Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

The Heat-Resistant Agglutinin Family Includes a Novel Adhesin from Enteroaggregative Escherichia coli Strain 60A

Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.     

Fasting and differential chemotherapy protection in patients

Chronic calorie restriction has been known for decades to prevent or retard cancer growth, but its weight-loss effect and the potential problems associated with combining it with chemotherapy have prevented its clinical application. Based on the discovery in model organisms that short term starvation (STS or fasting) causes a rapid switch of cells to a protected mode, we described a fasting-based intervention that causes remarkable changes in the levels of glucose, IGF-I and many other proteins and molecules and is capable of protecting mammalian cells and mice from various toxins, including chemotherapy. Because oncogenes prevent the cellular switch to this stress resistance mode, starvation for 48 hours or longer protects normal yeast and mammalian cells and mice but not cancer cells from chemotherapy, an effect we termed Differential Stress Resistance (DSR). In a recent article, ten patients who fasted in combination with chemotherapy, reported that fasting was not only feasible and safe but caused a reduction in a wide range of side effects accompanied by an apparently normal and possibly augmented chemotherapy efficacy. Together with the remarkable results observed in animals, these data provide preliminary evidence in support of the human application of this fundamental biogerontology finding, particularly for terminal patients receiving chemotherapy. Here we briefly discuss the basic, pre-clinical and clinical studies on fasting and cancer therapy.Key words: fasting, cancer, chemotherapy, calorie restriction, stress resistanceAfter decades of slow progress in the identification of treatments effective on a wide range of malignancies, cancer treatment is now turning to personalized therapies based in part on pharmacogenomics. By contrast, aging research is moving in the opposite direction by searching for common ways to prevent, postpone and treat a wide range of age-related diseases, based on the modulation of genetic pathways that are conserved from yeast to mammals.¹ In fact, it may be a solid evolutionary and comparative biology-foundation, which makes this ambitious goal of biogerontologists a realistic or at least a promising one. On the other hand, the progress of biogerontology is viewed by many clinicians as too fundamental and far from translational applications. In most cases, it is not clear how aging research will be translated into FDA approved drugs or treatments that have effects that are superior to those already available or being developed. For example, it is not clear how the long-term 20–30% reduction in calorie intake (dietary restriction, DR) that we and many others before us have shown to be effective in extending the life span of model organisms will make humans live longer or healthier.^1–3 Furthermore, despite the fact that long-term DR was confirmed to reduce cancer and cardiovascular disease in monkeys⁴ and to be effective in preventing obesity, type 2 diabetes, inflammation, hypertension and atherosclerosis, as indicated by the early results in humans studies,⁵ it is highly unlikely to be adopted in its more extreme and effective version by even a small portion of the population. For example, the 20 to 40% chronic reduction in daily calorie intake shown to be effective in retarding cancer growth in mice would not be feasible for cancer therapy for multiple reasons: (1) the effects of chronic DR in patients with a clinically evident tumor is expected to delay but not stop the progression of the disease^6–8 and this delay may only occur for a portion of the malignancies,⁹ (2) although weight loss and cachexia in the early stages of treatment are less prevalent than commonly thought,^10–12 the ∼15% loss of BMI and ∼30% long-term loss of body fat caused by a moderate (20%) calorie restriction¹³ may be tolerated by only a very small portion of cancer patients receiving treatment, (3) Because this long-term restriction is accompanied by delayed wound healing and immunologic impairment in rodents,^1,14,15 it is not clear what risks it may impose on cancer patients receiving treatment.¹⁶ Our studies of DSR, which were triggered by our fundamental findings that switching yeast cells to water protected them against a wide range of toxins, started as a way to address these concerns but also as an attempt to achieve a much more potent therapeutic effect than that achieved by DR.^17,18 Because starvation-induced protection can increase many fold when combined with modulation of pro-aging pathways and since it is in principle blocked by the expression of any oncogene, it has the potential to provide a method to allow common chemotherapy to selectively kill cancer cells, independently of the type of cancer.^19–21 The DSR experiments in mammals were also based on our hypothesis that stress resistance and aging regulatory pathways were conserved from yeast to mammals.We found that fasting for 48 or more hours or in vitro starvation conditions that mimic fasting protected mice and/or normal cells but not cancer cells from various chemotherapy drugs and other deleterious agents.²¹ This effect was shown to depend in part on the reduction of circulating IGF-I and glucose levels.^21,22 Although a differential regulation of cell division in normal and cancer cells^23,24 is likely to contribute to DSR, much of it appears to be dependent on protective systems which are normally maintained in an inactive or low activity state even in non-dividing cells.^1,25 In fact, in non-dividing yeast and mice, deficiencies in glucose or IGF-I signaling that match those observed after starvation promote resistance to doxorubicin, a chemotherapy drug that specifically targets muscle cells in the heart.^21,22As expected, many clinicians were skeptical of our hypothesis that cancer treatment could be improved not by a “magic bullet” but by a “not so magic DSR shield” as underlined by Leonard Saltz, an oncologist at Memorial Sloan-Kettering Cancer Center: “Would I be enthusiastic about enrolling my patients in a trial where they''re asked not to eat for 2.5 days? No.”²⁶ However, ten oncologists did allow their patients, suffering from malignancies ranging from stage II breast cancer to stage IV esophageal, prostate and lung malignancies to undergo a 48–140 hours pre-chemotherapy and a 5–56 hours post chemotherapy water-only fast. The six patients who received chemotherapy with or without fasting reported a reduction in fatigue, weakness and gastrointestinal side effects while fasting²⁷ (Fig. 1). A trend for a reduction of many additional side effects was also reported by the group of patients who always fasted before chemotherapy.²⁷ In those patients whose cancer progression was assessed, chemotherapy was effective and in some cases it was highly effective.²⁷ A clinical trial sponsored by the V-Foundation for Cancer Research, aimed at testing the safety and efficacy of a 24 hour fast in combination with chemotherapy, is in its safety stage. Because it was originally limited to patients diagnosed with bladder cancer the clinical trial progressed slowly. However, its recent expansion to include patients receiving platinum-based chemotherapy (breast, ovarian, lung cancer), is expected to expedite it. Conclusive results for the effect of a 3–4 day fast on chemotherapy-dependent side effects and possibly therapeutic index are not expected to become available for several years. Even if a more modest effect than the 1,000-fold differential protection against oxidative stress and chemotherapy observed in normal and cancer-like yeast cells was achieved in humans, this method could result in long-term survival for many patients with metastatic cancers, particularly those in which malignant cells have not acquired multidrug resistance.Open in a separate windowFigure 1Average self-reported severity of symptoms in patients that have received chemotherapy with or without fasting.     

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.     

Diaphragm adaptations in patients with COPD

Inspiratory muscle weakness in patients with COPD is of major clinical relevance. For instance, maximum inspiratory pressure generation is an independent determinant of survival in severe COPD. Traditionally, inspiratory muscle weakness has been ascribed to hyperinflation-induced diaphragm shortening. However, more recently, invasive evaluation of diaphragm contractile function, structure, and biochemistry demonstrated that cellular and molecular alterations occur, of which several can be considered pathologic of nature. Whereas the fiber type shift towards oxidative type I fibers in COPD diaphragm is regarded beneficial, rendering the overloaded diaphragm more resistant to fatigue, the reduction of diaphragm fiber force generation in vitro likely contributes to diaphragm weakness. The reduced diaphragm force generation at single fiber level is associated with loss of myosin content in these fibers. Moreover, the diaphragm in COPD is exposed to oxidative stress and sarcomeric injury. This review postulates that the oxidative stress and sarcomeric injury activate proteolytic machinery, leading to contractile protein wasting and, consequently, loss of force generating capacity of diaphragm fibers in patients with COPD. Interestingly, several of these presumed pathologic alterations are already present early in the course of the disease (GOLD I/II), although these patients appear not limited in their daily life activities. Treatment of diaphragm dysfunction in COPD is complex since its etiology is unclear, but recent findings indicate the ubiquitin-proteasome pathway as a prime target to attenuate diaphragm wasting in COPD.     

Heat-Resistant Agglutinin 1 Is an Accessory Enteroaggregative Escherichia coli Colonization Factor

Enteroaggregative Escherichia coli (EAEC) is an important cause of acute and persistent diarrhea. The defining stacked brick adherence pattern of Peruvian EAEC isolate 042 has previously been attributed to aggregative adherence fimbriae II (AAF/II), which confer aggregative adherence on laboratory E. coli strains. EAEC strains also show exceptional autoaggregation and biofilm formation, other phenotypes that have hitherto been ascribed to AAF/II. We report that EAEC 042 carries the heat-resistant agglutinin (hra1) gene, also known as hek, which encodes an outer membrane protein. Like AAF/II, the cloned EAEC 042 hra1 gene product is sufficient to confer autoaggregation, biofilm formation, and aggregative adherence on nonadherent and nonpathogenic laboratory E. coli strains. However, an 042 hra1 deletion mutant is not deficient in these phenotypes compared to the wild type. EAEC strain 042 produces a classic honeycomb or stacked brick pattern of adherence to epithelial cells. Unlike wild-type 042, the hra1 mutant typically does not form a tidy stacked brick pattern on HEp-2 cells in culture, which is definitive for EAEC. Moreover, the hra1 mutant is significantly impaired in the Caenorhabditis elegans slow kill colonization model. Our data suggest that the exceptional colonization of strain 042 is due to multiple factors and that Hra1 is an accessory EAEC colonization factor.Enteroaggregative Escherichia coli (EAEC) was originally identified as the etiologic agent of persistent diarrhea in developing countries but is gaining increasing prominence for its role in a wider spectrum of diarrheal syndromes. EAEC strains have been implicated in acute as well as persistent diarrhea among adults and children (reviewed in references 25 and 40). A recent meta-analysis found that EAEC is significantly associated with disease in every group at high risk for diarrhea, including young children, human immunodeficiency virus-positive individuals, and visitors to developing countries (24). In addition to its association with disease in epidemiological studies in developing countries, EAEC has also been identified as a principal cause of diarrheal disease in Germany, the United Kingdom, and the United States (11, 26, 51).Aggregative adherence is the defining characteristic of EAEC (38). EAEC strains adhere to the intestinal epithelium, and to epithelial cells in culture, in a characteristic two-dimensional “stacked brick” fashion. The pattern features bacteria adhering to the eukaryotic surface, other bacteria, and the solid substratum. Four types of fimbriae have so far been documented as conferring aggregative adherence (4, 14, 17, 37). Two noncontiguous plasmid loci containing the complete complement of genes encoding aggregative adherence fimbriae I (AAF/I) or AAF/II are sufficient to confer aggregative adherence on nonadherent E. coli (14, 49). The plasmid bearing type IV pili found in Serbian EAEC outbreak strain C1096 are also sufficient to confer a weak aggregative adherence phenotype on E. coli K-12 (17). AAF additionally play an essential role in production of a superfluous EAEC-associated biofilm, which could account for the association of these strains with persistent diarrhea in epidemiological studies (46).Some categories of diarrheagenic pathogens have a conserved set of adhesins which allow them to overcome flushing across the intestinal epithelium. Typical enteropathogenic E. coli isolates, for example, all possess bundle-forming pili and the outer membrane adhesin intimin, whereas atypical enteropathogenic E. coli isolates possess intimin but not bundle-forming pili (reviewed in reference 10). EAEC strains, by contrast, are considerably heterogeneous. While many EAEC strains carry genes encoding one of the known aggregative adherence fimbriae, some EAEC do not harbor any known AAF even though they do demonstrate aggregative adherence (4, 7, 13, 14). This, and the presence of multiple adhesins in most mucosal colonizers (53), points to the likelihood of other EAEC adhesins. Imuta et al. recently implicated a TolC secreted factor in adherence (27), and Montiero-Neto et al. (33) described a 58-kDa nonstructural adhesin in O111:H12 EAEC. However, the former factor is only a contributor to aggregative adherence and the latter adhesin is not found in other EAEC. Overall, nonstructural EAEC adhesins have received little attention.The outer membrane protein Tia was originally characterized as an invasin and later shown to confer adhesive properties on enterotoxigenic E. coli (ETEC) (20, 21). Fleckenstein et al. (21) observed that a tia gene probe hybridized to DNA from non-ETEC strains, one of which was EAEC strain 042. As the Southern blot data published by Fleckenstein et al. showed bands of different intensities, as well as size, between ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, which carries tia, and EAEC strain 042, we hypothesized that the probe was recognizing a similar, rather than identical, gene (21).We have determined that EAEC strain 042 harbors a gene encoding the heat-resistant agglutinin 1 (hra1), a hemagglutinin originally reported from an O9:H10:K99 porcine ETEC strain. Hra1 has also been reported from uropathogenic E. coli strains and neonatal meningitis E. coli strain RS218, in which context it is otherwise known as Hek (19, 48). (The hek nomenclature was introduced after hra1, to delineate the form of the gene found in invasive human pathogens from that of a porcine isolate [19].) A role for the outer membrane protein Hra1/Hek in adherence by neonatal meningitis E. coli has recently been defined (19).Although hra1/hek has been reported from multiple pathogens, its role in colonization and virulence has only been conclusively studied in the neonatal meningitis E. coli strain RS218 (19). In this paper, we demonstrate that the EAEC hra1 gene is sufficient to confer colonization-associated phenotypes, including aggregative adherence and biofilm formation, on laboratory E. coli strains. Intriguingly, we find that although it confers these phenotypes on K-12 and is expressed in 042, hra1 is not required for in vitro colonization-associated phenotypes demonstrated by 042. The hra1 gene is, however, essential for the formation of a true stacked brick pattern in EAEC and for optimal in vivo colonization in a Caenorhabditis elegans model.     

增强UV-B辐射对蚕豆叶片微粒体膜一些性质的影响

温室条件下,用0（Control）、8．65kJm－2d－1（TI）及11．2KJm－2d－1（t2）不同剂量的UV－B辐射处理蚕豆幼苗。Ca2 ．ATPase及Mg2 －ATPase的活性在辐射处理期间下降。在处理21d,T1和T2微粒体膜的MDA含量明显高于对照,同时IUFA急剧下降,且呈明显的剂量效应。14及21d时,膜磷脂的含量也明显下降。脂氧合酶（Lox）活性在第7及14天与对照相比都显著升高,而21d后迅速下降。结果表明,增强UV－B对微粒体膜的伤害可能是一方面导致正常酶合成与分解之间的平衡失调,另一方面导致了膜脂过氧化作用。