Calibration of Abbott AxSYM Ferritin kit using the WHO Human Liver Ferritin International Standard 80/602

A commercial ferritin kit (Abbott AxSYM Ferritin) was calibrated using the WHO Human Liver Ferritin International Standard 80/602. The reconstituted WHO freeze-dried standard was diluted to obtain six concentration levels ranging from 10-840 micrograms/l. In the analysis of the data, logarithmic transformation of the results was performed in order to stabilize the variance. The AxSYM kit yielded slightly higher values than the WHO Ferritin Standard (p < 0.05). The relation between the AxSYM kit and the WHO Ferritin Standard (untransformed values) was described by a proportionality: FerritinAxSYM = 1.057 x FerritinWHO. WHO Ferritin Standard values of 12 and 15 micrograms/l (used as cut-off values for absent or small body iron reserves) yielded calculated AxSYM values of 12.7 and 15.9 micrograms/l. A WHO Ferritin Standard value of 30 micrograms/l (used threshold value for the presence of stainable bone marrow haemosiderin iron) yielded a calculated AxSYM value of 31.7 micrograms/l.     

Ureteral obstructions and leaks after renal transplantation: outcome of percutaneous antegrade ureteral stent placement in 44 patients

Gene E-L, a chimeric lysis construct from bacteriophages phi X174 and MS2 lysis proteins E and L, respectively, was subjected to internal deletions to create a series of new E-L clones with altered lysis or killing properties. The lytic activities of the parental genes E. L. E-L and the internal truncated forms of E-L were investigated in this study to characterize the different lysis mechanisms, based on differences in the architecture of the different membrane spanning domains. Electron microscopy and release of marker enzymes for the cytoplasmic and periplasmic spaces revealed that two different lysis mechanisms can be distinguished depending on penetrating of the proteins either the inner membrane or the inner and outer membranes of Escherichia coli. Several candidates, which share efficient lysis properties, have biotechnological applications in terms of cell disruption.     

The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage-dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL-2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS-sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram-1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.     

Activation of c-myc gene expression by tumor-derived p53 mutants requires a discrete C-terminal domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3' end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

Static versus dynamic predictions of protective stepping following waist-pull perturbations in young and older adults

The purposes of this study were: (1) to determine the frequency of protective stepping for balance recovery in subjects of different ages and fall-status, and (2) to compare predicted stepping based on a dynamic model (Pai and Patton, 1997. Journal of Biomechanics 30, 347 354) involving displacement and velocity combinations of the center of mass (COM) versus a static model based on displacement alone against experimentally induced stepping. Responses to three different magnitudes of forward waist pulls were recorded for 13 young, 18 older-non-fallers and 18 older-fallers. The COM phase plane trajectories derived from motion analysis were compared with the model-predicted threshold values for stepping. We found that the older fallers had the highest percentage of stepping trials (52%), followed by older-non-fallers (17.3%), and young (2.7%) at the lowest perturbation level. Younger subjects stepped less often than the elderly at the middle level. Everyone consistently stepped at the highest level of perturbation. Overall, the dynamic model showed better predictive capacity (65%) than the static model (5%) for estimating the initiation of stepping. Furthermore, the threshold for step initiation derived from the dynamic model could consistently predict when a step must occur. However, it was limited, especially among older fallers at the low perturbation level, in that it considered some steps 'unnecessary' that were presumably triggered by fear of falling or other factors.     

Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.     

Antimicrobial drug residues in milk and meat: causes, concerns, prevalence, regulations, tests, and test performance

This paper presents a historical review of antimicrobial use in food animals, the causes of residues in meat and milk, the types of residues found, their regulation in Canada, tests used for their detection, and test performance parameters, with an emphasis on immunoassay techniques. The development of residue detection methods began shortly after the introduction of antimicrobials to food animal production in the late 1940s. From initial technical concerns expressed by the dairy industry to the present public health and international trade implications, there has been an ongoing need for reliable, sensitive, and economical methods for the detection of antimicrobial residues in food animal products such as milk and meat. Initially there were microbial growth inhibition tests, followed by more sensitive and specific methods based on receptor binding, immunochemical, and chromatographic principle. An understanding of basic test performance parameters and their implications is essential when choosing an analytical strategy for residue testing. While each test format has its own attributes, none test will meet all the required analytical needs. Therefore the use of a tiered or integrated system employing assays designated for screening and confirmation is necessary to ensure that foods containing violative residues are not introduced into the food chain.     

Acute endothelium-mediated vasodilation is not impaired at the onset of diabetes

Vascular injury and impaired vascular function are central to the increased mortality associated with diabetes. Hyperglycemia in diabetes has been suggested to play a role in this process, in part by impairing the function of the vascular endothelium. It has been difficult, however, to isolate the direct effect of glucose in both humans and in animal models of diabetes. This was evaluated in the present study in 7 rats that were chronically instrumented with a Transonic flow probe at the iliac bifurcation of the abdominal aorta, a nonoccluding catheter inserted immediately anterior to the flow probe, and a femoral vein catheter. Acute infusions of acetylcholine and sodium nitroprusside (1 and 10 microg/min IA) increased hindquarter blood flow significantly by approximately 27 and 10 mL/min over baseline, respectively, at the high dose. Streptozotocin (70 mg/kg IV) was administered, but normoglycemia was maintained with continuous intravenous insulin infusion to control for potential streptozotocin side effects. Diabetes was induced 5 to 7 days later by stopping the insulin infusion. Hindlimb blood flow (measured 24 hours per day) decreased during the diabetic period and was accompanied by an increase in mean arterial pressure, suggesting a vasoconstrictor response. However, the responses to acetylcholine and sodium nitroprusside were not altered significantly on either day 2 or day 6 of the diabetic period. This suggests that neither endothelium-mediated vasorelaxation nor responsiveness to nitric oxide is impaired during the initial phase of diabetes and that diabetic hyperglycemia does not have a significant, direct effect to impair endothelium-mediated relaxation in insulin-dependent diabetes mellitus. The mechanism for the change in baseline blood flow and its potential influence on endothelial function, however, are not known.     

Decreased cholesterol efflux from fibroblasts of a patient without Tangier disease, but with markedly reduced high density lipoprotein cholesterol levels

A 51-yr-old woman without clinical evidence of Tangier disease, but with an extremely low high density lipoprotein (HDL) cholesterol level, was studied. No defect in the major structural protein of HDL, apolipoprotein AI (apo AI), was detected. A preponderance of small HDL particles in the patient's plasma suggested defective uptake of cellular cholesterol. Efflux of [3H]cholesterol from patient fibroblasts to normal apo AI was decreased 50%. Cholesterol efflux to HDL was also decreased, but efflux to trypsin-modified HDL was not. The patient's cells partitioned more exogenously provided [3H]cholesterol into free cholesterol and synthesized greater amounts of phosphatidylcholine than did normal or Tangier fibroblasts. Her fibroblasts did not differ from normal fibroblasts in sterol synthesis rate, cellular cholesterol and cholesterol ester content, or incorporation of oleate into cholesterol ester. The data indicate the presence of a defect in apolipoprotein-dependent cellular cholesterol efflux that differs from that seen in Tangier disease. These findings are the first evidence that other low HDL cholesterol syndromes, besides Tangier disease, may also be associated with cholesterol efflux abnormalities. The identification of mutant genes responsible for apolipoprotein-mediated efflux abnormalities should provide valuable insights into cellular mechanisms involved in the reverse cholesterol transport pathway.     

Hospitalization for serious blood and skin disorders following use of co-trimoxazole

AIMS: The objective of this study was to quantify the risk of serious blood and skin disorders associated with co-trimoxazole. METHODS: We conducted a population-based cohort study of serious blood and skin disorders requiring hospitalization among otherwise healthy users of co-trimoxazole at Group Health Cooperative and Puget Sound (GHC). RESULTS: During the years 1987 to 1993 we found six cases of co-trimoxazole-associated blood disorders and three cases of co-trimoxazole-associated skin disorders yielding risks of 5.6/100,000 (95% CI 2.6-12.2) and 2.8/100,000 (95% CI 0.9-8.2) respectively. In all cases found there was prompt recovery after discontinuation of co-trimoxazole. We found no cases of toxic epidermal necrolysis. CONCLUSIONS: We conclude that the risk of blood and skin disorders associated with the use of co-trimoxazole leading to hospitalization is low.