Multisite, double-blind, placebo-controlled trial of porcine secretin in autism.

OBJECTIVE: To examine the efficacy of intravenous porcine secretin for the treatment of autistic disorder. METHOD: Randomized, double-blind, placebo-controlled, crossover design. Fifty-six subjects with autistic disorder received either a secretin or placebo infusion at baseline and the other substance at week 4. Subjects were given the Autism Diagnostic Observation Schedule (ADOS) and other pertinent developmental measures at baseline and at weeks 4 and 8 to assess drug effects. RESULTS: For the primary efficacy analysis, change of ADOS social-communication total score from week 0 to week 4, no statistically significant difference was obtained between placebo (-0.8 +/- 2.9) and secretin groups (-0.6 +/- 1.4; t54 = 0.346, p < .73). The other measures showed no treatment effect for secretin compared with placebo. CONCLUSION: There was no evidence for efficacy of secretin in this randomized, placebo-controlled, double-blind trial.

Survival of microorganisms under the extreme conditions of the Atacama Desert.

Spores of Bacillus subtilis, conidia of Aspergillus niger, versicolor and ochraceus and cells of Deinococcus radiodurans have been exposed in the dark at two locations (at about 23 degrees S and 24 degrees S) in the Atacama Desert for up to 15 months. B. subtilis spores (survival approximately 15%) and A. niger conidia (survival approximately 30%) outlived the other species. The survival of the conidia and spores species was only slightly poorer than that of the corresponding laboratory controls. However, the Deinococcus radiodurans cells did not survive the desert exposure, because they are readily inactivated at relative humidities between 40 and 80% which typically occur during desert nights. Cellular monolayers of the dry spores and conidia have in addition been exposed to the full sun light for up to several hours. The solar fluences causing 63% loss in viability (F37-values) have been determined. These F37-values are compared with those determined at other global locations such as Punta Arenas (53 degrees S), Key Largo (25 degrees N) or Mainz (50 degrees N) during the same season. The solar UVB radiation kills even the most resistant microorganisms within a few hours due to DNA damages. The data are also discussed with respect to possible similarities between the climatic conditions of the recent Atacama Desert and the deserts of early Mars.

The autism diagnostic observation schedule-generic: a standard measure of social and communication deficits associated with the spectrum of autism.

The Autism Diagnostic Observation Schedule-Generic (ADOS-G) is a semistructured, standardized assessment of social interaction, communication, play, and imaginative use of materials for individuals suspected of having autism spectrum disorders. The observational schedule consists of four 30-minute modules, each designed to be administered to different individuals according to their level of expressive language. Psychometric data are presented for 223 children and adults with Autistic Disorder (autism), Pervasive Developmental Disorder Not Otherwise Specified (PDDNOS) or nonspectrum diagnoses. Within each module, diagnostic groups were equivalent on expressive language level. Results indicate substantial interrater and test-retest reliability for individual items, excellent interrater reliability within domains and excellent internal consistency. Comparisons of means indicated consistent differentiation of autism and PDDNOS from nonspectrum individuals, with some, but less consistent, differentiation of autism from PDDNOS. A priori operationalization of DSM-IV/ICD-10 criteria, factor analyses, and ROC curves were used to generate diagnostic algorithms with thresholds set for autism and broader autism spectrum/PDD. Algorithm sensitivities and specificities for autism and PDDNOS relative to nonspectrum disorders were excellent, with moderate differentiation of autism from PDDNOS.

Predicting dysphoria in adolescence from actual and perceived peer acceptance in childhood.

Predicted dysphoria in midadolescence using actual and perceived peer acceptance of 68 4th and 5th graders (48% male, 30% minority). Main effect, additive, and interactive models for predicting dysphoria were examined. Perceived acceptance predicted later dysphoria, after controlling for initial levels of dysphoria, supporting the main effect model. Actual acceptance did not uniquely contribute to prediction of later dysphoria, and actual acceptance did not moderate the prediction of dysphoria from perceived acceptance. Sex differences in dysphoria were significant, but sex did not moderate the predictive links between perceived acceptance and dysphoria.

A Double-Blind, Placebo-Controlled Trial of Secretin for the Treatment of Autistic Disorder.

OBJECTIVE: This study examines the efficacy of intravenous porcine secretin for the treatment of autism. METHODS: Using a randomized, double-blind, placebo-controlled crossover design, 20 subjects with autistic disorder received either a secretin or placebo infusion at baseline and the other substance at week 4. Subjects were given the Autism Diagnostic Interview-Revised, the Autism Diagnostic Observation Schedule-Generic (ADOS-G), and other pertinent developmental measures at baseline and at weeks 4 and 8 to assess drug effects. RESULTS: For the primary efficacy analysis, change of ADOS-G social-communication total score from week 0 to week 4, no statistically significant difference was obtained between placebo (-1.0 +/- 2.4) and secretin groups (-0.7 +/- 1.4; t 0.34, df 18, P less than.74). No significant differences were obtained for the other measures, including when all 20 subjects were compared by paired t-test from baseline to 4 weeks after secretin infusion. CONCLUSION: There was no evidence for efficacy of secretin in this preliminary randomized controlled trial. These data were collected as part of a multicenter study with the University of California-Irvine and the University of Utah.

Biochemical constraints for survival under Martian conditions.

A wide variety of terrestrial organisms, the so-called "anhydrobiotes," has learned to survive in a state of extreme dehydration in dry environments. Strategies for survival include the accumulation of certain polyols and nonreducing saccharides, which help to prevent damage to membranes and proteins, but at low water partial pressure DNA is also progressively damaged by various lesions, including strand breaks and cross-linking to proteins. These lesions, if they are not too numerous, can be repaired before the first replication step after rehydration, but long-term exposure to dry conditions finally diminishes the chances of survival as these lesions accumulate. If an organism has no chance to repair the accumulated DNA damage during intermittent periods of active life, survival will not exceed a few decades. The restriction of survival by dryness-induced DNA lesions is corroborated by new data on conidia of Aspergillus and the free plasmid pBR 322. Our results will be discussed with respect to the chance of finding dormant life or biochemical fossils on the surface of Mars.

ERA-experiment "Space Biochemistry".

The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory. Accordingly, it could be correlated with the increase in DNA damages. The purple membranes, amino acids and urea were not measurably effected by the dehydrating condition of open space (in the dark). Plasmid DNA, however, suffered a significant amount of strand breaks under these conditions. The response of these biomolecules to high fluences of short wavelength solar UV-light is very complex. Only a brief survey can be given in this paper. The data on the relatively good survival of some of the microorganisms call for strict observance of COSPAR Planetary Protection Regulations during interplanetary space missions.

Extreme dryness and DNA-protein cross-links.

Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125 J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32P by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

Response of soluble and membrane-bound F1 ATPase of Rhodospirillum rubrum to the photoaffinity label 8-azido ATP.

Soluble ATPase (F1 ATPase) and membrane-bound ATPase (chromatophores) of Rhodospirillum rubrum have been photoaffinity labeled with 8-azidoadenosine 5'-triphosphate (8-N3ATP). The specific binding of 8-N3ATP to nucleotide binding sites of the chromatophore-bound enzyme is established by competition experiments and it is suggested that hydrolysis and phosphorylation involve the same nucleotide binding sites. The experimental results also indicate that the function of the coupling factor F1 is influenced by its environment.

Kinetic studies on bacterial plasma membrane ATPase (F1). Nucleotide-induced long term inactivation of ATP hydrolyzing activity is linked to the formation of multiple "tight" enzyme nucleotide complexes.

ADP and the ATP analogs Nb-S6ITP (6-[(3-carboxy-4-nitrophenyl)thio]-9-beta-D-ribofuranosylpurine 5'-triphosphate) and AMP-P(NH)P (adenyl-5'-yl imidodiphosphate) interact with soluble plasma membrane ATPase (F1) from Micrococcus species in two ways: (i) at short incubation times, these inhibitors exhibit the kinetics of competitive inhibition, (ii) at long incubation times, these inhibitors induce an inactivation of the ATPase which can be reversed only in the case of AMP-P(NH)P. Kinetic treatment of the long term inactivation by ADP or Nb-S6ITP reveals a pseudo-first order process via the formation of an enzyme-inhibitor complex for which a Km analogous constant is obtained that is identical with the corresponding Ki value of the competitive inhibition. The long term inactivation by ADP and Nb-S6ITP involves the successive "tight" binding of 6 +/- 1 nucleotides/F1 molecule. One additional ADP molecule/F1 complex which is also "tightly" bound has no effect on the ATPase activity. The long term inactivation by ADP and Nb-S6ITP is inhibited at higher inhibitor concentrations according to a kinetics analogous to a substrate excess inhibition. Evidence is presented indicating that the mechanism of ATP hydrolysis by F1 and the long term inactivation by ADP or Nb-S6ITP are related processes. The mechanism of long term inactivation by AMP-P(NH)P appears to be different from that of ADP or Nb-S6ITP.

F1-ATPase from Micrococcus sp. ATCC 398. Purification by ion-exchange chromatography and further characterization. (Auto)proteolysis and dissociative effects.

The preparation of highly purified F1-ATPase from Micrococcus sp. ATCC 398 by application of DEAE-Sepharose CL-6B chromatography as final step is described. This enzyme consists of five subunits of different molecular weight: alpha (65000), beta (55000),gamma (35000), delta (20000), and epsilon (17000). Disc electrophoresis on 5% polyacrylamide gels removes the epsilon-polypeptide yielding an active ATPase complex with four different subunits: alpha, beta, gamma, delta. Additionally, by variation of the ionic strength delta can (partly) removed allowing the isolation by disc electrophoresis of an active ATPase complex which consists only of three different subunits alpha, beta, and gamma. If the DEAE-Sepharose chromatography is carried out in the absence of diisopropyl phosphofluoridate (auto)proteolysis yields both an active ATPase with the subunits alpha+ (mol. wt 61000), beta, gamma, and delta and an inactive protein complex with the subunits alpha+, beta, gamma, delta, and two additional polypeptides a (mol. wt 38000) and b (mol. wt 23000). The latter two polypeptides are supposedly fragments of alpha+-chains which have become partially cleaved by (auto)proteolysis.

Membrane-bound F1 ATPase from Micrococcus Sp. ATCC 398E. Purificationa and characterization ny affinity chromatography.

A chemically reactive ATP analogue, 6-[(3-carboxy-4-nitrophenyl)thio]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) has been synthesized. It has the ability to form stable thioether bonds between the 6-position of the purine ring and aliphatic mercapto groups. The nucleotide moiety of the reagent has been covalently bound to agarose, via iminobispropylamine and N-acetyl-homocysteine as space with the purpose of producing an affinity chromatography material. The affinity matrix binds solubilized F1 ATPase from a crude extract of Micrococcus sp. membranes. Afterwards the enzyme can be selectively eluted from the column at a defined ATP concentration. This method is superior to the conventional purification with respect to speed and convenince of the preparation. The affinity chromatography leads in a one-step process to the same purity to enzyme, substituting several steps of the conventional method. In addition, the affinity matrix was used for binding studies. Although the presence of Mg2+ ions is a prerequisite for the hydrolysis of nucleoside 5'-triphosphates, evidence is presented indicating that the binding of the nucleoside triphosphates to highly purified F1 ATPase from Micrococcus sp. appears not to be influenced by Mg2+ ion concentrations so far examined.

Me2+-(13 S) ATPase from Micrococcus sp. ATCC 398E. The effect of trypsin on the purified enzyme.

By trypsin treatment of highly purified ATPase (EC 3.6.1.3) from Micrococcus sp. ATCC 398E, two enzyme modifications have been obtained. (i) ATPase Ta, which has about the same activity as untreated ATPase. (ii) A protein complex Ti, which lacks ATPase activity, but nevertheless binds ATP as shown by affinity chromatography. Trypsin primarily shortens the alpha-chains of the "native" enzyme to alpha-chains and removes the gamma-subunit, thus yielding ATPase Ta. The formation of the protein complex Ti appears to be due to additional cleavage of one alpha-chain into at least two more fractions.

Response of membrane-bound ATPase of Micrococcus luteus to heat and ultraviolet light.

It is shown that the properties of ATPase (EC 3.6.1.3) of Micrococcus luteus depend only to some extent on the state of the membrane to which it is attached. Its interaction with the membrane appears to be largely controlled by polar forces. It is shown, however, that the UV-sensitivity of the membrane-bound ATPase is also significantly influenced by the state of membrane lipids.