Dynamcis of the thymidine triphosphate pool during the cell cycle of synchronized 3T3 mouse fibroblasts

To investigate whether resting cells of 3T3 mouse fibroblasts carry out de novo synthesis of deoxyribonucleoside triphosphates, we determined the turnover of the thymidine triphosphate pool of G₀ cells obtained by starvation of cultures for platelet-derived growth factor. These cells were contaminated by less than 1% S-phase cells. In the absence of deoxyribonucleosides in the medium one million G₀ cells contained 5 pmole of dTTP with a turnover of 0.09 pmole/min. S-phase cells in comparison contained a 20 times larger dTTP pool with a more than 200-fold faster turnover. Our results suggest that G₀ cells carry out a slow but finite de novo synthesis of deoxyribonucleoside triphosphates to satisfy the cells' requirement for DNA repair and mitochondrial DNA synthesis.     

Frequency content asymmetry of the isokinetic curve between ACL deficient and healthy knee

The torque-time curve patterns of concentric isokinetic knee extension in anterior cruciate ligament (ACL) deficient patients usually present mid-range irregularities associated with the level of anterior tibial translation. The purpose of this study was to compare the smoothness in isokinetic torque production between the ACL deficient and the healthy knee. Thirty ACL deficient soccer players performed bilaterally five trials of maximum concentric knee extension-flexion at 60 degrees /s on a Biodex dynamometer. The three middle trials (a total of six curves) were retained and submitted to further data processing. Maximum frequency values contained within the 90%, 95% and 99% level of the signal power were calculated for each extension and flexion curve. The frequency content of the ACL deficient side proved to be statistically higher compared to the intact side at all levels of the power spectrum. The percentage differences in the frequency content were 18.8%, 10.6% and 40.0% for knee extension, and 49.5%, 24.5% and 16.3% for knee flexion, for the respective power levels. This indicated higher oscillations and, therefore, more unstable mechanical output of the injured knee. An overall biological interpretation of the present results is based on the notion that disturbed motion is generally connected to poor level of joint functionality.     

Genomic organization and identification of a novel alternative splicing variant of mouse mitochondrial thioredoxin reductase (TrxR2) gene

Eukaryotic mitochondria are equipped with a complete thioredoxin system, composed of thioredoxin and thioredoxin reductase, which has been implicated in the protection against the reactive oxygen intermdiates generated during the respiratory process in this organelle. Like its cytosolic counterpart, mammalian mitochondrial thioredoxin reductase is a homodimeric selenoprotein. We report here the genomic organization of the mouse mitochondrial thioredoxin gene (TrxR2) that spans 53 kb and consists of 18 exons ranging from 20 to 210 bp. All splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly at the same position as the human TrxR2 gene, the only mammalian mitochondrial thioredoxin reductase gene whose genomic structure has been elucidated to date. In addition, we have identified a novel mRNA splicing variant lacking intron 14 resulting in a protein subunit with a shorter interface domain. This new splicing variant provides a framework for further analysis of this important enzyme as its predicted homodimeric conformation can now be expanded to a putative heterodimeric structure as well as a small subunit homodimer with the obvious implications at the regulatory level.     

d-Galactose Uptake by Fenugreek Cotyledons : Effect of Water Stress

The uptake of d-galactose was studied in detached fenugreek (Trigonella foenum-graecum L.) cotyledons. Uptake kinetics and treatment with p-chloromercury-benzenesulfonic acid indicated that at low concentrations d-galactose was taken up by a carrier. At higher concentrations a diffusion-like component existed. Proton flux and pH studies, treatment with α-naphthaleneacetic acid, and uptake experiments under water stress conditions suggested that d-galactose was not taken up via H⁺ contransport. However, d-galactose uptake was under metabolic control. Uptake kinetics under water stress conditions suggested that moderate water stress either increased the K_m of the carrier or decreased the V_max. However, prolonged stress transformed the carrier-mediated uptake into a diffusion uptake transport. The uptake of d-galactose by fenugreek cotyledons was very low before and just after germination, was maximum after 35 hours imbibition, and started decreasing thereafter. The different uptake rates of d-galactose with imbibition times were attributed to the operation of the carrier. At low uptake rates the carrier did not operate. Treatment with cycloheximide suggested that the carrier was synthesized de novo just after germination and stopped operating when all galactomannan hydrolysis was over. Results were discussed in the context of control of endosperm galactomannan hydrolysis by the cotyledons of fenugreek embryo.     

Synthesis and biochemical investigation of scyphostatin analogues as inhibitors of neutral sphingomyelinase

The sphingolipid ceramide is considered to be an important intracellular mediator. However, many aspects of its action and the role of several different ceramide generating sphingomyelinases are still unclear. Recently, we reported on the synthesis of the first selective irreversible inhibitor of the neutral sphingomyelinase (N-SMase), as well as the identification of Manumycin A and some of its analogues as irreversible inhibitors of N-SMase. For the development of pharmacologically interesting competitive inhibitors of N-SMase, structure-activity studies are essential. Herein we show the synthesis and enzymatic investigation of two scyphostatin analogues 3a and 3b, revealing the importance of the primary hydroxy group in compound 2 for N-SMase inhibition.     

An intermittent mode of formation for the trace fossil Cruziana as a serial repetition of Rusophycus: the case of Cruziana tenella (Linnarsson )

Cruziana is one of the most recognizable trace fossils ascribed to arthropods. It ranges throughout the Phanerozoic and encompasses a diverse set of morphologies. The distinct features of Cruziana have incited fierce debate regarding its mode of formation. Here, we discuss critical aspects of trace fossil formation, namely epibenthic versus endobenthic origin and the ethology of the producer. Cruziana has largely been interpreted as a continuous ploughing trace fossil. It has been suggested, however, that at least some Cruziana could be structures resulting from the concatenation of Rusophycus‐type elements, although this claim remains unexplored. Cruziana tenella from the lower Cambrian of south‐central Sweden illustrates this intermittent mode of formation with a series of Rusophycus eutendorfensis leading into vertically undulating Cruziana that, at end stages of development, reflect a relatively equal depth distribution throughout the trace fossil and a great number of intergrading morphologies. In this study, a morphological evaluation of the intergrading morphologies of Cruziana tenella and Rusophycus eutendorfensis and a short morphometric analysis of the elements comprising Cruziana tenella suggests that at least in some cases Cruziana could be formed in intervals, as the serial overlap of distinct shallow Rusophycus could produce an apparently continuous cruzianaeform morphology. A comparison with possible evidence of intermittent formation on Cruziana semiplicata is made to illustrate the possibility of extending this mode of formation to larger Cruziana. An argument for the rise in the early Cambrian of a primitively intermittent mode of formation is made on the basis of energy efficiency.     

Detection of KRAS Exon 2 Mutations in Circulating Tumor Cells Isolated by the ISET System from Patients with RAS Wild Type Metastatic Colorectal Cancer

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n = 3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P = .950) or OS (P = .383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.