Localization of the genes for histatins to human chromosome 4q13 and tissue distribution of the mRNAs.

A cDNA coding for histatin 1 was isolated from a human submandibular-gland library and sequenced. This cDNA was used to probe RNAs isolated from a variety of tissues to investigate tissue-specific regulation and to determine whether histatins might play a role other than in the oral cavity. The same probe was also used for Southern blot analysis of human genomic DNA restricted with various enzymes, and it showed that the genes coding for histatins are on the same chromosome. In situ hybridization of the cDNA probe to metaphase chromosome spreads was performed to determine chromosomal location of the genes for histatins. A genomic fragment isolated using the cDNA probe was also hybridized to chromosome spreads, and the same chromosome was identified. The genes for histatins are located on chromosome 4, band q13. We have shown that three histatin mRNAs are expressed in human parotid and submandibular glands but in none of the other tissues studied. These results suggest that histatins are specific to salivary secretions.     

Metabolic epoxidation of trans-4-acetylaminostilbene: a protective mechanism against its activation to a mutagen

Trans-4-acetylaminostilbene is activated by liver preparations to mutagens for Salmonella typhimurium. Since this compound is metabolized to the trans-alpha,beta-epoxide and since many epoxides are ultimate mutagens, this epoxide was tested for direct mutagenicity. It was, however, found to be non-mutagenic, and, in contrast to the parent compound, the epoxide was no longer activated by liver preparations to mutagens. The same was found for the beta-ketone and for the threo-alpha,beta-dihydrodiol, which are formed metabolically from trans-4-acetylaminostilbene and from its alpha,beta-epoxide. 4-Acetylaminobibenzyl showed a very weak mutagenic activity in the presence of the liver preparation. Thus, it is important to realize that where epoxides are formed from compounds which are known to be metabolized to mutagens, they are not necessarily responsible for the mutagenicity. Epoxidation may even prevent the possibility of bioactivation to mutagens.     

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.     

Radiobiological problems in space

Summary An overview is presented on radiation problems in space, with emphasis on aspects of major interest for manned space exploration. A classification of the radiation hazards is presented and strategies for their evaluation are discussed. Space radiation problems are compared with characteristic aspects of radiation research in other disciplines, in order to provide further insight into those aspects that are unique to space.The research described in this paper was carried out while the author was a NASA VisitingThe research described in this paper was carried out while the author was a NASA VisitingThe research described in this paper was carried out while the author was a NASA VisitingThe research described in this paper was carried out while the author was a NASA Visiting     

The Rotifera of Hummock-Hollow Formations in a Poor (Mesotrophic) Fen in Newfoundland

Rotifers encountered from a poor (mesotrophic) fen, Avalon Peninsula of Newfoundland, are listed. Sixty-four species and varieties, including 25 Bdelloidea and 39 Monogononta, were recorded. All but 4 of the reported species and varieties are new records for the Province of Newfoundland, while 27 are new records for Canada; of these, 13 are new for North America. A seasonal quantitative study of rotifer communities in the hummock-hollow formations on the fen was carried out from August 1973 to October 1974. An average of 354 rotifers/cm² (17 species) was found per formation. Monogononta almost totally disappeared during winter, while the Bdelloidea decreased somewhat in numbers. The tops of hummocks contained mainly bdelloid species. The number of monogononts increased both in species and individuals on the slopes of hummocks and was greatest in the hollows, although the total number of rotifers was less in these areas than in the hummock tops. Some physical and biological factors affecting the rotifer communities were examined. Rotifers in these sites are not subjected to dessication. Predation was not an important factor. A quantitative sampling method was devised for procuring relatively accurate samples from living Sphagnum, and for extracting rotifers from these samples.     

Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.     

Immobilized electric eel acetylcholinesterase. I. Kinetics of acetylcholinesterase trapped in polyacrylamide membranes.

Techniques are described for the trapping of electric eel acetylcholinesterase in polyacrylamide gel. The activity of the trapped enzyme was substantially reduced, the effect being due to inhibition by acrylamide, but the emzyme immobilized in polyacrylamide was considerable more stable than that in free solutionma kinetic study was made of the hydrolysis of acetylthiocholine, covering a range of membrane thicknesses, enzyme concentrations, substrate concentrations and temperatures. The results were interpreted with reference to the theoretical treatment of Sundaram, Tweedale and Laidler, and of Kobayaski and Laidler, and provided support for those treatments; Clear evidence was obtained for diffusion control with the thicker membranes. An activation energy was obtained for the diffusion of the substrate within the membrane, by combining the temperature results for thick and thin membranes at low substrate concentrations. The results lead to the conclusion that the in vivo kinetics of acetylcholinesterase are largely diffusion-free in muscle filaments, but are substantially diffusion-controlled in fibrils and fibers.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Expression and purification of a convenient Ca-calmodulin-dependent protein kinase II GST-fusion substrate

Abundant and convenient protein substrates are extremely useful tools for studying protein kinases. However, few such substrates exist for alpha-Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and those that are available are generally small and expensive peptides that are cumbersome to use. The GST-fusion expression system was used to express a 10 amino acid substrate of CaMKII PLRRTLSVAA in bacteria. Using glutathione-agarose affinity chromatography, we obtained milligram quantities of the highly purified recombinant GST-fusion protein. The GST-fusion protein was tested for its efficacy and specificity as a substrate for CaMKII in phosphorylation assays using recombinant enzyme and radiolabeled [gamma-32P]ATP. The reaction products of these phosphorylation assays were resolved by electrophoresis in SDS-polyacrylamide gels and quantified by phosphoimage analysis. It was found that compared to a phosphorylation-null substrate, GST-PLRRTLAVAA, in which the phosphorylated target serine residue was mutated to an alanine, the GST-PLRRTLSVAA substrate was phosphorylated by CaMKII with an apparent K(m) of 18 microM, indicating that the latter is a highly effective substrate for this enzyme.     

Expression and purification of a convenient Ca2+-calmodulin-dependent protein kinase II GST-fusion substrate

Abundant and convenient protein substrates are extremely useful tools for studying protein kinases. However, few such substrates exist for alpha-Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and those that are available are generally small and expensive peptides that are cumbersome to use. The GST-fusion expression system was used to express a 10 amino acid substrate of CaMKII PLRRTLSVAA in bacteria. Using glutathione-agarose affinity chromatography, we obtained milligram quantities of the highly purified recombinant GST-fusion protein. The GST-fusion protein was tested for its efficacy and specificity as a substrate for CaMKII in phosphorylation assays using recombinant enzyme and radiolabeled [gamma-32P]ATP. The reaction products of these phosphorylation assays were resolved by electrophoresis in SDS-polyacrylamide gels and quantified by phosphoimage analysis. It was found that compared to a phosphorylation-null substrate, GST-PLRRTLAVAA, in which the phosphorylated target serine residue was mutated to an alanine, the GST-PLRRTLSVAA substrate was phosphorylated by CaMKII with an apparent K(m) of 18 microM, indicating that the latter is a highly effective substrate for this enzyme.     

Methods for starch-gel electrophoresis of sarcoplasmic proteins. An investigation of the relative mobilities of the glycolytic enzymes from the muscles of a variety of species

1. Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given. 2. Methods are described for detecting enzyme activities on the starch gel after electrophoresis, by using pieces of filter paper. 3. Compositions of incubation mixtures suitable for detecting any of the enzymes of glycolysis, and certain other enzymes, are given. 4. A comparison of the various enzymes in extracts of several muscles from one rabbit was made; most differences are quantitative only. 5. A detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made. Each species was found to have a characteristic pattern of proteins on the starch gel, and the mobilities of individual enzymes varied considerably. 6. Potential uses and extensions of the methods are discussed.     

Laronidase for treating mucopolysaccharidosis type I

Mucopolysaccharidoses are a group of inherited metabolic diseases caused by the absence or deficiency of the lysosomal enzymes that are needed for breaking down glycosaminoglycans (GAGs). Over time, GAGs collect in cells, blood and connective tissues, and increased amounts are excreted in the urine. The result is permanent and includes progressive cell damage that affects the individual's appearance, physical abilities, organ and system functioning and, in certain cases, mental development. Enzyme replacement therapies are currently in use or are being tested for at least three different subtypes (I, II and VI). The aim of the present study was to evaluate the effectiveness and safety of laronidase for treating mucopolysaccharidosis type I. A systematic review of the literature was conducted. A computerized electronic search was then conducted using the CENTRAL, Pubmed, EMBASE, and LILACS databases, to identify any randomized controlled trials. The last date of the search was June 2006. There was no possibility of combining the results, because only one study was included. In the pivotal placebo-controlled trial conducted over a 26-week period, there was a reduction in the urinary excretion of GAGs among treated patients. Regarding adverse events, there were no laronidase-related serious adverse events or deaths. Laronidase seems to be a promising agent for treating mucopolysaccharidosis type I, as shown by the reduction in the urinary excretion of GAGs and the associated improvements in vital capacity and in the performance of defined physical tasks.     

Obese Children,Adults and Senior Citizens in the Eyes of the General Public: Results of a Representative Study on Stigma and Causation of Obesity

Obese individuals are blamed for their excess weight based on causal attribution to the individual. It is unclear whether obese individuals of different age groups and gender are faced with the same amount of stigmatization. This information is important in order to identify groups of individuals at risk for higher stigmatization and discrimination. A telephone interview was conducted in a representative sample of 3,003 participants. Experimental manipulation was realized by vignettes describing obese and normal-weight children, adults and senior citizens. Stigmatizing attitudes were measured by semantic differential. Causal attribution was assessed. Internal factors were rated with highest agreement rates as a cause for the vignette''s obesity. Lack of activity behavior and eating too much are the most supported causes. Importance of causes differed for the different vignettes. For the child, external causes were considered more important. The overweight vignette was rated consistently more negatively. Higher educational attainment and personal obesity were associated with lower stigmatizing attitudes. The vignette of the obese child was rated more negatively compared to that of an adult or senior citizen. Obesity is seen as a controllable condition, but for children external factors are seen as well. Despite this finding, they are faced with higher stigmatizing attitudes in the general public, contradicting attribution theory assumptions. Internal and external attribution were found to be inter-correlated. Obese children are the population most at risk for being confronted with stigmatization, making them a target point in stigma-reduction campaigns.     

Identification and characterization of a novel biotin biosynthesis gene in Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae cells generally cannot synthesize biotin, a vitamin required for many carboxylation reactions. Although sake yeasts, which are used for Japanese sake brewing, are classified as S. cerevisiae, they do not require biotin for their growth. In this study, we identified a novel open reading frame (ORF) in the genome of one strain of sake yeast that we speculated to be involved in biotin synthesis. Homologs of this gene are widely distributed in the genomes of sake yeasts. However, they are not found in many laboratory strains and strains used for wine making and beer brewing. This ORF was named BIO6 because it has 52% identity with BIO3, a biotin biosynthesis gene of a laboratory strain. Further research showed that yeasts without the BIO6 gene are auxotrophic for biotin, whereas yeasts holding the BIO6 gene are prototrophic for biotin. The BIO6 gene was disrupted in strain A364A, which is a laboratory strain with one copy of the BIO6 gene. Although strain A364A is prototrophic for biotin, a BIO6 disrupted mutant was found to be auxotrophic for biotin. The BIO6 disruptant was able to grow in biotin-deficient medium supplemented with 7-keto-8-amino-pelargonic acid (KAPA), while the bio3 disruptant was not able to grow in this medium. These results suggest that Bio6p acts in an unknown step of biotin synthesis before KAPA synthesis. Furthermore, we demonstrated that expression of the BIO6 gene, like that of other biotin synthesis genes, was upregulated by depletion of biotin. We conclude that the BIO6 gene is a novel biotin biosynthesis gene of S. cerevisiae.     

A lung function information system

A lung function information system (LFIS) was developed for the data analysis of pulmonary function tests at different locations. This system was connected to the hospital information system (HIS) for the retrieval of patient data and the storage of the lung function variables of patients to generate follow-up reports and to support financial and administrative management. The application programs were developed in such a way that high flexibility was obtained with respect to the patient-computer-technician interaction. The sampled data are stored on a disc to correct earlier decisions, perform recalculations and reanalyse the data for research purposes. When the measurements performed on a patient are authorized, the sampled data are deleted, except for when they are needed for future research. A distributed computer system was chosen to combine the benefits of a centralized system with those of several stand-alone systems. The main tasks of the central unit are to store collected data and computer programs, generate a final lung function report on laser printer and provide a connection to the HIS. In the satellite computers, which are located close to the lung function equipment, the signals and raw data are processed. Furthermore, the satellite computers were in use for program development and several research projects, and for the offline data processing of the lung function measurements from two other hospitals by means of a modem connection. The LFIS improved the quantity and quality of data acquisition. It resulted in an increased capacity of about 50% concerning spirometry, and facilitated time-consuming complex analyses. It also avoided miscalculations and mistakes in reports previously experienced with hand calculations.     

An Experimental Ninth-Grade Anthropology Course

A ninth-grade course in anthropology was taught for three years at the North Shore Country Day School in Winnetka, Illinois. Initially, the first quarter concerned ethnography and the principles of cultural and social anthropology; the second centered around evolution, human origins, and the development of civilization; and the third dealt with comparative modern civilizations. However, the first year convinced us that this was too much material, so the third quarter was dropped and the remainder of the material was spread over the entire year. Ninth graders are a good audience for anthropology because they are interested in ethnography, can compare ethnographic situations to their own situations, and most especially are learning and practicing at being adults so that cultural and social techniques are of great immediate interest.     

Regulation of glycolysis in the mouse blastocyst during delayed implantation

The rate of oxidation of glucose is reduced in mouse embryos in the prolonged free living phase associated with delayed implantation and increases when the embryos are reactivated by estrogen. To determine how these changes in metabolism are regulated, several aspects of glucose metabolism were evaluated in dormant and reactivated blastocysts: 1) Embryos were exposed to 14C-pyruvate in vitro and evolved 14CO2 was measured. It was found that the rate of production of CO2 was equal in the two types of blastocysts, suggesting that aerobic pathways are fully functional during delayed implantation. 2) Production of lactate in the presence of O2 was measured and a decrease of 30% was found in delayed implanting embryos, suggesting that the overall regulatory mechanism for glucose metabolism resides in the glycolytic portion of the pathway. 3) Capacity for uptake and phosphorylation of glucose was evaluated using 3H-2-deoxyglucose and was found to be equal in the two types of embryos. 4) Total amounts of the rate-controlling enzymes for glycolysis (i.e., hexokinase and phosphofructokinase) in lysates of delayed and reactivated embryos were found to be equal, indicating that amounts of these enzymes are not limiting in delayed implantation. 5) Lactate production, measured under anaerobic conditions, was found to be equal, demonstrating that it is not the capacity for glycolysis but a difference in the degree of allosteric inhibition that is responsible for reduced glucose oxidation in delayed implantation. 6) Levels of ATP, ADP, and hexose-6-phosphates were found to be consistent with allosteric inhibition of the glycolytic pathway at phosphofructokinase during delay and a release of this inhibition with reactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new technique for measuring kinetic and mass transfer parameters in flocs of Saccharomyces cerevisiae

Yeast flocs are very fragile and cause problems when manipulations are needed. A new equipment for measuring kinetic and mass transfer parameters was developed and tested: the specific O uptake rate, q, was calculated for a highly flocculent strain of Saccharomyces cerevisiae and its validation was confirmed by comparing the obtained kinetic parameters with data reported in literature.