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.     

Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway.     

BACE1-/- mice exhibit seizure activity that does not correlate with sodium channel level or axonal localization

Background

BACE1 is a key enzyme in the generation of the Aβ peptide that plays a central role in the pathogenesis of Alzheimer's disease. While BACE1 is an attractive therapeutic target, its normal physiological function remains largely unknown. Examination of BACE1^-/- mice can provide insight into this function and also help anticipate consequences of BACE1 inhibition. Here we report a seizure-susceptibility phenotype that we have identified and characterized in BACE1^-/- mice.

Results

We find that electroencephalographic recordings reveal epileptiform abnormalities in some BACE1^-/- mice, occasionally including generalized tonic-clonic and absence seizures. In addition, we find that kainic acid injection induces seizures of greater severity in BACE1^-/- mice relative to BACE1^+/+ littermates, and causes excitotoxic cell death in a subset of BACE1^-/- mice. This hyperexcitability phenotype is variable and appears to be manifest in approximately 30% of BACE1^-/- mice. Finally, examination of the expression and localization of the voltage-gated sodium channel α-subunit Na_v1.2 reveals no correlation with BACE1 genotype or any measure of seizure susceptibility.

Conclusions

Our data indicate that BACE1 deficiency predisposes mice to spontaneous and pharmacologically-induced seizure activity. This finding has implications for the development of safe therapeutic strategies for reducing Aβ levels in Alzheimer's disease. Further, we demonstrate that altered sodium channel expression and axonal localization are insufficient to account for the observed effect, warranting investigation of alternative mechanisms.     

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.     

Expression of caspase-3 and -7 does not correlate with the extent of apoptosis in primary breast carcinomas

Association between the rate of apoptosis and expression of the several relevant molecules (Bcl-2, pro- and active caspase-3, and caspase-7) was studied in 61 primary breast carcinomas. The rate of apoptosis detected both morphologically and by the TUNEL assay appeared to be high in 18 (30%), moderate in 14 (23%), and low in 29 (48%) carcinomas. High apoptotic index was strongly associated with advanced tumor grade and estrogen receptor positive (ER+) status but not with other investigated clinical or morphological parameters. Among the molecules studied, only the Bcl-2 protein expression demonstrated strong (inverse) correlation with the apoptotic index (p = 0.032). The data of this expected correlation was served as internal control in the study. Interestingly, high levels of the anti-apoptotic protein Bcl-2 was frequently co-incident with increased expression of pro-apoptotic molecules, such as active caspase-3 (p = 0.004) and caspase-7 (p = 0.001). However, expression of caspase-3 or caspase-7 did not show correlation with the extent of apoptosis or any clinico-morphological features, except overrepresentation of ER+ status in tumors expressing caspase-3 (p = 0.009). Thus, these findings indicate a general dysregulation of spontaneous apoptosis in primary breast tumors.     

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.     

Tissue factor expression and serum level in patients with melanoma does not correlate with disease progression.

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.     

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.     

Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance

Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.     

Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase.

Certain phosphocreatine preparations contain a contaminant that inhibits phosphofructokinase and pyruvate kinase assays. The contaminant can be separated from phosphocreatine by anion exchange chromatography. After appropriate purification, phosphocreatine has no effect on phosphofructokinase or pyruvate kinase; thus, there is no evidence that it serves muscle as a regulator of these enzymes. Although the inhibitory preparations of phosphocreatine contain inorganic phosphate and trace amounts of more negatively charged phosphorylated contaminants, the inhibitor is not inorganic phosphate or pyrophosphate. The nature of the inhibitor remains to be determined.     

Light level does not alter ethylene sensitivity in radish or pea

Ethylene accumulation occurs in many plant growth environments. In some instances, low photosynthetic photon flux (PPF) is also a stress factor. Ethylene helps regulate the shade-avoidance mechanism and synthesis rates can be altered by light. We thus hypothesized that ethylene sensitivity in whole plants may be altered in low light. Radish (Raphanus sativus) and pea (Pisum sativum) plants were selected as models due to their rapid growth, use in previous studies and difference in growth habit. We first characterized radish and pea sensitivity to ethylene. Radish vegetation was less sensitive to ethylene than pea vegetation. Pea reproductive yield was highly sensitive. Plants grown under low light levels are typically etiolated and less robust than plants grown under higher light. In a second series of studies we examined the interaction of ethylene (50 ppb pea, 200 ppb radish) with PPFs from 50 to 400 μmol m^?2 s^?1. There was no statistically significant interaction between ethylene sensitivity and PPF, indicating that high PPF does not mitigate the detrimental effects of chronic low-level ethylene exposure. This also suggests there is no crosstalk between the shade avoidance pathway and the primary ethylene signaling pathway.     

Characterization of a phosphoglycerate kinase deficiency variants not associated with hemolytic anemia.

The properties of a variant phosphoglycerate kinase (PGK) found in a large German clan were examined. The normal and variant enzymes, isolated by affinity chromatography, have the same molecular weight, specific activity, substrate affinity, and nearly identical pH-optima. Using immunoinactivation and immunodiffusion, the same specific activity for both forms was again determined. Since the enzymatic activity in older and younger erythrocytes varied only slightly, and since the specific activity of the variant was normal, the variant seems to be stable in vivo. This suggests that the decreased enzyme content is due to a decreased synthesis rate. The variant PGK described here is distinctly different from the known PGK variants and has been designated as "PGK München."     

Vanadate inhibits 2,3-bisphosphoglycerate dependent phosphoglycerate mutases but does not affect the 2,3-bisphosphoglycerate independent phosphoglycerate mutases

There are two types of phosphoglycerate mutases. The 2,3-bisphosphoglycerate dependent phosphoglycerate mutases are inhibited by vanadate. In contrast, the 2,3-bisphosphoglycerate independent mutases are not affected. The effect of vanadate varies with pH, and can be reversed by dilution, EDTA and norepinephrine. The differential effect of vanadate on the two types of phosphoglycerate mutases supplies a novel way to easily differentiate both types of enzymes. In addition, it may contribute to the clarification of the mechanism of action of the 2,3-bisphosphoglycerate independent phosphoglycerate mutases.     

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.     

Electrophoresis of phosphoglycerate kinase

A technique for the visualization of phosphoglycerate kinase on starch gel after electrophoresis is described. Three bands of activity were found in hemolysates prepared from normal red cells. When ATP, a substrate of the enzyme, was incorporated into the gel, only a single band was found. This suggested that ATP complexed with the enzyme and/or produced configurational changes. Incidentally, it was found that ATP markedly altered the electrophoretic mobility of hemoglobin. Red cells of 92 Caucasian males, 121 Caucasian females, 114 Negro males, 10 Negro females, 4 Oriental males, and 4 Oriental females were examined. No evidence of an electrophoretic polymorphism of this enzyme was found. Patterns of activity similar to those found in red cells were found in liver, heart, kidney, and skeletal muscle.This work was supported, in part, by Public Health Service Grant No. 07449 from the National Heart Institute, NIH. Presented, in part, at the annual meeting of the American Society of Human Genetics, Austin, Texas, October 12, 1968.     

Frequency of radiation-induced micronuclei in neuronal cells does not correlate with clonogenic survival

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 相似文献