The effect of galactose supplementation on endurance cycling performance.

OBJECTIVES: This study tested the hypothesis that supplementation with galactose before and during endurance exercise would spare carbohydrate (CHO), optimize fat utilization and improve performance compared with a typical sports drink formulation. SUBJECTS: Nine well-trained cyclists undertook three trials, each consisting of 120 min at 65 VO(2max) followed immediately by a set work, self-paced time trial (TT). Three treatments, allocated as a randomized balanced design, consisted of the following: (a) 8% (w/w) solution of galactose (Gal); (b) 8% solution of 50% galactose/50% glucose (Gluc/Gal); and (c) 8% solution of 80% glucose/20% fructose (Gluc/Fru). These were consumed as 0.67 g CHO per kg body wt 45-min pre-exercise; 1.0 g CHO per kg body wt per h for the first 120 min of exercise; 0.33 g CHO per kg body wt during the TT. Blood samples were collected before and during exercise; respiratory gas samples were collected only during fixed workload exercise. RESULTS: Mean TT power output was significantly less in Gal compared with Gluc/Gal (P=0.030). Blood glucose and insulin concentrations were lower, and free fatty acids higher in Gal compared with Gluc/Gal and Gluc/Fru. Respiratory exchange ratio was not significantly different between trials. CONCLUSIONS: Ingestion of an 8% galactose-only solution (12.5 ml per kg body wt per h) is detrimental to endurance performance compared with equivalent volumes of iso-osmotic solutions containing 50% galactose/50% glucose or 80% glucose/20% fructose. This may reflect the inability of the liver to convert galactose into glucose at a rate required to support strenuous exercise intensity.

NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans.

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.albicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42 degrees C. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1-regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis.

Evaluation of the CaMAL2 promoter for regulated expression of genes in Candida albicans.

An expression vector (CIp10-MAL2p) for use in Candida albicans has been constructed in which a gene of interest can be placed under the control of the CaMAL2 maltase promoter and stably integrated at the CaRP10 locus. Using this vector to express the Candida URA3 gene from the CaMAL2 promoter, we have demonstrated tight regulation of CaURA3 expression by carbon source. Thus under conditions when the CaMAL2 promoter is not induced, expression of Candida URA3 was unable either to complement a C. albicans ura3 mutation or to confer sensitivity to 5-fluoroorotic acid, a compound which is highly toxic to URA3 strains. Since Candida albicans is an obligate diploid organism, analysis of gene function requires manipulation of both copies of any gene of interest. Our expression vector provides a strategy by which the remaining copy of a gene of interest can be placed under CaMAL2 promoter control in a strain where the first copy has been deleted, permitting analysis of gene function by manipulation of carbon source. CIp10-MAL2p should therefore provide a useful means for functional analysis of genes in C. albicans. We have used this strategy with C. albicans DPB2 to demonstrate that the gene is essential and that loss of function leads cells to adopt a hypha-like morphology as they cease proliferation.

Characterization of an HPr kinase mutant of Staphylococcus xylosus.

The Staphylococcus xylosus gene hprK, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His(6) fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of S. xylosus resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of S. xylosus. The hprK mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of S. xylosus according to the availability of preferred carbon sources.

Identification of a new repetitive element in Staphylococcus aureus.

The Staphylococcus aureus repeat (STAR) element is a sequence identified in two intergenic regions in S. aureus. The element is found in 13 to 21 copies in individual S. aureus strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.

The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation.

Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesin (PIA), which is composed of linear beta-1,6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica) locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Sequence comparison with the S. epidermidis ica genes revealed 59 to 78% amino acid identity. Deletion of the ica locus results in a loss of the ability to form biofilms, produce PIA, or mediate N-acetylglucosaminyltransferase activity in vitro. Cross-species hybridization experiments revealed the presence of icaA in several other Staphylococcus species, suggesting that cell-cell adhesion and the potential to form biofilms is conserved within this genus.

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase.

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.

Secretion of the lantibiotics epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on epidermin production and their regulation by EpiQ.

The closely related lantibiotics epidermin and gallidermin are produced by Staphylococcus epidermidis Tu3298 and S. gallinarum Tu3928, respectively. The epidermin biosynthetic genes involved in maturation, regulation, and immunity have been identified previously. How epidermin or gallidermin is secreted, however, has remained unclear. Here, we characterize two additional genes, epiH and epiT, as well as the homologous gallidermin genes gdmH and gdmT. EpiT and GdmT are similar to one-component ABC transporters that are involved in the secretion of proteins or peptides. EpiH and GdmH are hydrophobic proteins without conspicuous similarities to other proteins. Comparison of the gene sequences revealed that epiT is incomplete, having an internal deletion that causes a frame shift and a second deletion at the 3'-end, while gdmT is intact. Introduction of epiT and epiH into the heterologous host S. carnosus (pTepi14) bearing the maturation and regulation genes had no significant effect on the rather low level of epidermin production. The presence of the homologous gdmT and gdmH, however, resulted in a strong increase (seven- to tenfold) in the production level, which is very likely to be due to increased efficiency of epidermin secretion. Both gdmT and gdmH were necessary for this effect, indicating that the two gene products cooperate in some way. In the epidermin-producing wild-type strain Tu3298, which contains epiH and the disrupted epiT, the addition of gdmT alone led to a two-fold increase in epidermin production. Both gdmT and gdmH and the corresponding epi genes were activated by the transcriptional regulator EpiQ; this is in accordance with the presence of putative EpiQ operator sites in the promoter regions.

The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating.

The yeast Saccharomyces cerevisiae gene CCH1 (ORF YGR217w) shows high homology with animal calcium channel alpha1-subunit genes. Knock-out mutants were constructed of Cch1 and of Mid1 which is known to mediate Ca2+ influx in response to the alpha-mating pheromone. Cch1 is involved in calcium influx and the late stage of the mating process. The cch1 mutant sensitivity against the alpha-mating pheromone can be reduced by the addition of extra calcium. The product of this gene is likely to interact with the MID1 gene product in Ca influx or its control.

The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae, a c-jun homologue, is involved in oxygen metabolism.

The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H2O2-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H2O2 promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.

Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis.

Epidermin is produced by Staphylococcus epidermidis Tü3298 which harbors the 54-kb plasmid, pTü32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C, D, Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., Götz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57-68]. We isolated a number of stable mutants from strain Tü3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT181mcs and pCU1 led to their classification as epiA, epiB, epiC or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that epiC and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S. carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D, and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTü32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of Gly10 by Glu.

Analysis of genes involved in the biosynthesis of lantibiotic epidermin.

The structural gene of the lanthionine-containing peptide antibiotic epidermin is located on a 54-kb plasmid of Staphylococcus epidermidis [Schnell et al. (1988) Nature 333, 276-278]. A 13.5-kb DNA region neighbouring the epidermin structural gene (epiA) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, epiC and epiD shared no homology with previously described proteins stored in data bases. They were located 3' adjacent to epiA. Using epiB as a probe, a 5-kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3-kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3' to epiA, epiB, epiC and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis.

Analysis of genes involved in biosynthesis of the lantibiotic subtilin.

Lantibiotics are peptide-derived antibiotics with high antimicrobial activity against pathogenic gram-positive bacteria. They are ribosomally synthesized and posttranslationally modified (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). The most important lantibiotics are subtilin and the food preservative nisin, which both have a very similar structure. By using a hybridization probe specific for the structural gene of subtilin, spaS, the DNA region adjacent to spaS was isolated from Bacillus subtilis. Sequence analysis of a 4.9-kb fragment revealed several open reading frames with the same orientation as spaS. Downstream of spaS, no reading frames were present on the isolated XbaI fragment. Upstream of spaS, three reading frames, spaB, spaC, and spaT, were identified which showed strong homology to genes identified near the structural gene of the lantibiotic epidermin. The SpaT protein derived from the spaT sequence was homologous to hemolysin B of Escherichia coli, which indicated its possible function in subtilin transport. Gene deletions within spaB and spaC revealed subtilin-negative mutants, whereas spaT gene disruption mutants still produced subtilin. Remarkably, the spaT mutant colonies revealed a clumpy surface morphology on solid media. After growth on liquid media, spaT mutant cells agglutinated in the mid-logarithmic growth phase, forming longitudinal 3- to 10-fold-enlarged cells which aggregated. Aggregate formation preceded subtilin production and cells lost their viability, possibly as a result of intracellular subtilin accumulation. Our results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtilin transport.

Identification and characterization of a Saccharomyces cerevisiae gene (PAR1) conferring resistance to iron chelators.

o-Phenanthroline (1,10-phenanthroline) is a chemical known to chelate iron and other transition metal ions. This compound was added to solid yeast media to reduce the concentration of biologically available iron. Other essential divalent cations, like Zn2+ or Cu2+, which could also be bound, were supplemented. Growth of wild-type yeast strains was totally inhibited at specific concentrations of the chelator. However, several cells containing plasmids of a multicopy vector genomic library of S. cerevisiae could be selected by growth on these media. All of the resistant clones carried a single additional gene, PAR1 on their multicopy plasmids. Plasmid-directed overexpression of PAR1 increased the resistance of transformants to o-phenanthroline and additionally conferred resistance to 1-nitroso-2-naphthol, an iron(III)-binding molecule with different coordinating ligands. By supplementing the o-phenanthroline-containing media with several different metal ions, it could be proved that the selection plates really caused a specific iron limitation. These observations clearly demonstrated that the overexpressed PAR1 gene enables the cell to compete with iron-chelating organic molecules. PAR1 null mutants, constructed by insertion of the LEU2 gene into the open reading frame, showed a remarkable phenotype: they did not grow on slightly alkaline buffered media (pH greater than 7) and became hypersensitive to oxidative stress by hydrogen peroxide. Of several heavy metal ions, such as Fe3+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+, tested for supplementation of the alkaline growth deficiency, only iron, either added in the ferrous or ferric form, was able to restore cellular growth. It can be concluded from the DNA sequence that PAR1 encodes a highly acidic protein of 650 residues with mostly hydrophilic character. Some interesting repetitive amino acid motifs, such as (Asp-Asn)4 or Cys-Ser-Glu, may act as metal-binding sites. The possible role of PAR1 is discussed.

Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic.

Peptide antibiotics containing lanthionine and 3-methyllanthionine bridges, named lantibiotics are of increasing interest. A new lantibiotic, gallidermin, has been isolated from Staphyloccus gallinarum. Here we report the isolation of its structural gene which we name gdmA. In all lantibiotics so far studied genetically, three peptides can be formally distinguished: (i) the primary translation product, which we call the prepeptide; (ii) the propeptide lacking the leader sequence and (iii) the mature lantibiotic. Unlike the plasmid-coded epidermin, gdmA is located on the chromosome. The gdmA locus codes for a 52 amino acid residue prepeptide, consisting of an alpha-helical leader sequence of hydrophilic character, which is separated from the C-terminus (propeptide) by a characteristic proteolytic processing site (Pro-2 Arg-1 Ile1). Although pro-gallidermin differs from pro-epidermin (a recently isolated lantibiotic) only by a single amino acid residue exchange. Leu instead of Ile, the N-terminus of the prepeptide differs by an additional two exchanges.

Gallidermin: a new lanthionine-containing polypeptide antibiotic.

Gallidermin is a new member of the class of lanthionine-containing peptide antibiotics, which are summarized under the common name lantibiotics. The lantibiotic gallidermin is produced by Staphylococcus gallinarum (F16/P57) Tü3928, and it exhibits activities against the Propionibacteria, involved in acne disease. Gallidermin differs from the recently discovered tetracyclic 21-residue peptide antibiotic epidermin only in a Leu/Ile exchange in position 6. The isolation procedures for gallidermin included adsorption directly from the culture broth, ion-exchange chromatography of the amphiphilic and basic polypeptide followed by desalting, and final purification by reversed-phase HPLC. The structural elucidation of the polypeptide containing four thioether bridges involved mainly a combination of automated gas-phase sequencing, thermospray liquid chromatography/mass spectrometry and fast-atom-bombardment mass spectrometry.

Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings.

The genetic basis for the biosynthesis of large polypeptide antibiotics such as nisin has not been explained so far. We show here that the structural gene epiA encoding the antibiotic epidermin from Staphylococcus epidermidis is located on a 54-kilobase plasmid and codes for a 52-amino-acid prepeptide, which is processed to the tetracyclic 21-peptide amide antibiotic. The mature sequence of epidermin corresponds to the C-terminal 22-peptide segment of pre-epidermin and contains the precursor amino acids Ser, Thr and Cys, from which the unusual amino-acid constituents are derived. The more lipophilic epidermin is cleaved at a hydrophilic turn between Arg-1 and Ile+1 from the N-terminal segment-30 to -1, which probably assumes a partially amphiphilic alpha-helix conformation. We propose that the N-terminus (-30 to -1) plays a cooperative role during modification reactions and prevents toxicity of the mature epidermin to the producing strain before the antibiotic is cleaved off and secreted.

Requirements on resolution of digital imaging equipment in the cardiac catheterization laboratory.

We evaluated the requirements on spatial resolution of digital imaging equipment in the cardiac catheterization laboratory. Fifty cinefilms of the heart as the biological object and one film of a lead-ladder-pattern as an objective test were used. The patient films were examined for the visibility of the left ventricular angiogram, coronary arterial tree, coronary artery lesions, branching of septal arteries and the number of septal arteries. All films were viewed three times: with a 625 line TV-system, with a 1249 line TV-system and with a cineprojector. It was found that two application areas with different demands on the spatial and temporal resolution can be distinguished: 1) low spatial resolution and high temporal resolution, e.g. left ventriculography; and 2) high spatial resolution and low temporal resolution, e.g. coronary arteriography. For the diagnostic assessment of the state of the coronary system, the spatial resolution provided by the 1249 line TV-system was sufficient. Exceeding this resolution by using cinefilm quality provided no additional diagnostic information. A frame rate lower than 50 frames per second for coronary arteriography seems possible.