Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. fulciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (ACC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2(.)5 x 10(-9) mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and antimalarial drug therapy by demonstrating that even with lon mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control, In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein, Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Spl decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells, Mutation in two Spl consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the;basal level of Spl, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in,A-T cells. On the other hand full-length ATM cDNA increased the basal level of Spl binding in A-T cells, and in response to EGF Spl binding decreased, confirming that this is an ATR I-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Spl binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Spl regulation.

Early Australian experience with infliximab, a chimeric antibody against tumour necrosis factor-alpha, in the treatment of Crohn's disease: is its efficacy augmented by steroid-sparing immunosuppressive therapy?

Background: Tumour necrosis factor-alpha (TNF-alpha) plays an important role in the pathology of Crohn's disease. Infliximab, a chimeric antibody against TNF-alpha, has been shown in controlled clinical trials to be effective in two-thirds of patients with refractory or fistulating Crohn's disease. The factors that determine a clinical response in some patients but not others are unknown. Aims: To document the early Australian experience with infliximab treatment for Crohn's disease and to identify factors that may determine a beneficial clinical response. Methods: Gastroenterologists known to have used infliximab for Crohn's disease according to a compassionate use protocol were asked to complete a spreadsheet that included demographic information, Crohn's disease site, severity, other medical or surgical treatments and a global clinical assessment of Crohn's disease outcome, judged by participating physicians as complete and sustained (remission for the duration of the study), complete but unsustained (remission at 4 weeks but not for the whole study) or partial clinical improvement (sustained or unsustained). Results: Fifty-seven patients were able to be evaluated, with a median follow-up time of 16.4 (4-70) weeks, including 23 patients with fistulae. There were 21 adverse events, including four serious events. Fifty-one patients (89%) had a positive clinical response for a median duration (range) of 11 (2-70) weeks. Thirty patients (52%) had a remission at 4 weeks, 10 of whom had remission for longer than 12 weeks. Forty-two per cent of fistulae closed. Sustained remission (P = 0.065), remission at 4 weeks (P = 0.033) and a positive clinical response of any sort (P = 0.004) were more likely in patients on immunosuppressive therapy, despite there being more smelters in this group. Conclusion: This review of the first Australian experience with infliximab corroborates the reported speed and efficacy of this treatment for Crohn's disease. The excellent response appears enhanced by the concomitant use of conventional steroid-sparing immunosuppressive therapy.

Outcomes of cephalosporin treatment for serious infection due to apparently susceptible organisms producing extended-spectrum b-Lactamases: Implications for the clinical microbiology laboratory

Although extended-spectrum beta-lactamases (ESBLs) hydrolyze cephalosporin antibiotics, some ESBL-producing organisms are not resistant to all cephalosporins when tested in vitro. Some authors have suggested that screening klebsiellae or Escherichia coli for ESBL production is not clinically necessary, and when most recently surveyed the majority of American clinical microbiology laboratories did not make efforts to detect ESBLs, We performed a prospective, multinational study of Klebsiella pneumoniae bacteremia and identified 10 patients who were treated for ESBL-producing K. pneumoniae bacteremia with cephalosporins and whose infecting organisms were not resistant in vitro to the utilized cephalosporin. In addition, we reviewed 26 similar cases of severe infections which had previously been reported. Of these 36 patients, 4 had to be excluded from analysis. Of the remaining 32 patients, 100% (4 of 4) patients experienced clinical failure when MICs of the cephalosporin used for treatment were in the intermediate range and 54% (15 of 28) experienced failure when MICs of the cephalosporin used for treatment were in the susceptible range, Thus, it is clinically important to detect ESBL production by klebsiellae or E, coli even when cephalosporin MICs are in the susceptible range (less than or equal to 8 mug/ml) and to report ESBL-producing organisms as resistant to aztreonam and all cephalosporins (with the exception of cephamycins).

Treatment of lupus nephritis: art or science? [Editorial]

No Abstract

The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) - Results of an international collaborative study

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2–5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

Population screening for HFE-associated haemochromatosis: should we have to pay for our genes?

Haemochromatosis associated with mutations in the HFE gene is the most common inherited disorder in Caucasian populations. Early diagnosis and treatment allows for normal life expectancy whereas there is considerable morbidity and early mortality in those patients diagnosed late or untreated. Unfortunately, the development of symptoms and signs in haemochromatosis is usually associated with significant iron overload. For this reason, many clinicians and geneticists have advocated population screening. The recent identification of the HFE gene and the availability of a simple DNA-based diagnostic test have led to international debate as to the most cost-effective means of population screening for HFE-associated haemochromatosis. The present paper summarizes the evidence in favour of population screening and analyses the relative advantages of genotypic (DNA test) versus phenotypic (transferrin saturation) testing.

Effects of high-dose etidronate treatment on microdamage accumulation and biomechanical properties in beagle bone before occurrence of spontaneous fractures

We recently demonstrated that suppressed bone remodeling allows microdamage to accumulate and causes reductions in some mechanical properties. However, in our previous study, I year treatment with high-dose etidronate (EHDP) did not increase microdamage accumulation in most skeletal sites of dogs in spite of complete remodeling suppression and the occurrence of spontaneous fractures of ribs and/or thoracic spinous processes. This study evaluates the effects of EHDP on microdamage accumulation and biomechanical properties before fractures occur. Thirty-six female beagles, 1-2 years old, were treated daily for 7 months with subcutaneous injections of saline vehicle (CNT) or EHDP at 0.5 (E-low) or 5 mg/kg per day (E-high). After killing, bone mineral measurement, histomorphometry, microdamage analysis, and biomechanical testing were performed. EHDP treatment suppressed intracortical and trabecular remodeling by 60%-75% at the lower dose, and by 100% at the higher dose. Osteoid accumulation caused by a mineralization deficit occurred only in the E-high group, and this led to a reduction of mineralized bone mass. Microdamage accumulation increased significantly by two- to fivefold in the rib, lumbar vertebra, ilium, and thoracic spinous process in E-low, and by twofold in the lumbar vertebra and ilium in E-high. However, no significant increase in damage accumulation was observed in ribs or thoracic spinous processes in E-high where fractures occur following 12 months of treatment. Mechanical properties of lumbar vertebrae and thoracic spinous processes were reduced significantly in both E-low and E-high. These findings suggest that suppression of bone remodeling by EHDP allows microdamage accumulation, but that osteoid accumulation reduces production of microdamage. (Bone 29:271-278; 2001) (C) 2001 by Elsevier Science Inc. All rights reserved.

Rapid opiate detoxification and naltrexone treatment: past, present and future

This Article does not have an abstract

Non-Markovian homodyne-mediated feedback on a two-level atom: a quantum trajectory treatment

Quantum feedback can stabilize a two-level atom against decoherence (spontaneous emission), putting it into an arbitrary (specified) pure state. This requires perfect homodyne detection of the atomic emission, and instantaneous feedback. Inefficient detection was considered previously by two of us. Here we allow for a non-zero delay time tau in the feedback circuit. Because a two-level atom is a non-linear optical system, an analytical solution is not possible. However, quantum trajectories allow a simple numerical simulation of the resulting non-Markovian process. We find the effect of the time delay to be qualitatively similar to chat of inefficient detection. The solution of the non-Markovian quantum trajectory will not remain fixed, so that the time-averaged state will be mixed, not pure. In the case where one tries to stabilize the atom in the excited state, an approximate analytical solution to the quantum trajectory is possible. The result, that the purity (P = 2Tr[rho (2)] - 1) of the average state is given by P = 1 - 4y tau (where gamma is the spontaneous emission rate) is found to agree very well with the numerical results. (C) 2001 Elsevier Science B.V. All rights reserved.

Refusal of treatment

Patients' thoughts, feelings and desires are communicated in a variety of ways, and require sympathetic, critical interpretation. Patients need clear, evidence-based medical information so that they can make their own decisions about whether to consent to or refuse medical treatment. Treatment refusal may provide an opportunity to introduce patients to advance care planning. Unconscious motivations in doctors may obstruct good clinical decision-making. Although respect for the patient's responsibility to make healthcare decisions should be a condition of the clinical relationship, healthcare decision-making is a collaborative process.

The effect of heat treatment on the toughness, hardness and microstructure of low carbon white cast irons

The effect of destabilisation and subcritical heat treatment on the impact toughness, hardness, and the amount and mechanical stability of retained austenite in a low carbon white cast iron have been investigated. The experimental results show that the impact energy constantly increases when the destabilisation temperature is raised from 950 degreesC to 1200 degreesC. Although the hardness decreases, the heat-treated hardness is still greater than the as-cast state. After destabilisation treatment at 1130 degreesC, tempering at 200 to 250 degreesC for 3 hours leads to the highest impact toughness, and secondary hardening was observed when tempering over 400 degreesC. The amount of retained austenite increased with the increase in the destabilisation temperature, and the treatment significantly improves the mechanical stability of the retained austenite compared with the as-cast state. Tempering below 400 degreesC does not affect the amount of retained austenite and its mechanical stability. But the amount of retained austenite is dramatically reduced when tempered above 400 degreesC. The relationship between the mechanical properties and the microstructure changes was discussed. (C) 2001 Kluwer Academic Publishers.