Glutathione-induced radical formation on lactoperoxidase does not correlate with the enzyme's peroxidase activity

Lactoperoxidase (LPO) is believed to serve as a mediator of host defense against invading pathogens. The protein is more abundant in body fluids such as milk, saliva, and tears. Lactoperoxidase is known to mediate the oxidation of halides and (pseudo)halides in the presence of hydrogen peroxide to reactive intermediates presumably involved in pathogen killing. More recently, LPO has been shown to oxidize a wide diversity of thiol compounds to thiyl free radicals, which ultimately lead to the formation of a protein radical characterized by DMPO-immunospin trapping. In the same study by our group the authors claimed that a consequence of this protein radical formation was the inactivation of LPO (Guo et al., J. Biol. Chem.279:13272-13283; 2004). Here we demonstrate that although thiyl radical formation does lead to LPO radical production, the formation of this radical is unrelated to the enzyme's activity. We suggest the source of this misleading interpretation to be the binding of GSH to ELISA plates, which interferes with ABTS and guaiacol oxidation. In addition, DMPO-GSH-nitrone adducts bind to ELISA plates, leading to ambiguities of interpretation since we have demonstrated that DMPO-GSH nitrone does not bind to LPO, and only LPO-protein-DMPO-nitrone adducts can be detected by Western blot.     

Fas aggregation does not correlate with Fas-mediated apoptosis.

Cross-linking of cell surface Fas molecules by Fas ligand or by agonistic anti-Fas Abs induces cell death by apoptosis. We found that a serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK), dramatically enhances Fas-mediated apoptosis in the human T cell line Jurkat and in various B cell lines resistant to Fas-mediated apoptosis. The enhancing effect of TLCK is specific to Fas-induced cell death, with no effect seen on TNF-alpha or TNF-related apoptosis-inducing ligand-induced apoptosis. TLCK treatment had no effect on Fas expression levels on the cell surface, and neither promoted death-inducing signaling complex formation nor decreased expression levels of cellular inhibitors of apoptosis (FLICE inhibitory protein, X chromosome-linked inhibitor of apoptosis, and Bcl-2). Activation of the Fas-mediated apoptotic pathway by anti-Fas Ab is accompanied by aggregation of Fas molecules to form oligomers that are stable to boiling in SDS and beta-ME. Fas aggregation is often considered to be required for Fas-mediated apoptosis. However, sensitization of cells to Fas-mediated apoptosis by TLCK or other agents (cycloheximide, protein kinase C inhibitors) causes less Fas aggregation during the apoptotic process compared with that in nonsensitized cells. These results show that Fas aggregation and Fas-mediated apoptosis are not directly correlated and may even be inversely correlated.     

New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila.

Programmed chromosome breakage occurs in many ciliated protozoa and is accompanied by efficient new telomere formation. In this study, we have investigated the relationship between programmed chromosome breakage and telomere formation in Tetrahymena thermophila. Using specially constructed DNA clones containing the breakage signal Cbs in transformation studies, we have determined the locations of telomere addition around the breakage sites. They occur at variable positions, over 90% of which are within a small region (less than 30 bp) starting 4 bp from Cbs. This distribution is independent of the nucleotide sequence in the region or of the orientation of Cbs. In five of six cases determined, these sites occur at or before a T, and in the remaining case, the site occurs at or before a G. When sequences devoid of G or T are placed in this region, telomere addition still occurs within the region to maintain a similar distance relationship with Cbs. This efficient and healing process appears to be associated specifically with Cbs-directed breakage, since it does not occur when DNA ends are generated by restriction enzyme digestion. These results suggest a strong mechanistic link between chromosome breakage and telomere formation.     

Tempo does not correlate with mode in the fossil record

The dominating view of evolution based on the fossil record is that established species remain more or less unaltered during their existence. Substantial evolution is on the other hand routinely reported for contemporary populations, and most quantitative traits show high potential for evolution. These contrasting observations on long‐ and short‐time scales are often referred to as the paradox of stasis, which rests on the fundamental assumption that periods of morphological stasis in the fossil record represent minimal evolutionary change. Investigating 450 fossil time series, I demonstrate that the nonaccumulating morphological fluctuations during stasis travel similar distances in morphospace compared to lineages showing directional change. Hence, lineages showing stasis are commonly undergoing considerable amounts of evolution, but this evolution does not accumulate to produce large net evolutionary changes over time. Rates of evolutionary change across modes in the fossil record may be more homogenous than previously assumed and advocated, supporting the claim that substantial evolution is not exclusively or causally linked to the process of speciation. Instead of exemplifying minimal evolution, stasis likely represents information on the dynamics of the adaptive landscape on macroevolutionary time scales, including the persistence of adaptive zones and ecological niches over millions of years.     

Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exhibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function, we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.     

The extent of oxidative mitogenesis does not correlate with the degree of aldehyde formation of the T lymphocyte membrane

Chemical oxidation of T lymphocytes with periodate or the combined action of the enzymes neuraminidase and galactose oxidase (NAGO) results in T cell activation. The latter process includes the production of interleukin 2 (IL 2) and the induction of IL 2 receptors. Because membrane-bound aldehydes act in the transmission of the oxidative mitogenic signal, we designed a comparative study in human thymocytes and peripheral blood leukocytes in order to determine a possible correlation between the degree of the membrane aldehydes generated chemically or enzymatically and the extent of the resulting activation. The differences between periodate- and NAGO-induced aldehydes were demonstrated by flow cytometry of cells stained with a novel fluoresceinated hydrazide and by an electrophoretic procedure performed with biocytin hydrazide and 125I-streptavidin. In both cellular systems, periodate oxidation resulted in stronger formation of aldehydes than NAGO oxidation. However, the IL 2 receptor induced by NAGO formation and the resultant activation were significantly higher than those induced by periodate. The degree of aldehyde formation on peripheral blood leukocytes was also considerably higher than that of thymocytes, yet similar patterns of [3H]thymidine uptake were observed in the mitogenic assays of both cellular systems. The data indicate that no correlation exists between the extent of aldehyde formation and the degree of oxidative mitogenesis. It is thus suggested that relatively few (or maybe only one) membrane-bound aldehyde-containing molecules act in the transmission of the oxidative mitogenic signal.     

Infection with Wolbachia does not influence crossing-over in Drosophila melanogaster

The influence of endosymbiotic bacterium Wolbachia on the recombination processes in the sex chromosome of Drosophila melanogaster on the region between white and cut genes has been studied. The analysis of the crossing-over frequency in various variants of strain crossing, infected and noninfected, with bacteria has been carried out. The results have shown the absence of the influence of infection with Wolbachia on the frequency of crossing-over in the studied region of the X chromosome of D. melanogaster.     

Background level of 8-oxo-2'-deoxyguanosine in lymphocyte DNA does not correlate with the concentration of antioxidant vitamins in blood plasma.

Antioxidant vitamins, being effective free radical scavengers, can protect cellular DNA from oxidative damage. Therefore, in the present study we report on the relationship between basal level of 8-oxo-2'-deoxyguanosine in human lymphocyte DNA and the concentration of antioxidant vitamins (A, C and E). The average level of 8-oxo-2'-deoxyguanosine in lymphocytes of the studied group (15 males and 20 females) was 9.57 per 10(6) dG molecules. The endogenous level of ascorbic acid (vitamin C) in the plasma was, on average, 56.78 microM, while the mean concentrations of retinol (vitamin A) and alpha-tocopherol (vitamin E) were 1.24 uM and 25.74,uM, respectively. No correlations were found between individual 8-oxo-2 micro-deoxyguanosine levels in lymphocyte DNA and endogenous concentration of the vitamins.     

The Y chromosome and sperm length in D. melanogaster

Contrary to earlier reports, the sperm length of D. melanogaster males with two Y chromosomes is the same as in males with one Y chromosome.     

Rates of DNA sequence evolution are not sex-biased in Drosophila melanogaster and D. simulans

To determine whether male- or female-biased mutation rates have affected the molecular evolution of Drosophila melanogaster and D. simulans, we calculated the male-to-female ratio of germline cell divisions ([symbol: see text]) from germline generation data and the male-to-female ratio of mutation rate ([symbol: see text]) by comparing chromosomal levels of nucleotide divergence. We found that the ratio of germline cell divisions changes from indicating a weak female bias to indicating a weak male bias as the age of reproduction increases. The range of [symbol: see text] values that we observed, however, does not lead us to expect much, if any, difference in mutation rate between the sexes. Silent and intron nucleotide divergence were compared between nine loci on the X chromosome and nine loci on the second and third chromosomes. The average levels of nucleotide divergence were not significantly different across the chromosomes, although both silent and intron sites show a trend toward slightly more divergence on the X. These results indicate a lack of sex- or chromosome-biased molecular evolution in D. melanogaster and D. simulans.      

The TX; Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. I. Analysis of spontaneous events and results of male exposure

The translocation X; Y test is a selective system in Drosophila melanogaster designed to detect and distinguish among sex chromosome nondisjunction, chromosome breakage, and X-Y interchange. In the test, only exceptional progeny survive. This enables the investigator to score thousands of progeny with relative ease. The distribution of spontaneous events occurring in individual TX; Y males are analyzed in this paper. Evidence is obtained suggesting that the clusters of two products arising from a single nondisjunction can significantly affect the distribution of recovered chromosome gain or chromosome loss events. Non-parametric statistical methods are therefore recommended for the analysis of TX; Y data. In addition, use of the TX; Y test following exposures of pre-adult males to X-rays, heat shock, cold shock, colchicine, dimethyl sulfoxide (DMSO), and trifluralin are presented. Significant increases in nondisjunction (both gain and loss) were obtained following exposures to heat shock, cold shock, DMSO and trifluralin. Significant increases in chromosome breakage and X-Y interchange were obtained after exposures to X-rays and heat shock. These results indicate that the TX; Y test is an efficient method for detecting aneuploidy. Further work is needed, however, to fully validate this system for the routine screening of aneuploidy-inducing agents.     

Steroid hormone toxicity in human fibroblasts does not correlate with high affinity receptor content.

Human diploid skin fibroblasts derived from normal individuals and those with the testicular feminization syndrome (TFM) have been shown to be killed to the same degree by dihydrotestosterone in spite of the absence of high affinity cellular androgen receptors in the TFM fibroblasts. Furthermore, several different normal fibroblast strains from various anatomical sites all showed similar amounts of androgen-induced cytotoxicity even though their respective receptor contents differed by as much as ten-fold. These results suggest that steroid-induced cytotoxicity in human fibroblasts is not correlated with receptor content, unlike murine lymphoid cells in which the receptor content has been shown to be closely related to their ability to survive hormone exposure.     

Thermal stability of proteins does not correlate with the energy of intramolecular interactions

Small monomeric proteins from mesophilic and thermophilic organisms were studied. They have close structural and physical and chemical properties but vary in thermal stability. A thermodynamic analysis of heat unfolding was made and integral enthalpy of unfolding (DeltaH(unf)), heat capacity of hydration (DeltaC(p)(hyd)) and enthalpy of hydration (DeltaH(hyd)) and of the buried surface area (DeltaASA) of nonpolar and polar groups as well as the enthalpy of disruption of intramolecular interaction (DeltaH(int) in gas phase) at 298 K were determined. The absence of correlation between protein thermostability and energetic components suggests that regulatory mechanism of protein thermal stabilization has entropic nature.     

Diphosphate concentration does not correlate with the level of inorganic diphosphatase inEscherichia coli

Partial inhibition of DNA synthesis stimulates the production of inorganic diphosphatase inEscherichia coli but the changes in diphosphate (PP _i) level observed did not correlate with the enzyme activity. An accumulation ofPP _I was observed in the presence of inhibitors of RNA synthesis or nucleotide synthesis. In the former case the level of the enzyme did not change but in the latter case it increased. Thus the amount of inorganic diphosphatase alone does not determine the concentration ofPP ₁ inE. coli.     

Hyperthermia increases chromosome breakage and loss induced by fission neutrons in Drosophila melanogaster

Pretreatment with hyperthermia did significantly increase the breakage and chromosome loss induced by 0.85-MeV fission neutrons from the JANUS biological research reactor in spermatozoa, early and late spermatids and spermatocytes. Radiation-induced breakage of chromosomes was also enhanced by hyperthermia in early spermatogonia.     

Cellular level of 8-oxo-2'-deoxyguanosine in DNA does not correlate with urinary excretion of the modified base/nucleoside

We assessed a relationship between the level of 8-oxodG in leukocyte DNA measured with the high performance liquid chromatography with electrochemical detection (HPLC/EC) technique and urinary excretion of the modified nucleoside/base analysed with a recently developed methodology involving HPLC prepurification followed by gas chromatography with isotope dilution mass spectrometric detection. No correlation was found between these markers of oxidative DNA damage commonly used in epidemiological studies. Several possible explanations of this finding are discussed.     

Inviability of hybrids between D. melanogaster and D. simulans results from the absence of simulans X not the presence of simulans Y chromosome.

Interspecific crosses between D. melanogaster and D. simulans or its sibling species result in unisexual inviability of the hybrids. Mostly, crosses of D. melanogaster females x D. simulans males produce hybrid females. On the other hand, only hybrid males are viable in the reciprocal crosses. A classical question is the cause of the unisexual hybrid inviability on the chromosomal level. Is it due to the absence of a D. simulans X chromosome or is it due to the presence of a D. simulans Y chromosome? A lack of adequate chromosomal rearrangements available in D. simulans has made it difficult to answer this question. However, it has been assumed that the lethality results from the absence of the D. simulans X rather than the presence of the D. simulans Y. Recently I synthesized the first D. simulans compound-XY chromosome that consists of almost the entire X and Y chromosomes. Males carrying the compound-XY and no free Y chromosome are fertile. By utilizing the compound-XY chromosome, the viability of hybrids with various constitutions of cytoplasm and sex chromosomes has been examined. The results consistently demonstrate that the absence of a D. simulans X chromosome in hybrid genome, and not the presence of the Y chromosome, is a determinant of the hybrid inviability.