Normal to cancer microbiome transformation and its implication in cancer diagnosis

Microbial communities coexisting with humans are collectively known as microbiome. It influences almost every aspect of an individual's body function. Microbiome is idiosyncratic for body condition and its alteration is indicative for several abnormalities. This article discusses about recent ideas for developing microbiology based cancer indicators using alterations in microbiome. It is noteworthy that large exploratory studies are required to identify cancer indicator microorganisms from complex and diverse microbiome constituents. This complexity also warrants that these markers should be used in conjunction with other routine cancer indicators. The present article concludes that such studies can spur development of novel microbiome based cancer diagnostics.     

Mitochondrial recombination in crosses of iso- and anisomitochondrial saccharomyces cerevisiae

Summary Mitochondrial mutants resistant to erythromycin, neomycin and monomycin were isolated. Mitochondria were transmitted from different natural strains to the cells of the same nuclear genotype. In bifactorial crosses of such isochromosomal and anisomitochondrial yeasts we tested random samples of diploid colonies. The distribution of mitochondrial markers in parent and recombinant classes has been shown to occur unequally. The asymmetry of parent and the polarity of recombinant classes were observed to differ in different mitochondrial mutants.Anisomitochondrial strain crosses proved that mitochondrial origin essentially influenced both the parent and recombinant classes distribution and the susceptibility of the transmission to the effect of mating type locus. One can distinguish between homo- and heterosexual cross combinations in terms of recombination polarity.The new type of mitochondria was found to occur with high frequency of transmission to the zygote progeny of markers resistant to erythromycin but not of markers resistant to neomycin. The problem of sex in mitochondria is discussed.     

CSF acid-base regulation and ventilation in iso- and hypocapnic Hacetate acidosis

Intravenous infusion in conscious rabbits of Hacetate decreases both arterial CO2 partial pressure PaCO2 and cerebrospinal fluid (CSF) HCO3- more than observed with HCl or HNO3 infusion. These acids did not affect CSF HCO3- in isocapnic conditions, and this study asks whether Hacetate infusion will do so. Arterial, central venous, and cisterna magna catheters were implanted in pentobarbital-anesthetized rabbits and all subsequent measurements were performed in the conscious state. Hacetate was infused intravenously over 6 h to decrease plasma HCO3- the same amount in a group allowed to decrease its PaCO2 in response to the acid (hypocapnic) and one in which PaCO2 was maintained at control levels (isocapnic). CSF HCO3- decreased significantly in isocapnia, although the change was less than in hypocapnia. Stoichiometrically by 6 h the measured CSF HCO3- change was balanced by an increase in acetate in hypocapnia and the sum of an increase in acetate and a decrease in chloride in isocapnia. Mechanistically, net acetate entry into CSF appears to involve an exchange for chloride as proposed for NO3-/Cl- and a process that lowers CSF HCO3-. This process could be competitive replacement of HCO3- by acetate in the CSF production mechanism or nonionic diffusive entry of Hacetate into CSF with subsequent titration of HCO3-. The decreases in CSF HCO3- result from the acetate mechanism and the hypocapnic effect on Cl- and HCO3-. The greater ventilatory response results from the greater CSF acidification or a specific effect of acetate per se.     

Yeast mutants defective in iso-2-cytochrome c.

The structural gene CYC7 for yeast iso-2-cytochrome c was previously identified by isolating a mutant, cyc7-1-1, totally lacking iso-2-cytochrome c and demonstrating that revertants of this mutant contained iso-2-cytochrome c with an altered primary structure (Downie et al., 1977). In this paper we describe a variety of different types of mutants that completely or partially lack iso-2-cytochrome c due to mutations in either the structural gene, CYC7, or unlinked “regulatory” genes. The iso-2-cytochrome c-deficient mutants were isolated by benzidine staining of over 3 × 10⁵ colonies from ?^? strains (cytoplasmic petites) that lacked iso-1-cytochrome c due to the deletion cyc1-1 and that contain abnormally high levels of iso-2-cytochrome c due to a chromosomal translocation, CYC7-1, adjacent to the normal structural gene CYC7 +. The cytochrome c content of mutants not staining with the benzidine reagents was estimated by low temperature spectroscopy, and 139 mutants containing significantly decreased levels of iso-2-cytochrome c were analyzed genetically by complementation with previously identified cyc mutants. In this way 50 mutants at the cyc2 and cyc3 loci were identified along with a group of 62 mutants of the structural gene cyc7. The different types of mutants of the structural gene which were uncovered and which were more or less anticipated included those that completely lacked iso-2-cytochrome c, those that were suppressible by UAA or UAG suppressors, those that lacked iso-2-cytochrome c but had increased levels after growth at lower temperatures, and those that exhibited visibly altered c_a absorption bands of iso-2-cytochrome c. Iso-2-cytochrome c mutants with altered primary structures were obtained from intragenic revertants of several of these mutants, confirming our earlier conclusion that cyc7 is the structural gene. In addition we observed an unexpected class of mutants that lacked iso-2-cytochrome c when in the ?^? state but contained approximately the CYC7-1 parental level when in the ?⁺ state. Two of these mutants, cyc7-1-47 and cyc7-1-49, were shown to contain altered iso-2-cytochromes c. The different contents of the abnormal iso-2cytochromes c suggest that cytochrome c has different environments in ?⁺ and ?^? mitochondria and that the ?⁺ condition may stabilize certain altered proteins.     

Normal and cancer cell metabolism: lymphocytes and lymphoma

Recent studies of normal and neoplastic lymphocytes have revealed overlapping metabolic rewiring in activated T?cells and Myc-transformed lymphocytes. Myc expression is attenuated in normal lymphocytes that return to the basal state, but Notch-activated or Myc-transformed lymphocytes persistently express Myc, which activates genes involved in glucose and glutamine metabolism. Although this difference could provide a therapeutic window for the treatment of cancers, the overlapping metabolic profiles suggest a potential for immunosuppression by metabolic inhibitors.     

Structural intermediates in folding of yeast iso-2 cytochrome c

The kinetic properties of the folding reactions of iso-2 cytochrome c from Saccharomyces cerevisiae have been investigated by stopped-flow and temperature-jump methods. Three different structural probes are compared: (1) absorbance changes in the visible reflecting changes in heme environment, (2) ultraviolet absorbance changes due to the exposure of aromatic groups to solvent, and (3) tryptophan fluorescence attributable principally to the average distance between the tryptophan residue (donor) and the heme (quencher). In addition, two probes either indicative of or correlated with function, ascorbic acid reducibility and the 695-nm absorbance band, have been used to monitor specifically the rate of formation of the native protein on refolding. The fastest phase observed (tau 3) has a measurable relative amplitude only when monitored by visible absorbance changes, suggesting that this reaction involves changes in heme environment in the absence of significant changes in the heme to tryptophan distance or in the extent to which aromatic groups are exposed to solvent. Different slow phases are observed when complete refolding is monitored by visible or ultraviolet absorbance (tau 1a) as opposed to tryptophan fluorescence (tau 1b), the fluorescence changes being complete on a time scale 4-8-fold faster than for absorbance. A mid-range kinetic phase (tau 2) is detected by all three structural probes. When ascorbic acid reducibility or 695-nm absorbance changes are used to monitor the rate of formation of the native protein, two phases are detected: tau 2 and tau 1a. Taken together these results demonstrate that kinetic phase tau 1b results in the formation of a structural intermediate in folding with fluorescence close to that of the native protein but with distinct absorbance properties.     

Interactions between yeast iso-1-cytochrome c and its peroxidase

Isothermal titration calorimetry was used to study the formation of 19 complexes involving yeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprised combinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinations were designed to probe the crystallographically defined interface between Cc and CcP. The data show that the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changing charged residues to alanine increases the enthalpy of complex formation by a constant amount, but the decrease in stability depends on the location of the amino acid substitution. Deleting methyl groups has a small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistent with model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize the complex. Double-mutant cycles were used to determine the coupling energies for nine Cc-CcP residue pairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that many of the substitutions induce a rearrangement of the complex.     

Folding of yeast iso-1-AM cytochrome c

We describe a specific modification of iso-1 cytochrome c which results in blocking a single free sulfhydryl group. The derivative differs from the unmodified protein by the introduction of a small, uncharged group, thus maintaining the same charge balance as the native protein. The modified protein, obtained by treatment of iso-1 cytochrome c with iodoacetamide, has an activity indistinguishable from that of the unmodified protein in the lactate dehydrogenase-cytochrome c reductase system from yeast and has the same stability toward denaturation by guanidine hydrochloride. The kinetics of fluorescence changes associated with the guanidine hydrochloride induced folding-unfolding transition for modified iso-1 cytochrome c (iso-1-AM) have been investigated throughout the transition zone by using stopped-flow mixing. The results are compared to those for the yeast isozyme, iso-2 cytochrome c. The main features of the fluorescence-detected folding kinetics are similar, as might be expected for homologous proteins; however, the limiting value of the fraction of fast refolding protein (alpha 2) below the transition zone is smaller for iso-1-AM (approximately 0.7) than for iso-2 (approximately 0.9).     

Deletions and replacements of omega loops in yeast iso-1-cytochrome c

omega (omega)-loops are protein secondary structural elements having small distances between segment termini. It should be possible to delete or replace certain of these omega-loops without greatly distorting the overall structure of the remaining portion of the molecule. Functional requirements of regions of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae were investigated by determining the biosynthesis and activity in vivo of mutant forms in which four different omega-loops were individually deleted, or in which one omega-loop was replaced with five different segments. Deletions encompassing amino acid positions 27-33 and 79-83 either prevented synthesis of the holoprotein, or produced highly labile iso-1-cytochromes c, whereas deletions encompassing positions 42-45 and 48-55 allowed partial synthesis and activity. These two latter regions, therefore, are not absolutely required for any biosynthetic process such as heme attachment, mitochondrial import, or for enzymatic interactions. All replacements in Loop A (residue positions 24-33) with same size (10 amino acid residues), longer (13 and 15 amino acid residues), or shorter segments (6 amino acid residues), resulted in strains having at least partial levels of iso-1-cytochrome c; however, the relative activities ranged from zero to almost the normal level. Thus, Loop A does not appear to be essential for such biosynthetic steps as heme attachment and mitochondrial import. In contrast, the full range of relative activities suggest that this region interacts with physiological partners to carry out efficient electron transport.     

pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.     

Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5-13C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degrees C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in slow exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters: delta H++ = 28 kcal/mol for both I and III; delta S++ = 42 cal/(mol.K) for I, and delta S++ = 41 cal/(mol.K) for III. The remaining tyrosine (V) has a larger enthalpy and entropy of activation: delta H++ - 36 kcal/mol, delta S++ = 72 cal/(mol.K). Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS)     

Massively parallel resequencing of the isogenic Drosophila melanogaster strain w1118; iso-2; iso-3 identifies hotspots for mutations in sensory perception genes

We used the Illumina reversible-short sequencing technology to obtain 17-fold average depth (s.d.~8) of ~94% of the euchromatic genome and ~1-5% of the heterochromatin sequence of the Drosophila melogaster isogenic strain w1118; iso-2; iso-3. We show that this strain has a ~9 kb deletion that uncovers the first exon of the white (w) gene, ~4 kb of downstream promoter sequences, and most of the first intron, thus demonstrating that whole-genome sequencing can be used for mutation characterization. We chose this strain because there are thousands of transposon insertion lines and hundreds of isogenic deficiency lines available with this genetic background, such as the Exelixis, Inc., and the DrosDEL collections. We compared our sequence to Release 5 of the finished reference genome sequence which was made from the isogenic strain y1; cn1 bw1 sp1 and identified ~356,614 candidate SNPs in the ~117 Mb unique sequence genome, which represents a substitution rate of ~1/305 nucleotides (~0.30%). The distribution of SNPs is not uniform, but rather there is a ~2-fold increase in SNPs on the autosome arms compared with the X chromosome and a ~7-fold increase when compared to the small 4th chromosome. This is consistent with previous analyses that demonstrated a correlation between recombination frequency and SNP frequency. An unexpected finding was a SNP hotpot in a ~20Mb central region of the 4th chromosome, which might indicate higher than expected recombination frequency in this region of this chromosome. Interestingly, genes involved in sensory perception are enriched in SNP hotspots and genes encoding developmental genes are enriched in SNP coldspots, which suggests that recombination frequencies might be proportional to the evolutionary selection coefficient. There are currently 12 Drosophila species sequenced, and this represents one of many isogenic Drosophila melanogaster genome sequences that are in progress. Because of the dramatic increase in power in using isogenic lines rather than outbred individuals, the SNP information should be valuable as a test bed for understanding genotype-by-environment interactions in human population studies.     

pH dependence of folding of iso-2-cytochrome c

Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes. Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes. At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding. The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps. The rates of slow folding also depend on the final pH. At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes. At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude. At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude. In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH. The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of the yeast iso-1-cytochrome c mRNA

The nucleotide sequence of the yeast iso-1-cytochrome c (CYC1) mRNA is presented. The mRNA was enriched by hybridization to cloned CYC1 DNA attached to a solid matrix: either nitrocellulose filters or diazobenzyloxymethyl cellulose powder. The sequence of the 5'-end of the mRNA was determined by the extension of a CYC1-specific dodecanucleotide primer; the sequence of the 3'-end was determined using a decanucleotide d(pT8-G-A) primer. The CYC1 mRNA begins 61 nucleotides 5' to the AUG initiation codon, extends through the coding sequence to 172 to 175 nucleotides 3' to the UAA termination codon, followed by the poly(A) tail. There are no intervening sequences. Some of the sequences that the CYC1 mRNA shares in common with other eukaryotic mRNAs are discussed.