Production and characterization of recombinant heteropolymers of human ferritin H and L chains

Vertebrate ferritins are iron storage proteins composed by 24 subunits of one or more types. The recombinant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers with various proportions of H- and L-type chains cannot be predicted. Present study shows that unfolded human ferritin H- and L- type chains renature under similar conditions to form homopolymers indistinguishable from the native ones and that, when mixed, the unfolded H and L chains renature to form heteropolymers with restricted heterogeneity and with the expected H:L ratios. Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunological reactivity, and stability to guanidine denaturation varied as predicted, based on the homopolymers. In contrast, the rate of iron uptake, monitored by the variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; further increments in H chains had no additional effect. This finding indicates that, under the present conditions, only a limited number of H chains are needed for the maximum rate of ferritin iron uptake. Variations of L- and H-type chains in vivo may thus have biological relevance.     

Serum ferritin as a component of the insulin resistance syndrome

Six human cancer cell lines exhibiting a large range of sensitivity to 5-fluorouracil (5-FU) were evaluated for thymidylate synthase (TS) and p53 gene expression, TS and dihydropyrimidine dehydrogenase (DPD) activity, as well as cell cycle parameters, S-phase fraction (SPF), bromodeoxyuridine labelling index (LI) and S-phase duration (SPD). All these parameters were investigated for 7 days in asynchronously growing cell populations and compared with the cell sensitivity to 5-FU. No significant correlation was found between S-phase parameters and TS gene expression and/or activity. TS activity was higher in proliferating cells; however, it was not significantly higher in rapidly growing cell lines with short SPD. Neither TS gene expression nor activity was found to correlate with 5-FU sensitivity. On the another hand, a statistically significant correlation (P < 0.0001) was observed between LI and SPD and 5-FU sensitivity. The present results suggest that cell cycle parameters such as SPD and/or LI could be better parameters for 5-FU sensitivity prediction than TS gene expression and/or activity. This could be especially informative in cases of concomitant radio-chemotherapy as S-phase parameters are already proposed for hyperfractionated radiotherapy planning.     

How to evaluate study methodology in published clinical research

Postmortem, magnetic resonance, and event-related potential studies suggest the presence of temporal lobe abnormalities in schizophrenia. Analyses using convergent measurements of brain structure and function, however, have rarely been done in the same patients. We recently developed a protocol using high-spatial-resolution magnetic resonance scans, auditory P300 event-related potentials, and thought disorder scales to examine temporal lobe structure and function in the same patients. We report a case of schizophrenia that showed left-lateralized volume reduction in the superior temporal gyrus, hippocampus, and parahippocampal gyrus (also on right), with associated P300 amplitude reduction and thought disorder marked by word-finding difficulties and perseverations.     

Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer

BACKGROUND & AIMS: Germline mutations in four DNA mismatch repair genes are known to cause susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). The rapidly increasing information about these mutations needs to be collected and appropriately stored to facilitate further studies on the biological and clinical significance of the findings. METHODS: The International Collaborative Group on HNPCC has established a database of DNA mismatch repair gene mutations and polymorphisms. In this report, 126 predisposing mutations were analyzed. RESULTS: A majority of the mutations affected either the Mut L homologue (MLH) 1 (n = 75) or the Mut S homologue (MSH) 2 (n = 48) and were quite evenly distributed, with some clustering in MSH2 exon 12 and MLH1 exon 16. Most MSH2 mutations consisted of frameshift (60%) or nonsense changes (23%), whereas MLH1 was mainly affected by frameshift (40%) or missense alterations (31%). Although most mutations were unique, a few common recurring mutations were identified. Of the families studied (n = 202), 82% met the Amsterdam criteria and 15% did not; the general mutation profile was similar in both groups. CONCLUSIONS: The construction of mutation profiles will facilitate the development of diagnostic strategies in HNPCC.     

Reproducibility of 1H-NMR integrals: a collaborative study

Respiratory distress syndrome (RDS) is characterized by intrapulmonary fibrin deposition, which can adversely affect surfactant function, and stimulate fibroblast proliferation, which may contribute to the development of bronchopulmonary dysplasia (BPD). We speculated that the premature lung may have impaired regulation of thrombin, thus making preterm infants susceptible to fibrin formation within the lung. Therefore, we studied the effect of stretch, which simulates fetal breathing movements (FBMs), on the generation and inhibition of a key hemostatic enzyme-thrombin-by rat fetal mixed lung cells (FMLCs). Our results showed that stretch induced glycosaminoglycan production with increased antithrombin activity due to an increase in the concentration of active chondroitin sulfate. Stretch downregulated secretion of tissue factor procoagulant activity, which may lead to decreased thrombin generation on the surface of FMLCs. Overall, stretch enhanced the local control of thrombin by FMLCs. These results suggest that premature infants, who will have experienced less FBM, may have impaired thrombin regulation. Impaired thrombin regulation likely contributes to increased fibrin deposition and, potentially, the development of BPD.     

Performance of panel-based criteria to evaluate the appropriateness of colonoscopy: a prospective study

One in every three people on Earth is believed to be infected with Mycobacterium tuberculosis, leading to seven to eight million cases of active tuberculosis (TB) per year and approximately three million deaths annually. this epidemic, like those of most infectious diseases, creates scientific challenges and opportunities as it raises the demand for public health solutions. The currently available weapons for fighting TB are inadequate. The ultimate goal of biomedical TB research is to lessen the public health burden of this disease by developing improved diagnostic, therapeutic, and intervention strategies. Achieving this goal requires a base of knowledge about the biology of M. tuberculosis and related mycobacteria, their interactions with human and animal hosts, and the nature of an effective host-protective immune response. TB researchers are applying this accumulating base of knowledge to developing rapid, easy-to-use diagnostic assays appropriate for low-as well as high-income countries, improving the current complicated therapeutic regimen, identifying potential new drugs to combat multidrug-resistant TB, and creating more effective vaccines.     

The path to a clinical pathway: collaborative care for the patient with an ostomy

A colostomy and ileostomy clinical pathway was developed at a southeastern teaching hospital in 1990 in response to excessive lengths of stay and costs at our hospital compared with national data for this patient group. A multidisciplinary clinical pathway team was formed and charged with the development, implementation, and ongoing monitoring of the clinical pathway tool and its effect on the outcomes of the population of patients with colostomies and ileostomies. Through this multidisciplinary collaboration, length of stay and cost have been reduced while quality care indicators have been maintained. This article presents the sample pathway we developed and describes the pathway development process, documentation, the variance analysis process, and the outcomes achieved with implementation. A urostomy/urinary diversion pathway that was developed after variance analysis review of the colostomy and ileostomy clinical pathway is also presented.     

Purification of a RNA-binding protein from rat liver. Identification as ferritin L chain and determination of the RNA/protein binding characteristics

In cultured rat hepatocytes the degradation of phosphoenolpyruvate carboxykinase mRNA might be regulated by protein(s), which by binding to the mRNA alter its stability. The 3'-untranslated region of phosphoenolpyruvate carboxykinase mRNA as a potential target was used to select RNA-binding protein(s) from rat liver by the use of gel retardation assays. A cytosolic protein was isolated, which bound to the phosphoenolpyruvate carboxykinase mRNA 3'-untranslated region and other in vitro synthesized RNAs. The protein was purified to homogeneity; it had an apparent molecular mass of 400 kDa and consisted of identical subunits with an apparent size of 24.5 kDa. Sequence analysis of a tryptic peptide from the 24.5-kDa protein revealed its identity with rat ferritin light chain. Binding of ferritin to RNA was abolished after phosphorylation with cAMP-dependent protein kinase and was augmented after dephosphorylation with alkaline phosphatase. Weak binding was observed in extracts from okadaic acid-treated cultured hepatocytes compared with untreated cells. Preincubation of ferritin with an anti-phosphoserine or an anti-phosphothreonine antibody attenuated binding to RNA, while an anti-phosphotyrosine antibody generated a supershift indicating that phosphoserine and phosphothreonine but not phosphotyrosine residues were in close proximity to the RNA-binding region. Ferritin is the iron storage protein in the liver. Binding of ferritin to RNA was diminished in the presence of increasing iron concentrations, whereas the iron chelator desferal was without effect. It is concluded that ferritin might function as RNA-binding protein and that it may have important functions in the general regulation of cellular RNA metabolism.     

Glassy Carbon as an Absolute Intensity Calibration Standard for Small-Angle Scattering

Absolute calibration of small-angle scattering (SAS) intensity data (measured in terms of the differential scattering cross section per unit sample volume per unit solid angle) is essential for many important aspects of quantitative SAS analysis, such as obtaining the number density, volume fraction, and specific surface area of the scatterers. It also enables scattering data from different instruments (light, X-ray, or neutron scattering) to be combined, and it can even be useful to detect the existence of artifacts in the experimental data. Different primary or secondary calibration methods are available. In the latter case, absolute intensity calibration requires a stable artifact with the necessary scattering profile. Glassy carbon has sometimes been selected as this intensity calibration standard. Here we review the spatial homogeneity and temporal stability of one type of commercially available glassy carbon that is being used as an intensity calibration standard at a number of SAS facilities. We demonstrate that glassy carbon is sufficiently homogeneous and stable during routine use to be relied upon as a suitable standard for absolute intensity calibration of SAS data.     

Evaluation of a recombinant 27-kDa outer membrane protein of Coxiella burnetii as an immunodiagnostic reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.     

Impact of a collaborative immunisation programme in an inner city practice

OBJECTIVES: To describe the impact of a collaborative immunisation programme, between an inner city practice and the Eastern Health Board (EHB). DESIGN: An observational study using a computer database formed from practice and EHB records. SETTING: One Dublin inner city practice with three partners located in an area with a deprived socio-economic profile. SUBJECTS: All patients in the practice aged more than six months and less than five years identified both from practice registers and opportunistically during study period. RESULTS: 342 children, older than six months and less than five years were identified at start and 464 (a 36% increase) by end of the programme. Uptake changed for DPT from 30% before, to 57% after the programme (p < 0.0005), for DT from 15% to 13%, for Hib from 7% to 50% (p < 0.0005) and for MMR (over 15 months) from 53% to 75% (p < 0.0005). Uptake of the DPT, Hib and MMR was 35% among GMS eligible, 51% among GMS ineligible (p < 0.005). CONCLUSION: A collaborative immunisation programme significantly improved practice uptake rates. These improved rates still do not attain declared national targets. To achieve these targets, radical overhaul of the immunisation service is required.     

A generalizability study of a new standardized rating form used to evaluate students'' clinical clerkship performances

Monitoring the depth of sedation in patients under intensive care is difficult. Clinical assessment by the different scoring systems produces insufficient information, especially once deeply sedated patients become unresponsive to any external stimulation. Recently, the bispectral index (BIS), the result of computerized bispectral electroencephalographic monitoring, was found to be the best predictor of depth of anaesthesia during surgical intervention. This report concerns BIS monitoring in 18 randomly selected, deeply sedated, surgical patients in the intensive care unit, who were unresponsive to standard clinical stimulation (Ramsay sedation score). A wide range of BIS was observed, with 15 of the patients having a BIS below 60, indicating a state of deep sedation (or possibly over-sedation). Therefore, further studies using BIS monitoring in patients under intensive care are needed to determine if this method can guide sedation and prevent oversedation in this context and, most importantly, to analyse its final cost-benefit ratio.     

Non-compliance with treatment in patients suffering from schizophrenia: a study to evaluate possible contributing factors

In the decade since the clinical arrival of laparoscopic cholecystectomy, there have been gratifying improvements in imaging technology and instrumentation, and innovative techniques have evolved. Laboratory-simulator devices are available for basic skills exercises and can at least reasonably mimic the appearance of the gallbladder and some other organs or anatomic regions. Unfortunately, there is no satisfactory method to practice dealing with certain structural abnormalities or disease processes. Because of that, some operations will be particularly difficult and the outcomes will be favorable only with careful planning and capable execution. The experiences and skill level of the surgeon can be enhanced by appropriate mental preparation. As a result, the surgeon will have the opportunity to accomplish the task, both laparoscopically and safely, under circumstances that initially were thought to be inappropriate or impossible for laparoscopy.     

Evaluation of the home-based maternal record: a WHO collaborative study

Thirteen centres in eight countries (Egypt, India, Pakistan, Philippines, Senegal, Sri Lanka, Democratic Yemen and Zambia) participated in the WHO collaborative study to evaluate the home-based maternal record (HBMR). The evaluation showed that use of the HBMR had a favourable impact on utilization of health care services and continuity of the health care of women during their reproductive period. When adapted to local risk conditions, their cut-off points and the available resources, the HBMR succeeded in promoting self-care by mothers and their families and in enhancing the timely identification of at-risk cases that needed referral and special care. The introduction of the HBMR increased the diagnosis and referral of at-risk pregnant women and newborn infants, improved family planning and health education, led to an increase in tetanus toxoid immunization, and provided a means of collecting health information in the community. The HBMR was liked by mothers, community health workers and other health care personnel because, by using it, the mothers became more involved in looking after their own health and that of their babies. Apart from local adaptation of the HBMR, the training and involvement of health personnel (including those at the second and tertiary levels) from the start of the HBMR scheme influenced its success in promoting maternal and child health care. It also improved the collection of community-based data and the linking of referral networks.