Involvement and identification of a lysine in the PPi-site of pyrophosphate-dependent phosphofructokinase from Giardia lamblia

There exists a great deal of overlap between many myelodysplastic syndromes and myeloproliferative disorders. This is most evident in the spectrum of disorders classified under the term chronic myeloid leukemia. These include chronic granulocytic leukemia, atypical chronic myeloid leukemia and chronic myelomonocytic leukemia. Current classification often does not clearly separate these entities since they share many features, both clinically and hematologically. We report here a case that satisfies criteria for both chronic myelomonocytic leukemia and atypical chronic myeloid leukemia, appearing to fluctuate between the two. This lends further evidence for the heterogeneity of these disorders and the need for better definition. An improved classification scheme would allow for more accurate reporting and research into etiology and treatment. The complex cytogenetic abnormalities of the case are unique and to our knowledge have not been reported previously. Also, this case report underscores the importance of cytochemical stains when such disorders are under consideration.     

Synthesis of deoxyglucose-1-phosphate, deoxyglucose-1,6-bisphosphate, and other metabolites of 2-deoxy-D-[14C]glucose in rat brain in vivo: influence of time and tissue glucose level

When the kinetics of interconversion of deoxy[14C]glucose ([14C]DG) and [14C]DG-6-phosphate ([14C]DG-6-P) in brain in vivo are estimated by direct chemical measurement of precursor and products in acid extracts of brain, the predicted rate of product formation exceeds the experimentally measured rate. This discrepancy is due, in part, to the fact that acid extraction regenerates [14C]DG from unidentified labeled metabolites in vitro. In the present study, we have attempted to identify the 14C-labeled compounds in ethanol extracts of brains of rats given [14C]DG. Six 14C-labeled metabolites, in addition to [14C]DG-6-P, were detected and separated. The major acid-labile derivatives, DG-1-phosphate (DG-1-P) and DG-1,6-bisphosphate (DG-1,6-P2), comprised approximately 5 and approximately 10-15%, respectively, of the total 14C in the brain 45 min after a pulse or square-wave infusion of [14C]DG, and their levels were influenced by tissue glucose concentration. Both of these acid-labile compounds could be synthesized from DG-6-P by phosphoglucomutase in vitro. DG-6-P, DG-1-P, DG-1,6-P2, and ethanol-insoluble compounds were rapidly labeled after a pulse of [14C]DG, whereas there was a 10-30-min lag before there was significant labeling of minor labeled derivatives. During the time when there was net loss of [14C]DG-6-P from the brain (i.e., between 60 and 180 min after the pulse), there was also further metabolism of [14C]DG-6-P into other ethanol-soluble and ethanol-insoluble 14C-labeled compounds. These results demonstrate that DG is more extensively metabolized in rat brain than commonly recognized and that hydrolysis of [14C]DG-1-P can explain the overestimation of the [14C]DG content and underestimation of the metabolite pools of acid extracts of brain. Further metabolism of DG does not interfere with the autoradiographic DG method.     

In-situ emergency paediatric surgery in the intensive care unit

The role of surgery in the intensive care unit (ICU) remains unclear. Although previously shown not to increase morbidity for patent ductus arteriosus ligation, Broviac catheter insertion, and recently, general neonatal and paediatric surgery, there remains a reluctance to operate on sick patients in the ICU (in-situ surgery, ISS). A retrospective study of 25 critically ill children and neonates who underwent ISS was performed. Surgery was aided by operating loupes and a high-intensity headlight. ISS was not associated with any morbidity, and although a 36% mortality occurred in this small series, in no case was this due to ISS. ISS avoids the risks of transfer to the operating theatre and the potential delays in theatre access. Our results suggest that ISS in a tertiary-level paediatric surgical hospital is safe and does not impact adversely on clinical outcome.     

Whimpers and bangs: of death and dying in the nervous system

Mechanisms of cell death in general and in the nervous system in particular have received increasing attention over the past several years. Recent studies have begun to address the pathogenetic and therapeutic implications of these mechanisms vis-a-vis human disease. This review is aimed at putting the rapidly evolving vocabulary and body of knowledge regarding cell death in the perspective of neurologic disease of childhood.     

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.     

Cardiovascular evaluation of the athlete. Issues regarding performance, screening and sudden cardiac death

Recent studies have reported ECG anomalies and a high prevalence of exercise-related arrhythmias among well trained, apparently healthy endurance athletes with superior levels of cardiorespiratory fitness. The occurrence of sudden and premature cardiac deaths in amateur and professional athletes, who appear to embody all of the virtues of health and fitness, ahs raised our consciousness regarding the underlying atherosclerotic or nonatherosclerotic causes, and the need for, and extent of, preparticipation screening in competitive athletes. It appears that strenuous physical activity may trigger acute cardiovascular events in some athletes. Coronary artery disease is the most frequent autopsy finding in those over the age of 35 years who die suddenly. In contrast, structural cardiovascular abnormalities, including hypertrophic cardiomyopathy and malformations of the coronary arteries, are the major cause of sudden death in younger athletes. This article reviews these issues, with specific reference to the assessment of cardiorespiratory fitness, legal and prohibited performance-altering medications, the pathophysiological basis of exertion-related untoward events, the athlete at risk, limitations of conventional screening programmes and contemporary recommendations to identify latent cardiovascular disease in athletic populations.     

Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension

Anorexigens such as aminorex fumarate and dexfenfluramine are associated with the development of severe pulmonary hypertension (PH), which clinically and histopathologically is considered indistinguishable from idiopathic or primary pulmonary hypertension (PPH). For the current study, we asked whether anorexigen-associated PH is characterized by monoclonal pulmonary endothelial cell proliferation (such as in PPH) or, alternatively, is associated with a polyclonal endothelial cell proliferation as found in secondary PH. Analysis of clonality by the human androgen receptor assay was performed in microdissected endothelial cells of plexiform lesions of two patients with anorexigen-associated PH. The four plexiform lesions of Patient 1 and the six of Patient 2 with anorexigen-associated PH exhibited a monoclonal expansion of pulmonary endothelial cells, with a mean clonality ratio of 0.03 +/- 0.01 SE. Our results indicate that appetite suppressant-associated PH is identical to PPH not only in clinical and histopathologic features but also, at a molecular level, in terms of the monoclonal nature of the endothelial cell proliferation. The anorexigens may accelerate the growth of pulmonary endothelial cells in patients with predisposition to develop PPH.     

Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.