Effects of fasted vs fed-state exercise on performance and post-exercise metabolism: A systematic review and meta-analysis.

The effects of nutrition on exercise metabolism and performance remain an important topic among sports scientists, clinical, and athletic populations. Recently, fasted exercise has garnered interest as a beneficial stimulus which induces superior metabolic adaptations to fed exercise in key peripheral tissues. Conversely, pre-exercise feeding augments exercise performance compared with fasting conditions. Given these seemingly divergent effects on performance and metabolism, an appraisal of the literature is warranted. This review determined the effects of fasting vs pre-exercise feeding on continuous aerobic and anaerobic or intermittent exercise performance, and post-exercise metabolic adaptations. A search was performed using the MEDLINE and PubMed search engines. The literature search identified 46 studies meeting the relevant inclusion criteria. The Delphi list was used to assess study quality. A meta-analysis and meta-regression were performed where appropriate. Findings indicated that pre-exercise feeding enhanced prolonged (P = .012), but not shorter duration aerobic exercise performance (P = .687). Fasted exercise increased post-exercise circulating FFAs (P = .023) compared to fed exercise. It is evidenced that pre-exercise feeding blunted signaling in skeletal muscle and adipose tissue implicated in regulating components of metabolism, including mitochondrial adaptation and substrate utilization. This review's findings support the hypothesis that the fasted and fed conditions can divergently influence exercise metabolism and performance. Pre-exercise feeding bolsters prolonged aerobic performance, while seminal evidence highlights potential beneficial metabolic adaptations that fasted exercise may induce in peripheral tissues. However, further research is required to fully elucidate the acute and chronic physiological adaptations to fasted vs fed exercise.

Planetary contamination I: the problem and the agreements.

On the contents page of the 24 March issue, the title of the first article should read "Planetary Contamination I: The Problem and the Agreements: N. H. Horowitz, R. P. Sharp, R. W. Davies."

Planetary contamination I: the problem and the agreements.

The sterility requirements for landed spacecraft tentatively adopted in the COSPAR resolution of 1964 are so severe as to pose a major obstacle to planetary exploration. This by itself would not justify modification of the re quirements, since preservation of the biological integrity of Mars is essential for proper exploration of the planet. However, when the physical and biological assumptions underlying the COSPAR recommendations are com pared with actual conditions on Mars, as established by recent observations, it becomes apparent that the COSPAR as sumptions are unrealistic in important respects. Specifically, the belief that eolian erosion on Mars can effect the release of spores trapped in the interior of solids in periods of time that are short compared with the time scale of the unmanned space program is unsup ported by either observation or theory. On the contrary, the analysis suggests that rates of eolian erosion on Mars are very low. Similarly, present knowledge of the Martian environment opposes the view that terrestrial microorganisms would readily contaminate the planet. The combination of dryness, lack of oxygen, and high ultraviolet flux makes the surface of Mars peculiarly unsuit able for the multiplication of terrestrial organisms. Recent studies give little sup port to the proposal that significant areas of geothermal activity exist on Mars. These various findings suggest that the COSPAR-recommended constraints could be substantially relaxed without compromising to any significant degree the biological condition of Mars. In particular, a distinction needs to be made between microorganisms trapped in solids and those on exposed sur faces of landed spacecraft. Surface sterility is an unconditional require ment, in the sense that it is imposed by considerations unrelated to the nature of the Martian environment. Sterilization of the interior of solids to the extreme level recommended by COSPAR, however, is based on the as sumption that entrapped organisms con stitute a substantial hazard to the ecology of Mars. This assumption now seems unjustified, and the need for a high degree of interior sterility is doubt ful. Current spacecraft-sterilization pol icies should be revised accordingly.

Secretion of a bacterial cellulase by yeast.

Gene fusions were constructed between a yeast expression plasmid and a Cellulomonas fimi DNA fragment encoding an endo-1,4-beta-D-glucanase or carboxymethylcellulase. Yeast transformed with the recombinant plasmids secreted carboxymethylcellulase activity. Secretion of active enzyme was greatly increased when the leader of a secreted yeast protein, the Kl toxin, was inserted immediately upstream of and in frame with the bacterial cellulase sequence. This is the first step in constructing a functional cellulase complex in Saccharomyces cerevisiae. It also provides an excellent system for the detailed examination of the determinants of protein secretion because of the ease with which secreted cellulase can be detected.

Predictive impact of rare genomic copy number variations in siblings of individuals with autism spectrum disorders.

Identification of genetic biomarkers associated with autism spectrum disorders (ASDs) could improve recurrence prediction for families with a child with ASD. Here, we describe clinical microarray findings for 253 longitudinally phenotyped ASD families from the Baby Siblings Research Consortium (BSRC), encompassing 288 infant siblings. By age 3, 103 siblings (35.8%) were diagnosed with ASD and 54 (18.8%) were developing atypically. Thirteen siblings have copy number variants (CNVs) involving ASD-relevant genes: 6 with ASD, 5 atypically developing, and 2 typically developing. Within these families, an ASD-related CNV in a sibling has a positive predictive value (PPV) for ASD or atypical development of 0.83; the Simons Simplex Collection of ASD families shows similar PPVs. Polygenic risk analyses suggest that common genetic variants may also contribute to ASD. CNV findings would have been pre-symptomatically predictive of ASD or atypical development in 11 (7%) of the 157 BSRC siblings who were eventually diagnosed clinically.

Proteinuria, not altered albumin metabolism, affects hyperlipidemia in the nephrotic rat.

It has been established previously that nephrotic hyperlipidemia is characterized by both an increase in lipid synthesis and a defect in removal of lipoproteins. The relationship between these defects and altered albumin metabolism is uncertain. One hypothesis is that hepatic lipogenesis increases in parallel with albumin synthesis. To test this hypothesis, albumin synthesis was increased in nephrotic rats fed an 8.5% protein diet (LPN) by increasing dietary protein to 40% (HPN). Proteinuria was modulated in half of the rats fed 40% protein by enalapril (HPE). Albumin synthesis was the same in both HPN and HPE, but proteinuria was reduced in HPE compared to HPN, and so were serum cholesterol and triglycerides (TG). To examine the effect of serum albumin on lipid clearance in the absence of proteinuria, plasma clearance of chylomicrons (CM) and VLDL was measured in Nagase analbuminemic rats (NAR) and found to be no different than in normal SD rats. When proteinuria was induced in NAR and in SD rats, a severe and identical defect in both CM and VLDL clearance was acquired in both groups and blood lipid levels were increased to a similar degree in both groups. Neither hyperlipidemia nor defective removal of lipoproteins from the circulation are linked to albumin synthesis or serum albumin concentration but result, at least in part, from proteinuria. Postheparin lipoprotein lipase (LPL) activity was reduced slightly in nephrotic animals compared to nonnephrotic controls, but the most striking finding was a highly significant decrease in postheraprin LPL activity in normal NAR compared to SD rats (P less than 0.001), suggesting that reduced LPL activity is not responsible for reduced clearance of CM and VLDL in nephrotic rats.

Reverse genetics of the mouse central nervous system: targeted genetic analysis of neuropeptide function and reverse genetic screens for genes involved in human neurodegenerative disease.

The development of gene targeting technology in mouse embryonic stem cells allows reverse genetics to be used to investigate the function of any cloned gene in the developing and adult brain. Promoter-trap, replacement and insertion vector strategies can be used to generate defined mutations in the chromosomal copy of a cloned gene in embryonic stem cells. These cells can be used to make chimaeric mice, some of which transmit the in vitro mutation via the germline to transgenic offspring. The phenotype of complete loss-of-function mutations (gene knock-outs) can be studied at molecular, cell biological, neurophysiological and behavioural levels, and allows inferences about gene function to be made. Precise small mutations can also be made using integrative vector or two-step replacement vector strategies, allowing specific questions to be asked about regulation and protein structure-function relationships. Reverse genetics can therefore be used as an alternative or additional approach to pharmacology for the study of molecular functions in the central nervous system. Reverse genetic studies of the involvement of particular molecules in neurological disease syndromes may be superior to pharmacological studies to the extent that the syndrome is determined by genetic predisposition. The general ways in which reverse genetics of the mouse can be used to ask questions about molecules in the central nervous system are illustrated by examples from ongoing work of this laboratory. Neuropeptides are an important class of transmitters in the brain, but only in very few cases have specific CNS functions been assigned to a particular neuropeptide. Targeted mutation of neuropeptide precursor and receptor genes offers a rapid way to learn about neuropeptide function. Complete loss-of-function mutations will provide information on any developmental roles of a neuropeptide and on overall behavioural and physiological effects of loss-of-function. More specific targeted mutations allow dissection of the individual roles of multiple neuropeptides that derive from a common precursor protein, and allow in vivo studies of the functional importance of particular amino acids. Experimental progress towards targeted mutation of the neurotensin receptor is described as an example. Recent technological improvements makes targeted mutation of a number of genes possible. This allows reverse genetic screening to be undertaken for genes involved in particular neurobiological phenomena: genes are identified on the basis of molecular criteria (e.g. expression pattern), and gene-targeting used to check their relevance to a phenotype. Neurodegenerative disease is an important aspect of the human phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)

Close relationship between certain nuclear and mitochondrial introns. Implications for the mechanism of RNA splicing.

We present the first indication of a direct relationship between a nuclear and a mitochondrial splicing system. The intron in the precursor of the large, nuclearly coded ribosomal RNA of two species of Tetrahymena possesses all the features of a class of fungal mitochondrial introns. Sequences conserved in mitochondrial introns of different fungal species are also found in the same order in these Tetrahymena nuclear introns, and the intron RNA can be folded to form a secondary structure similar to that proposed for mitochondrial introns by Davies et al. (1982). This "core" secondary structure brings the ends of the intron together. Furthermore, the first intron in the precursor of the large, nuclearly coded rRNA of Physarum polycephalum also has the characteristic conserved sequences and core RNA secondary structure. The limited sequence data available suggest that the intron in the large rRNA of chloroplasts in Chlamydomonas reinhardtii also resembles the mitochondrial introns. Tetrahymena large nuclear rRNA introns also have an internal sequence that can act as an adaptor by pairing with upstream and downstream exon sequences adjacent to the splice junctions to precisely align the splice junctions. These nuclear introns therefore fit the model of the role of intron RNA in the splicing process that was proposed by Davies et al. (1982), suggesting that the mechanisms of splicing may be very similar in these apparently diverse systems. It is therefore probable that the RNA secondary structures for which there is good evidence in the case of mitochondrial introns will be found to form the basis of active site structure and precise alignment in splicing and cyclization of the Tetrahymena intron "ribozyme".

Processing of mitochondrial RNA in Aspergillus nidulans.

Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review.

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.

Discovering genes with localised expression in the mouse brain: cDNAs specific to the substantia nigra.

Many important phenomena of normal brain physiology and disease are likely to be related to the function of genes expressed in localised regions of the brain. We show that subtracted libraries enriched in clones corresponding to rare mRNAs, which must include genes with very localised and neuron-specific expression, can easily be produced from single-stranded directional cDNA libraries after hybridization to excess photobiotinylated opposite-stranded cDNA (or RNA) from another brain region, followed by the removal of biotinylated molecules. We also demonstrate the use of heterologous probes from anatomically precise small regions of bovine brain to identify cDNA clones that putatively represent mRNAs present at significantly higher levels in a substantia nigra mRNA population enriched for pars compacta mRNA than in the total ventral midbrain or cerebellar mRNA population. Some of these cDNAs may identify genes that play important roles in the specific molecular biology of dopaminergic neurons, including susceptibility to Parkinson's disease.

Molecular cloning of the gene for dihydrofolate reductase from Lactobacillus casei.

The Lactobacillus casei gene for dihydrofolate reductase has been cloned in Escherichia coli using the multicopy vector pBR322. A restriction map of the cloned DNA has been prepared. The cloned DNA directs the synthesis of L. casei dihydrofolate reductase in E. coli and confers trimethoprim and methotrexate resistance.

Production of mutant dihydrofolate reductases of Lactobacillus casei for nuclear magnetic resonance spectroscopy.

Seven mutations (L4P, W21L, D26E, D26N, R57H, R57K and T63Q) affecting residues of dihydrofolate reductase of Lactobacillus casei, suspected of being important in substrate, inhibitor, or cofactor binding, were made by gapped-duplex site-directed mutagenesis. Expression of the L. casei dhfr gene required the removal of nucleotide sequences flanking the coding region. Temperature-inducible expression from the lambda pL promoter of plasmid pPLc28 allowed synthesis and subsequent affinity purification of five mutant proteins in amounts and purity sufficient for nuclear magnetic resonance (NMR) spectroscopic analysis (100 mg or more) from 10-liter cultures. W21L required the growth of 40-liter batches, and L4P was not found. Using a two-plasmid system with pcI857 providing lambda repressor and pMAC5-14 expressing the mutant gene, any auxotrophic strain of Escherichia coli can be used as a host, allowing isotopic labelling of each amino acid of any protein for rapid NMR peak assignment.

Cloning of a new bidirectionally selectable marker for Aspergillus strains.

Mutants that lack adenosine triphosphate sulfurylase (ATPsase; EC 2.7.7.4) are unable to use sulfate as sole source of sulfur and are also resistant to selenate. These mutants, denoted sC-, are readily obtained from any strain of Aspergillus niger or Aspergillus nidulans by the strong selection for selenate resistance. We have cloned the gene encoding ATPsase from A. nidulans by complementation of an sC mutant strain of A. nidulans with a gene library and show that plasmids containing this gene transform both A. niger and A. nidulans sC- strains, restoring their ability to grow on sulfate as sole sulfur source. The fact that strong selection for either sC+ or sC- can be applied provides a simple way of delivering genetically engineered constructs to any strain of A. niger including strains of industrial importance. In addition, this system is useful for gene replacements and other genomic DNA manipulations in Aspergillus species.

Vectors permitting visual monitoring of simple transposition events.

The construction and use of two novel transposon(Tn)-delivery vectors is described. These vectors carry Inc.W or Inc.N broad-host-range transfer functions cloned next to the narrow-host-range replicon of pBR329. The host specificities of pSLX10 and pSLX23 both complement and extend the host specificities of existing Tn delivery vectors. Plasmids pSLX10 and pSLX23 were shown to transfer at high frequency in intergeneric matings. The lux genes which are present on each vector permit the visual monitoring of transconjugants which have retained a Tn element, but are devoid of plasmid molecules. pSLX10 and pLSX23 were efficiently used to generate a range of auxotrophic mutants in various strains of Pseudomonas as well as to clone genes from Serratia liquefaciens. These vectors may have general applicability to identify and clone genes in a wide range of Gram-negative bacteria.

A novel vector allowing the expression of genes in a wide range of gram-negative bacteria.

The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described. The vector utilizes the regulatory region from IS50. The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn5 by creating EcoRI and Sa/I or PstI restriction sites by in vitro mutagenesis. This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Different strains containing the cat gene behind the regulatory elements of IS50 were able to tolerate high concentrations (300 micrograms/ml) of chloramphenicol in the medium. The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI. Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans.

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.

The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR.

The regulatory gene, alcR, of Aspergillus nidulans, encodes a protein that induces the expression of the alcA and aldA genes. The alcR gene is inducible, autoregulated, and subject to carbon catabolite repression. We report the complete nucleotide sequence of the alcR gene and its 5' and 3' non-coding regions. In the 5' flanking region of the alcR gene, several repeats and inverted repeats were found, and small sequence similarities were also found with the 5' flanking regions of the alcA and aldA genes. One intron of small size interrupts the open reading frame. The start point of transcription was mapped 50 nucleotides upstream from the putative start codon, and a sequence CAATG was found 5' to the polyadenylation site of the transcript that could play a role in selection of the polyadenylation site. The putative alcR-encoded protein was identified in vivo as an inducible polypeptide of 96 kDa in a transformant carrying multiple copies of the alcR gene.

Cloning and molecular analysis of the ornithine carbamoyl transferase gene of Aspergillus niger.

We have cloned the gene encoding ornithine carbamoyl transferase (OCTase) from Aspergillus niger. The structure and complete nucleotide sequence of this gene have been determined. The gene encodes an mRNA of 1.3 kb. The transcription unit contains an open reading frame of 1110 nucleotides (nt) which shows strong homology to the OCTase of Aspergillus nidulans along most of its length. The N terminus, which shows little or no homology to other OCTases, is highly basic and is probably involved in mitochondrial targeting.

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase).

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.