Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance.

Expression vectors were designed and constructed to achieve optimum production of two different isozymes of rat glutathione S-transferase (GST) (EC 2.5.1.18) in COS cells, for studies of drug resistance. Promoter-enhancer elements from the simian virus 40 (SV40) early-region or the mouse alpha 2(I)-collagen gene, GST cDNAs encoding the rat Ya or Yb1 isozymes, and an SV40 replicative origin (ori) were positioned in the vector to express two GSTs at high levels in the same cell. The optimized construct yielded levels of both GST proteins (1% of postmitochondrial protein fraction) that were up to 1.3-fold greater than the sum of those produced individually by two single-unit expression constructs. The best production of the tandem recombinant gene products was observed when the genes were placed in a head to head orientation in close proximity (1 kilobase). With the recombinant genes configured in this way, the plasmid DNA was also amplified in COS cells to higher levels (30% increase over single-unit expression constructs), as ori elements were placed on both DNA strands. Cells expressing the recombinant GSTs were viably sorted by flow cytometry on the basis of a GST-catalyzed conjugation of glutathione to monochlorobimane. Sorted COS cells that expressed both GST Ya and Yb1 from recombinant genes in a tandem, head to head configuration were 25 or 70% more resistant to the alkylating agent chlorambucil than cells that expressed GST Ya or Yb1 alone.     

Chemopreventive efficacy and anti-lipid peroxidative potential of Jasminum grandiflorum Linn. on 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis

The aim of this study was to investigate the chemopreventive efficacy and anti-lipid peroxidative potential of Jasminum grandiflorum Linn. on 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinogenesis. Mammary tumors were developed by a single subcutaneous injection of 25 mg DMBA in 1 mL emulsion of sunflower oil and physiological saline. The tumor incidence and tumor volume that formed in the breast were determined. Oral administration of ethanolic extract of J. grandiflorum flowers (JgEt) at a dose of 300 mg/kg body weight for 14 weeks to DMBA-injected animals completely prevented the formation of tumors in the pre-initiation period. JgEt also exerted significant anti-lipid peroxidative effect and improved the antioxidant defense system in DMBA-treated rats. The results of this study clearly indicate that JgEt has potent chemopreventive efficacy in experimental mammary carcinogenesis and further studies are warranted to isolate and characterize the bioactive principle from JgEt.     

Assessment of renal artery stenosis: side-by-side comparison of angiography and duplex ultrasound with pressure gradient measurements.

AIMS: A ratio of distal renal pressure to aortic pressure (P(d)/P(a)) <0.90 can be considered a threshold for defining a significant renal artery stenosis (RAS). The aim of this study was to compare renal angiography (QRA) and colour duplex ultrasound (CDUS) to pressure measurements in assessing RAS. METHODS AND RESULTS: In 56 RAS, percent diameter stenosis (DS(angio)), minimal luminal diameter (MLD), Doppler-derived peak systolic velocity (PSV), end-diastolic velocity (EDV), and renal-to-aortic ratio (RAR) were obtained and compared with the P(d)/P(a) measured with a 0.014" pressure wire. P(d)/P(a) correlated with angiography- and CDUS-derived parameters. The best correlation was observed with EDV (R = -0.61). To identify stenosis associated with a P(d)/P(a) < 0.90, the diagnostic accuracy of DS(angio) > 50%, MLD < 2 mm, PSV > 180 cm/s, EDV > 90 cm/s and RAR > 3.5 were, respectively, 60%, 77%, 45%, 77% and 79%, yet, with a high proportion of false positives (38%, 15%, 55%, 11% and 15%, respectively) indicating an overestimation of the severity of the RAS by both QRA and CDUS. New cut-off values for QRA- and CDUS-derived indices were proposed. CONCLUSION: Generally accepted QRA and CDUS-derived indices of RAS severity overestimate the actual severity of RAS. This 'overdiagnosis' is likely the main cause of the disappointing results of renal angioplasty for renovascular hypertension.     

Oxalate measurement in the picomol range by ion chromatography: values in fasting plasma and urine of controls and patients with idiopathic calcium urolithiasis

Oxalate was measured by ion chromatography in the ultrafiltrate of heparinized plasma from peripheral venous blood, using a membrane with a cut-off molecular weight (Mr). The following criteria were established: sensitivity 0.7 mumol.l-1; intra- and inter-assay coefficients of variation 4% and 12%, respectively; precision of duplicate determinations (expressed as standard deviation) 0.08 mumol.l-1; overall recovery (oxalate added and diluted, respectively) 100.7%. These qualified the method for assessment of plasma oxalate in healthy human controls (males: n = 12) as well as patients with idiopathic renal calcium urolithiasis (males: n = 22; females: n = 16). Renal calcium urolithiasis patients were subclassified into those with normocalciuria and idiopathic hypercalciuria. In male and female controls the mean values (and range) of plasma oxalate were 1.98 (1.4-2.5) and 1.78 (0.7-2.9) mumol.l-1, respectively. In male controls ultrafiltration (membrane cut off Mr 10,000) revealed that 11-16% plasma oxalate was bound to constituents having an apparent Mr above 10,000, and that with use of membranes with smaller pore size, the ultrafilterability of oxalate decreases further. In renal calcium urolithiasis the following values were elicited (mumol.l-1): male normocalciuria 1.78 (0.8-4.0), idiopathic hypercalciuria 1.58 (1.2-2.2); female normocalciuria 1.69 (0.8-3.6), idiopathic hypercalciuria 1.21 (0.8-2.1). The difference from controls is significant in idiopathic hypercalciuria (males and females). In contrast, in fasting urine of renal calcium urolithiasis the oxalate excretion rate (5-45 mumol per 120 min) and oxalate clearance (21-328 ml per min) resemble those in controls, whereas in renal calcium urolithiasis the fractional oxalate clearance (30-357% of creatinine clearance) tended to higher values (p less than 0.01, in male idiopathic hypercalciuria versus controls). It is suggested that 1) ion chromatography allows the reliable assessment of ultrafiltrable plasma oxalate in health and disease states, 2) in renal calcium urolithiasis this technique may help to elucidate oxalate pathophysiology, especially the mode of renal handling of oxalate.     

Polymorphisms in the CRP gene moderate an association between depressive symptoms and circulating levels of C-reactive protein

Although many studies have found psychological depression associated with higher circulating levels of C-reactive protein (CRP), not all findings are consistent. Since DNA sequence variation in the CRP gene has also been shown to predict plasma CRP levels, we hypothesized that plasma CRP may covary with depressive symptomatology as a function of allelic variation in the CRP gene. We tested this hypothesis in 868 healthy community volunteers of European ancestry. Depressive symptomatology was measured using the Center for Epidemiological Studies-Depression (CESD) scale, and plasma CRP was assayed from whole blood. Three polymorphisms [rs1417938 (A/T), rs1800947 (C/G) and rs1205 (C/T)] were genotyped and three-locus haplotypes were generated. Regression models adjusting for demographic and lifestyle-related covariates showed no direct association of CESD depression scores with CRP. In regression models adjusting for age, gender, education, smoking status and statin use, one CRP haplotype (T-G-C) was associated with CRP level (p = 0.014) and a second haplotype (A-G-T) showed marginal association (p = 0.064, respectively). Neither haplotype was related to depressive symptoms. However, plasma CRP was predicted by the interaction of A-G-T haplotype with depressive symptomatology (p = 0.009). Higher CESD scores were associated positively with CRP levels among individuals with the A-G-T haplotype (p = 0.004). In secondary analyses, body mass index was found to partially account for the moderating effects of the A-G-T haplotype on the association of depression with circulating CRP. In conclusion, we found that haplotypic variation in the CRP locus moderates an association of depressive symptoms with circulating CRP, which is partially mediated by BMI.     

Meta-analysis and meta-regression of hypothalamic-pituitary-adrenal axis activity in functional somatic disorders

Dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is the most investigated biological risk marker in functional somatic disorders (FSDs), such as chronic fatigue syndrome (CFS), fibromyalgia (FM), and irritable bowel syndrome (IBS). Our aim was to assess whether there is an association between basal hypocortisolism and FSD and to identify potential moderators of this association. Meta-analysis on 85 studies revealed that although basal cortisol levels were generally lower in FSD subjects compared to controls, this association did not reach statistical significance (SMD −0.07, 95% CI −0.17 to 0.04, p = 0.241). However, when the three FSD were assessed separately, statistically significant basal hypocortisolism was observed in CFS subjects compared to controls (SMD −0.14, 95% CI −0.28 to 0.00, p = 0.047), but not in FM or IBS. When all potential moderators were entered into a meta-regression analysis, only type of FSD and female gender were significant independent predictors of basal hypocortisolism. In conclusion, we did not find evidence to consider all three main FSD as hypocortisolemic disorders, as significant reduction in basal cortisol compared to healthy controls was only found in CFS and in females with FM, but not in IBS.     

Antitumor initiating potential of rosmarinic acid in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis

Oral cancer accounts for 40%-50% of all cancers in India. Tobacco and alcohol are the major etiological factors contributing to its pathogenesis. The aim of the present study was to explore the key mechanism behind the inhibitory effects of rosmarinic acid against 7,12-dimethylbenz(a)anthracene (DMBA)-induced oral carcinogenesis by evaluating the status of biochemical markers (lipid peroxidation, antioxidants, and detoxification enzymes) and immunoexpression patterns of p53 and bcl-2 proteins. Oral tumors were developed by painting the buccal pouches of golden Syrian hamsters with 0.5% DMBA in liquid paraffin 3 times a week for 14 weeks. We noticed 100% tumor formation in hamsters treated with DMBA alone, and the tumors were histopathologically confirmed as well-differentiated squamous cell carcinoma. Oral administration of rosmarinic acid (100 mg/kg body wt) to DMBA-treated hamsters completely prevented the tumor formation. In addition, rosmarinic acid significantly returned the status of biochemical and molecular markers to near normal range in DMBA-treated hamsters. The results of the present study suggest that rosmarinic acid suppresses oral carcinogenesis by stimulating the activities of detoxification enzymes, improves the status of lipid peroxidation and antioxidants, and downregulates the expression of p53 and bcl-2 during DMBA-induced oral carcinogenesis.     

The management of completely resected childhood intracranial ependymoma: the argument for observation only

Introduction  The management of children with completely resected intracranial ependymomas is controversial. We favor deferring adjuvant radiotherapy in low-risk patients, whereas others recommend radiotherapy. Review  This article reviews the available evidence for and against deferring radiotherapy in children with low-risk completely resected childhood ependymomas.     

Specimen size and porosity can introduce error into microCT-based tissue mineral density measurements

The accurate measurement of tissue mineral density, rho(m), in specimens of unequal size or quantities of bone mineral using polychromatic microCT systems is important, since studies often compare samples with a range of sizes and bone densities. We assessed the influence of object size on microCT measurements of rho(m) using (1) hydroxyapatite rods (HA), (2) precision-manufactured aluminum foams (AL) simulating trabecular bone structure, and (3) bovine cortical bone cubes (BCt). Two beam-hardening correction (BHC) algorithms, determined using a 200 and 1200 mg/cm(3) HA wedge phantom, were used to calculate rho(m) of the HA and BCt. The 200 mg/cm(3) and an aluminum BHC algorithm were used to calculate the linear attenuation coefficients of the AL foams. Equivalent rho(m) measurements of 500, 1000, and 1500 mg HA/cm(3) rods decreased (r(2)>0.96, p<0.05 for all) as HA rod diameter increased in the 200 mg/cm(3) BHC data. Errors averaged 8.2% across these samples and reached as high as 29.5%. Regression analyses suggested no size effects in the 1200 mg/cm(3) BHC data but differences between successive sizes still reached as high as 13%. The linear attenuation coefficients of the AL foams increased up to approximately 6% with increasing volume fractions (r(2)>0.81, p<0.05 for all) but the strength of the size-related error was also BHC dependent. Equivalent rho(m) values were inversely correlated with BCt cube size (r(2)>0.92, p<0.05). Use of the 1200 mg/cm(3) BHC ameliorated the size-related artifact compared to the 200 mg/cm(3) BHC but errors with this BHC were still significant and ranged between 5% and 12%. These results demonstrate that object size, structure, and BHC algorithm can influence microCT measurements of rho(m). Measurements of rho(m) of specimens of unequal size or quantities of bone mineral must be interpreted with caution unless appropriate steps are taken to minimize these potential artifacts.     

Accuracy of single-photon emission computed tomography in differentiating frontotemporal dementia from Alzheimer's disease

Background

Alzheimer''s disease (AD) and frontotemporal dementia (FTD) are the commonest causes of presenile dementia. In the absence of a biological marker, diagnosis is reliant on clinical evaluation. Confirmation is often sought from neuroimaging, including single‐photon emission computed tomography (SPECT). Most previous SPECT studies lack pathological validation.

Aim

To examine the accuracy of SPECT in differentiating FTD from AD in patients with subsequent pathological confirmation.

Methods

Technetium‐99‐labelled hexamethyl propylene amine oxime SPECT images obtained at initial evaluation in 25 pathologically confirmed cases of FTD were examined. These images were visually rated by an experienced blinded nuclear medicine consultant and compared with those of 31 patients with AD, also with pathological validation.

Results

A reduction in frontal cerebral blood flow (CBF) was more common in FTD and was of diagnostic value (sensitivity 0.8, specificity 0.65 and likelihood ratio (LR) 2.25; 95% CI 1.35 to 3.77). A pattern of bilateral frontal CBF reduction without the presence of associated bilateral parietal CBF change is diagnostically more accurate (sensitivity 0.80, specificity 0.81 and +LR 4.13, 95% CI 1.96 to 8.71). Diagnostic categorisation (FTD or AD) on the basis of SPECT alone was less accurate than clinical diagnosis (based on neurology and detailed neuropsychological evaluation). One patient with FTD was initially clinically misdiagnosed as AD, owing to the lack of availability of full neuropsychological assessment. However, SPECT correctly diagnosed this patient, providing a diagnostic gain of 4%.

Conclusion

Technetium‐99‐labelled hexamethyl propylene amine oxime SPECT CBF patterns provide valuable information in the diagnosis of FTD and AD. These data can be better used as an adjunct to clinical diagnosis if pathology is to be correctly predicted in life.Frontotemporal dementia (FTD) is a cortical dementia distinct from other dementing illnesses. It typically presents with personality/behavioural change and decline in social conduct with early loss of insight.^1,2 In the absence of biological markers, the pathological detection of characteristic histological changes remains the gold standard of diagnosis. In life, diagnosis is primarily based on patterns of neurological and neuropsychological findings. However, differentiation from other dementias can be difficult and demands an astute qualitative analysis of various behaviours and neuropsychological test performances.³ With a paucity of experienced neuropsychologists, additional and independent diagnostic information is often sought through imaging, be it structural (CT and MRI) and/or functional (single‐photon emission computed tomography (SPECT) and positron emission tomography).SPECT is used to evaluate patients with dementia and can show purported characteristic changes in FTD and in Alzheimer''s disease (AD).^4,5,6,7,8,9 The technique provides a method of evaluating blood flow in various regions of the brain, which reflects areas of poor function by showing reductions in regional cerebral blood flow (CBF). It has been shown that posterior changes in regional CBF are common in AD,^4,5,6,7 whereas in FTD anterior changes are prevalent^7,8,9 and posterior changes rare.⁷However, CBF changes are neither wholly specific nor invariable in various dementing illnesses. Masterman et al¹⁰ looked at the value of bitemporal hypoperfusion in diagnosing AD, and found that, although a sensitive measure for detecting dementia (0.75), it was poorly specific for AD (0.55). Consequently, bitemporal hypoperfusion on SPECT can be a non‐specific finding in various forms of dementia and is not exclusive to AD. Starkstein et al¹¹ reported deficits in CBF in the frontal (especially orbitofrontal) and anterior temporal cortices in FTD. However, they provided neither the measure of the diagnostic accuracy of SPECT in FTD nor of the diagnostic gain it may provide. Most of these studies are also limited by the fact that the dementia groups are defined clinically. The clinical diagnostic accuracy of FTD in life varies hugely between 14–85%.^12,13,14A few studies have looked at the accuracy of clinical and SPECT findings in relation to the final pathological diagnoses.^{15,16,17,18,19} Although these studies found that SPECT findings do correlate with dementia type, they failed to enquire whether SPECT provides any additional diagnostic gain over clinical judgement. These studies are also severely limited by the small numbers of patients in the FTD groups.The aims of this study include evaluation of the diagnostic accuracy of SPECT in differentiating FTD from AD at initial assessment in a group of patients with final pathological confirmation of diagnosis. We also examined the diagnostic gain SPECT may provide over clinical diagnosis of FTD from among this group of patients with FTD and AD.