Chromosome spreading techniques for primary gastrointestinal tumors

The requirement for well spread out chromosomes for the cytogenetic analysis of primary gastrointestinal tumors led us to develop new techniques. These techniques involved two main procedures: (1) preliminary incubation with culture medium in the presence of collagenase, Dispase, and colcemid, for 3h, and (2) treatment with an extremely hypotonic solution (0.044 M KCl) for 30 min. The techniques were applied to 11 gastrointestinal malignancies (including 1 early gastric cancer and 1 metastatic liver lesion of colon cancer) and significant increases (P<0.01) in the number of metaphases of analyzable karyotypes were obtained, compared with a previous method in which the standard hypotonic molarity of KCL (0.075 M) was employed. The mean value for metaphase numbers of the analyzable karyotypes was 37.0±3.7% in the 5 gastric cancers and 44.7±4.8% in the 5 colon cancers and 1 metastatic lesion. These values were three times and more than twice, respectively, the values obtained by the previous method. A fluorescence in situ hybridization (FISH) study was carried out on one cologenic tumor, the α-satellite centromere-specific probe 17 being used. Deletion of the long arm of chromosome 17 was demonstrated. The method proposed here could yield a sufficient number of metaphases without the use of tissue culture that might cause alteration of karyotype. It can be employed with small biopsy specimens and in studies utilizing the FISH technique.     

Reference values for urinary steroids in Japanese newborn infants: gas chromatography/mass spectrometry in selected ion monitoring

Urinary steroid profile analysis using gas chromatography/mass spectrometry (GC/MS) has been reported for the diagnosis of abnormal steroidogenesis in newborn infants with some success. We tried to establish the reference values of 63 urinary steroids in Japanese newborn infant, using GC/MS in selected ion monitoring (SIM) that utilizes two characteristic mass ions for each steroid for definitive identification. We studied 36 healthy full-term newborn infants (1-56 days of age) on spot urine samples to define the reference values (mg/g creatinine, median and 10-90 percentile range) and to investigate the possible difference between daytime and nighttime levels. We also studied 23 healthy adult females (20-24 years of age) on 24-hour-urine for the comparison of the reference values of newborn infants. Fifty metabolites of DHEA, pregnenolone, 17-hydroxypregnenolone, androstenedione, progesterone, 17-hydroxyprogesterone, 21-deoxycortisone, corticosterone, 18-hydroxycorticosterone, aldosterone, 18-hydroxycortisol, 11-deoxycortisol, cortisone, cortisol, and estrogen in each infant were measurable without interference, but 13 metabolites of 11-hydroxyandrostenedione, pregnenolone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 21-deoxycortisol, 11-deoxycortisol and cortisol were unmeasurable in each infant due to the interference of fetal cortex steroids as confirmed by abnormal peak area ratios of two mass ions. All 63 metabolites in each control adult were measurable without interference. 16alpha-, 16beta-, and 15beta-hydroxy metabolites of 3beta-hydroxy-5-en-steroids, and 6beta-, 18-hydroxy and 11-oxo-metabolites of corticosteroids were significantly higher in full-term newborn infants than those in adults as previously reported. Urinary steroids showed little circadian variation in the newborn infants, indicating that spot urine can substitute for 24-hour urine.     

Physical activity attenuates the effect of increased left ventricular mass on the risk of ischemic stroke: The Northern Manhattan Stroke Study

OBJECTIVES: The goal of this study was to determine whether the risk of ischemic stroke associated with increased left ventricular mass (LVM) is modified by physical activity (PA). BACKGROUND: Increased LVM is associated with an increased risk for stroke. Physical activity can decrease the risk of stroke and may have variable effects on LVM. METHODS: We used a case-control study design in a multiethnic population in northern Manhattan, New York, to study 394 case subjects who had a first ischemic stroke and 413 stroke-free control subjects. All subjects were interviewed and two-dimensional echocardiograms obtained to determine LVM. RESULTS: A sharp increase in risk of ischemic stroke was seen in the highest quartile of LVM (odds ratio [OR]: 6.14 [95% confidence interval [CI]: 3.04 to 12.38]). Thus, increased LVM was defined by the highest quartile of LVM. In multivariate analysis, the effect of increased LVM on the risk of stroke was significantly decreased by the presence of any level of PA versus no PA (OR: 1.59 [95% CI: 0.99 to 2.57] p < 0.07 vs. 3.53 [95% CI: 1.94 to 6.42] p < 0.0001). Although PA decreased the risk of stroke in all patients, the effect was stronger in subjects with increased LVM than among those without increased LVM (p = 0.033). CONCLUSIONS: Increased LVM is associated with an increased risk of stroke, especially among sedentary patients. Physical activity decreases the risk of stroke among patients with increased LVM to a level comparable to that of patients without increased LVM. Recommending PA may be a nonpharmacologic tool to reduce the stroke risk, especially among patients with increased LVM.     

Clinical impact of complete atrioventricular block in patients with ST‐segment elevation myocardial infarction

Complete atrioventricular block (CAVB) is a common complication of ST‐segment elevation myocardial infarction (STEMI). Although STEMI patients complicated with CAVB had a higher mortality in the thrombolytic era, little is known about the impact of CAVB on STEMI patients who underwent primary percutaneous coronary intervention (PCI). The study aimed at evaluating the clinical impact of CAVB on STEMI patients in the primary PCI era. We consecutively enrolled 1295 STEMI patients undergoing primary PCI within 24 hours from onset. Patients were divided into two groups according to the infarct location: anterior STEMI (n = 640) and nonanterior STEMI (n = 655). The outcomes were all‐cause death and major adverse cardiocerebrovascular events (MACCE) with a median follow‐up period of 3.8 (1.7–6.6) years. Eighty‐one patients (6.3%) developed CAVB. The incidence of CAVB was lower in anterior STEMI patients than in nonanterior STEMI (1.7% vs 10.7%, p < .05). Anterior STEMI patients with CAVB had a higher incidence of all‐cause deaths (82% vs 20%, p < .05) and MACCE (82% vs 25%, p < .05) than those without CAVB. Although higher incidence of all‐cause deaths was found more in nonanterior STEMI patients with CAVB compared with those without CAVB (30% vs 18%, p < .05), there was no significant difference in the incidence of MACCE (24% vs 19%). Multivariate analysis showed that CAVB was an independent predictor for all‐cause mortality and MACCE in anterior STEMI patients, but not in nonanterior STEMI. CAVB is rare in anterior STEMI patients, but remains a poor prognostic complication even in the primary PCI era.     

NLRP3 Inflammasome Inhibitor OLT1177 Suppresses Onset of Inflammation in Mice with Dextran Sulfate Sodium-Induced Colitis

Digestive Diseases and Sciences - NLRP3 inflammasomes have been reported to have a key role in the initiation and perpetuation of inflammatory bowel diseases (IBD). Here we investigated the effects...     

Evolution of bacterial steroid biosynthesis and its impact on eukaryogenesis

Steroids are components of the eukaryotic cellular membrane and have indispensable roles in the process of eukaryotic endocytosis by regulating membrane fluidity and permeability. In particular, steroids may have been a structural prerequisite for the acquisition of mitochondria via endocytosis during eukaryogenesis. While eukaryotes are inferred to have evolved from an archaeal lineage, there is little similarity between the eukaryotic and archaeal cellular membranes. As such, the evolution of eukaryotic cellular membranes has limited our understanding of eukaryogenesis. Despite evolving from archaea, the eukaryotic cellular membrane is essentially a fatty acid bacterial-type membrane, which implies a substantial bacterial contribution to the evolution of the eukaryotic cellular membrane. Here, we address the evolution of steroid biosynthesis in eukaryotes by combining ancestral sequence reconstruction and comprehensive phylogenetic analyses of steroid biosynthesis genes. Contrary to the traditional assumption that eukaryotic steroid biosynthesis evolved within eukaryotes, most steroid biosynthesis genes are inferred to be derived from bacteria. In particular, aerobic deltaproteobacteria (myxobacteria) seem to have mediated the transfer of key genes for steroid biosynthesis to eukaryotes. Analyses of resurrected steroid biosynthesis enzymes suggest that the steroid biosynthesis pathway in early eukaryotes may have been similar to the pathway seen in modern plants and algae. These resurrected proteins also experimentally demonstrate that molecular oxygen was required to establish the modern eukaryotic cellular membrane during eukaryogenesis. Our study provides unique insight into relationships between early eukaryotes and other bacteria in addition to the well-known endosymbiosis with alphaproteobacteria.

The emergence of modern eukaryotic cells from their archaeal ancestor requires multiple evolutionary steps, most notably the acquisition of mitochondria, nucleus, endomembrane system, and bacterial-like cellular membranes. The acquisition of mitochondria by early eukaryotes before the last eukaryotic common ancestor (LECA) would have been a key step to exploit new resources within an aerobic environment (1). Similarly, the transformation of the eukaryotic cellular membrane system from an archaeal-type to bacterial-type membrane would have also been crucial to develop the dynamic cellular membrane system observed in modern eukaryotes (2). Such a dynamic membrane may have been the prerequisite for a pre-LECA cell to perform endocytosis and, hence, later acquire organelles such as mitochondria and chloroplasts. While the tetraether monolayer membrane in archaea is more rigid and is advantageous to harsh environments, such as high temperature and low pH conditions, the diester bilayer membrane found in bacteria and eukaryotes has been suggested to be adaptable to more general environments (3). Recent phylogenomic analyses of archaea and eukaryotes suggest that eukaryotes evolved from within the archaeal domain (4, 5). However, there is little similarity between the archaeal and the bacterial/eukaryotic cellular membranes. Both the stereochemistry of lipid molecules (i.e., phospholipids) and their lipid composition (e.g., fatty acid chain versus isoprenoid chain and the presence of sphingolipids and steroids) are fundamentally different between the two types of membranes. As such, the evolutionary history of the eukaryotic cellular membrane is a major gap in our understanding of eukaryogenesis (6).Here, we focus on the evolution of the steroid component of the eukaryotic cellular membrane. Steroids have indispensable roles in the process of eukaryotic endocytosis because they are embedded in the cellular membrane and are known to regulate membrane fluidity and permeability (7–9). Thus, steroids may also have been integral for the endocytic process to acquire mitochondria during eukaryogenesis. Steroids would also function as hormones to control various cellular processes, including differentiation, morphogenesis, and homeostasis to enable multicellularity (10). In addition to the vital biochemical role in modern eukaryotes, steroids also have a geobiological importance. They can serve as unique biological markers (biomarkers) for eukaryotes in the geological record and thus provide clues to trace the evolutionary history of eukaryotes on a geological time scale. Membrane-bound steroids are modified in a unique manner within each eukaryotic taxon. For instance, cholesterol is a major component of cellular membrane in metazoans, whereas stigmasterol is a major component in plants (11, 12). These taxon-specific modifications can be used as taxonomic markers in the geological record (13–15). No analogous steroid biosynthesis pathway has been observed in archaea and thus steroid biosynthesis is generally inferred to have evolved de novo within eukaryotes (16). Despite this inference, some bacteria are known to produce hopanoids that are structurally similar to steroids (17). Indeed, several genes that are involved in hopanoid and steroid biosynthesis are suggested to have been horizontally transferred from bacteria to eukaryotes (18, 19). These recent observations suggest a more complex evolutionary history of steroid biosynthesis in eukaryotes. Ancestral sequence reconstruction (ASR) enables us to experimentally analyze resurrected enzymes (20, 21) and thus infer evolutionary histories of steroid biosynthesis and their impacts on eukaryogenesis.     

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.