Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle Part IV. X-rays

Radiation-induction and rejoining of single-strand breaks (SSBs) in the DNA of synchronized HeLa S3 cells were investigated by alkaline sucrose density gradients. We could not find any significant differences in the extent of SSBs induced in cellular DNA and in the extent of their rejoining throughout the cell cycle, including mitosis. The cyclic variation curve of the content of non-protein sylfhydryls (NPSH) during the cell cycle is similar to that of X-ray survivals except in mitosis, although there was no close correlation between the content of apparent total sulfhydryls (APSH) and X-ray survivals.Radiation-induced mutants resistant to 8-azaguanine (8AG) occured in higher frequency in the radio-sensitive G₁S boundary phase thanin the radio-resistant G₁, S and early G₂ phases. Further, the pre-irradiation treatment with 50 mM cysteamine prevented reproductive death and induction of 8AG-resistant mutants by X-rays throughout the cell cycle. These findings seem to indicate that there is a close correlation between the extent of lethal radiation damage to the cells and their mutability, and that sulfhydryls may play an important role as a factor governing cellular radio-sensitivity.     

Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cδ, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCδ by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK in vascular cells and tissues.     

Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors

Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain. We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS antibody (5D4). However, it has not been clarified whether KS is expressed in brain tumors and is involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas (68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that KS-expression is significantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associated with the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.     

Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas sp. Strain RB2256

Sphingomonas sp. strain RB2256 was isolated from Resurrection Bay in Alaska and possibly represents the dominant bacterial species in some oligotrophic marine environments. Strain RB2256 has a high-affinity nutrient uptake system when growing under nutrient-limiting conditions, and growing cells are very small (<0.08 (mu)m(sup3)). These characteristics indicate that RB2256 is highly evolved for withstanding nutrient limitations and grazing pressure by heterotrophic nanoflagellates. In this study, strain RB2256 was subjected to nutrient starvation and other stresses (high temperature, ethanol, and hydrogen peroxide). It was found that growing cells were remarkably resistant, being able to survive at a temperature of 56(deg)C, in 25 mM hydrogen peroxide, or in 20% ethanol. In addition, growing cells were generally as resistant as starved cells. The fact that vegetative cells of this strain are inherently resistant to such high levels of stress-inducing agents indicates that they possess stress resistance mechanisms which are different from those of other nondifferentiating bacteria. Only minor changes in cell volume (0.03 to 0.07 (mu)m(sup3)) and maximum specific growth rate (0.13 to 0.16 h(sup-1)) were obtained for cells growing in media with different organic carbon concentrations (0.8 to 800 mg of C per liter). Furthermore, when glucose-limited, chemostat-grown cultures or multiple-nutrient-starved batch cultures were suddenly subjected to excess glucose, maximum growth rates were reached immediately. This immediate response to nutrient upshift suggests that the protein-synthesizing machinery is constitutively regulated. In total, these results are strong evidence that strain RB2256 possesses novel physiological and molecular strategies that allow it to predominant in natural seawater.     

Protective Capacity of L-Form Salmonella typhimurium against Murine Typhoid in C3H/HeJ Mice

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L-forming units. Although L-form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2–3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L-form whole-cell lysate (WCL), while delayed-type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T-cell responses nor DTH to heat-killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat-killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.     

Lipid rafts mediate chemotropic guidance of nerve growth cones

Axon guidance requires signal transduction of extracellular cues through the plasma membrane for directional motility. Here we present evidence that cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid rafts by various approaches targeting cholesterol or gangliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients, respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone in bath BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Raft manipulation also blocked Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse. Finally, guidance responses appeared to involve raft-dependent activation of p42/p44 MAPK and ligand-induced receptor recruitment to lipid rafts. Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance gradients, our findings indicate that localized signaling through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons.     

Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer

An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have determined the structure of the aptamer complexed with two argininamide molecules. Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat.     

Role of angiotensin II-mediated AMPK inactivation on obesity-related salt-sensitive hypertension

Salt-sensitive hypertension is a characteristic of the metabolic syndrome. Given the links to cardiovascular events, the mechanisms underlying sodium metabolism may represent an important therapeutic target for this disorder. Angiotensin II (AII) is a key peptide underlying sodium retention. However, 5'AMP-activated protein kinase (AMPK) has also been reported to participate in the regulation of ion transport. In this study we examined the relationship between AII and AMPK on the development of hypertension in two salt-sensitive mouse models. In the first model, the mice were maintained on a high-fat diet (HFD) for 12 weeks, in order to develop features similar to the metabolic syndrome, including salt-sensitive hypertension. HFD-induced obese mice showed elevated systolic blood pressure and lower sodium excretion in response to salt loading, along with an increase in AII contents and inactivation of AMPK in the kidney, which were significantly improved by the treatment of an angiotensin II antagonist, losartan, for 2 weeks. To clarify the effects of AII, a second group of mice was infused with AII via an osmotic pump, which led to higher systolic blood pressure, and decreases in urinary sodium excretion and the expression of AMPK, in a manner similar to those observed in the HFD mice. However, treatment with an AMPK activator, metformin, improved the changes induced by the AII, suggesting that AII induced sodium retention works by acting on AMPK activity. Finally, we evaluated the changes in salt-sensitivity by performing 2-week salt loading experiments with or without metformin. AII infusion elevated blood pressure by salt loading but metformin prevented it. These findings indicate that AII suppresses AMPK activity in the kidney, leading to sodium retention and enhanced salt-sensitivity, and that AMPK activation may represent a new therapeutic target for obesity-related salt-sensitive hypertension.