-20) which are two of the ligands of the 3Fe:3S center. These sequence features are compared to the sequences of various ferredoxin groups. Structure predictions for other suspected 7Fe ferredoxins are discussed.

Single crystals of cadmium, zinc metallothionein.

Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space group is P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 31.0 A and c = 120.0 A, and one molecule in the crystallographic asymmetric unit. The crystals are square bipyramids elongated on the tetragonal c-axis and are grown by repetitive seeding. The crystals are suitable for high resolution structure analysis. Assays of dissolved crystals show that the crystals have the same Cd and Zn content and amino acid composition as the native, as-isolated protein.

Involvement of the renal parenchyma in acute urinary tract infection: the contribution of 99mTc dimercaptosuccinic acid scan.

We performed 99mTc dimercaptosuccinic acid (DMSA) scan and ultrasonography in 146 children during the acute phase of a proven urinary tract infection (UTI). In 99 a micturating cysto-urethrography and in 83 an intravenous urography was also done. The occurrence of fever and increased WBC count, CRP and ESR were also studied. It appeared from this retrospective study that 47% of the kidneys had a cortical or patchy pattern of decreased uptake of 99mTc DMSA, as compared to 23% with abnormal findings on US. Vesico-ureteral reflux was present in 38% of the kidneys with parenchymal involvement on 99mTc DMSA scan. Although fever, leucocytosis and elevated CRP and ESR were significantly correlated with abnormal 99mTc DMSA scan, they were also observed in children without renal parenchymal involvement. Our results suggest that 99mTc DMSA scan is a sensitive method for the detection of parenchymal involvement during acute UTI. The exact nature of these lesions and their relation with scars need, however, to be defined.

Spectroscopic studies of ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.

The Fe(CN)3-(6) oxidation of the crystallographically characterized [[3Fe-3S], [4Fe-4S]] ferredoxin I of Azotobacter vinelandii has been studied using absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopies. A paramagnetic intermediate is observed en route to Fe-S cluster-free apoprotein, possessing an anisotropic g approximately equal to 2 EPR signal, surviving to temperatures greater than 77 K. This species is shown to result from 3-electron oxidation of the [4Fe-4S] cluster, without modification of the [3Fe-3S] cluster. However, it does not give rise to observable paramagnetic magnetic circular dichroism in the visible-near UV spectral region and is therefore neither an oxidized HIPIP [4Fe-4S] cluster nor an oxidized [3Fe-3S] cluster. We identify the paramagnetic species as a cysteinyldisulfide radical formed on dissociation of an oxidized cysteinate and an oxidized sulfide ion from the [4Fe-4S] cluster. This conclusion is consistent with the observed reaction stoichiometry, the spectroscopic results obtained, known EPR spectra of disulfide radicals, and the reconstitution of the native [4Fe-4S] cluster by dithiothreitol alone. This reaction, earlier interpreted as a HIPIP-type oxidation, is a previously uncharacterized oxidation reaction of [4Fe-4S] clusters.

Technetium-99m hexamethylpropylene amine oxime single photon emission tomography in febrile convulsions.

We report our initial experience with technetium-99m hexamethyl propylene amine oxime (99mTC-HMPAO) brain single photon emission tomography (SPET) in the investigation of 19 children presenting with febrile convulsions. Two patients with complex febrile convulsions showed focal SPET lesions contralateral to the neurological deficit. However, in 9 out of 17 patients with simple febrile convulsions, focally disturbed perfusion was shown. In 4 out of 6 patients with electroencephalogram (EEG) abnormalities on admittance, SPET revealed at least 2 focal lesions. The temporofrontal region was the one most commonly involved. The SPET findings presented here also suggest a temporal relationship with the febrile convulsions, with markedly fewer lesions if examined after 12 days. In our initial experience, perfusion SPET did not show any particular pattern helpful in the differential diagnosis of the child presenting with febrile convulsions. Physiopathologically, our findings may support the hypothesis that brain tissue is regionally more vulnerable to fever, in patients presenting with febrile convulsions.

New Ecdysteroids from Serratula tinctoria.

Six new ecdysteroids have been isolated from SERRATULA TINCTORIA; these are: the 2,22- and 3,22-diacetates of 20-hydroxyecdysone, 5beta-hydroxyrubrosterone, 3-epi-poststerone, 3-epi-rubrosterone, and 22-oxo-20-hydroxyecdysone. These minor compounds were found together with the known ecdysteroids, 20-hydroxyecdysone, its 2-, 3-, and 22-monoacetates, rubrosterone, poststerone, polypodine B (5beta,20-dihydroxyecdysone), pterosterone (25-deoxy-20,24-dihydroxyecdysone), and makisterone C (24-ethyl-20-hydroxyecdysone). All these ecdysteroids were isolated by a combination of several chromatographic techniques (liquid chromatography on alumina, DCCC, and HPLC), then identified using standard mass spectrometric and 2D (1)H-NMR techniques.