Influence of VEGF-A gene variation and protein levels in breast cancer susceptibility and severity

Vascular endothelial growth factor-A (VEGF-A) plays an important role in tumour angiogenesis and cancer progression. VEGF gene variation may influence VEGF levels and therefore cancer susceptibility and progression. We studied the role of VEGF single nucleotide polymorphisms and haplotypes in breast cancer susceptibility and severity. We also studied the relationships of VEGF SNPs with circulating VEGF levels in healthy volunteers and protein expression in breast cancers. Single nucleotide polymorphisms (SNPs) in the regulatory regions of the VEGF gene were genotyped by high throughput methods in approximately 500 breast cancer cases and 500 appropriate controls. Haplotype frequencies were inferred using methods based on the Expectation Maximisation algorithm. The effect of VEGF genotypes on serum and plasma VEGF levels were studied in another cohort of healthy individuals. A semi-quantitative assessment of VEGF protein expression on tissue micro arrays (TMA) constructed from approximately 300 breast cancer samples was performed and compared with VEGF genotypes and with histopathological parameters and survival in breast cancer. The -460T/+405C/-7C/936C haplotype in the VEGF gene was found to be associated with decreased breast cancer risk (p = 0.029). The -7C>T polymorphism may influence overall breast cancer survival (p = 0.027). Individual polymorphisms however did not affect breast cancer susceptibility. There was no association between the individual polymorphisms and circulating VEGF levels in healthy volunteers and VEGF expression on the breast cancer micro array. VEGF expression in breast cancers was however associated with high grade (p = 0.002) and ER negative tumours (p = 0.03).     

Dodecanoic acid derivatives: Synthesis, antimicrobial evaluation and development of one-target and multi-target QSAR models

In this study a series of dodecanoic acid derivatives (1–30) were synthesized and evaluated for in vitro antimicrobial activity against the panel of Gram positive, Gram negative bacterial and fungal strains. 4-Nitro phenyl dodecanoate (4) and quinolin-8-yl dodecanoate (5) emerged as most effective antibacterial agents, and 1-(4-benzylpiperazin-1-yl) dodecan-1-one (15) was found to be the most effective antifungal agent amongst the synthesized dodecanoic acid derivatives. Quantitative structure activity relationship (QSAR) studies performed by the development of one-target and multi-target models indicated that multi-target model was effective in describing the antimicrobial activity of dodecanoic acid derivatives as well demonstrated the importance of topological parameter, zero-order molecular connectivity index (⁰χ).     

Predictors and ocular outcomes of rescue treatment in preterm infants with treated retinopathy of prematurity—a retrospective study

AimTo determine predictors of rescue treatment among infants treated for retinopathy of prematurity and to evaluate their ocular outcomes at 18–24 months of corrected age.MethodsThis is a single centre retrospective study of infants who received treatment for type 1 ROP, using laser photocoagulation or anti VEGF agents. Multivariable logistic regression was used to generate a prediction model for rescue treatment of ROP. The primary outcome was an abnormal refractive outcome by 24 months of corrected age, among infants primarily treated with laser therapy.ResultsTwo hundred and eight infants (including 416 eyes) who received single (n = 151) or rescue (multiple) treatments (n = 57) were included. Ninety three percent of the infants were primarily treated with laser photocoagulation. Lower gestational age, small for gestational age, early packed red blood cell transfusion (within 2 weeks of postnatal age), and presence of Zone 1 retinopathy predicted the need for rescue treatment in treated infants [area under the receiver operating characteristic curve: 0.81 (0.73–0.89)]. The incidence of abnormal refractive outcome, assessed in a total of 174 infants, was found to be significantly higher in the rescue treatment group (67% versus 21%, adjusted odds ratio: 7.56 (3.3–17.2), P < 0.001). Myopia, very high myopia and use of spectacles was significantly higher in the rescue treatment group (P < 0.001 for each).ConclusionsRescue treatment for ROP was associated with an increased incidence of refractive errors and requirement of spectacles by 2 years of age. Larger prospective multicentre studies are required to confirm the findings from our study.Subject terms: Paediatrics, Retinal diseases     

Structure and stability of the dityrosine-linked dimer of gammaB-crystallin.

Oxidative damage to proteins leads to a variety of modifications such as racemization, carbonyl compound formation, new fluorophores, aggregation, crosslinking and insolubility, several of which are markers of pathogenesis. A particular modification that has been associated with abnormal and pathological situations is the dityrosine crosslink in proteins, thought to be responsible for the reduced solubility and elasticity of proteins, and plaque formation. Dityrosine crosslinking has been suspected to occur in the crystallins of the eye lens during cataract. We focus attention here on the generation, structure and conformational stability of such a dityrosine-linked protein of the eye lens. We find this crosslink to be readily generated photodynamically in the presence of sensitizers. Among the crystallins, crosslinking occurs most readily in the gamma-crystallins under these conditions. We have isolated, purified and studied the properties of the dityrosine-linked dimer of the eye lens protein gammaB-crystallin. While the dityrosine crosslink does not alter the secondary structure of the protein, it changes the tertiary structure in a subtle manner. This alteration destabilizes the dimer, which denatures more readily than the parent monomer, and also makes it precipitate more readily, a point of relevance to cataractogenesis of the eye lens. Comparison of these results with those reported on other dityrosine-dimerized proteins suggests that while the conformation of these proteins might not be altered in a major manner upon dityrosine linkage, the dimer is structurally less stable and displays reduced solubility, both of which are of pathological importance.     

Evaluation of existing limited sampling models for busulfan kinetics in children with beta thalassaemia major undergoing bone marrow transplantation.

Busulfan pharmacokinetic parameters are useful in predicting the outcome of allogeneic bone marrow transplantation (BMT). Standard pharmacokinetic measurements require multiple blood samples. Various limited sampling models (LSM) have been proposed for reducing the sample number required for these measurements, essentially for patients with malignant disorders undergoing BMT. This study was undertaken to evaluate the existing LSM for busulfan pharmacokinetics to find out the most suitable method for patients with thalassaemia major undergoing BMT. Busulfan levels in plasma samples were analysed by HPLC. The AUC calculated by non-compartmental analysis using the program 'TOPFIT' was compared with previously published LSMs. Our seven sample pharmacokinetic data for AUC calculation was compared with the published LSMs. The three sample models suggested by Chattergoon et al and Schuler et al showed significant agreement with AUC TOPFIT (R(2) = 0.98 and 0.94, respectively) in our clinical context. Other models resulted in significant over or under representation of observed values (Vassal's model R(2) = 0.61; Chattergoon's two sample model R(2) = 0.84; four sample model R(2) = 0.83; Schuler's two sample model R(2) = 0.79). By these data the three sample LSM proposed by Chattergoon et al and Schuler et al are suitable for calculation of the AUC in patients with thalassaemia major undergoing BMT conditioned with oral busulfan.     

Virulence of Mycobacterium tuberculosis for guinea pigs: a quantitative modification of the assay developed by Mitchison.

One of the more accepted methods of assay of virulence of tubercle bacilli is one developed by Mitchison in which guinea pigs were infected by the intramuscular route with 1.0 mg of tubercle bacilli freshly harvested from Lowenstein Jensen medium and in which virulence was based on a subjective score of the extent of gross disease in the animal 6 weeks after infection. Due to the practical difficulties involved in such an assay when routinely performed, the following modifications were made: frozen stocks of the culture were prepared prior to the date of infection, the inoculum was quantitated by means of colony forming units, and virulence was based on the number of tubercle bacilli recovered from the spleen of infected animals 6 weeks after infection. A significant correlation was obtained between the findings from the Mitchison assay of virulence and the modification. The latter assay is recommended for its quantitative and objective features.     

Dopamine Agonist Signalling in the Hypothalamus of Female Rats is Independent of Calcium-Dependent Kinases

We have previously demonstrated that dopamine agonist, SKF38396 (SKF), can substitute for progesterone in the facilitation of female reproductive behaviour in oestradiol benzoate-primed female rats and mice. We also reported that both progesterone- and SKF-initiated signalling were mediated by the cAMP-dependent protein kinase A signal transduction cascade. As the rapid effects of progesterone are also mediated by calcium-dependent kinases, calcium- and calmodulin-dependent kinase (CaMKII) and protein kinase (PKC), we sought to determine whether SKF-initiated signalling also recruited calcium as a second messenger. We measured the changes in the activation of CaMKII and PKC in the ventromedial nucleus (VMN) of the hypothalamus and preoptic area (POA) of the rat brain, which are the two regions implicated in the regulation of female reproductive behaviour in rodents. We measured the basal activities representing the activation of the kinases by in vivo treatments, as well as the total kinase activities assayed in the presence of exogenous cofactors in vitro . We report that, in contrast to progesterone-initiated signalling, there was no recruitment of calcium by SKF in the hypothalamus, as shown by the absence of changes in CaMKII activities in the VMN and POA. Furthermore, SKF-treatment resulted in a rapid increase in calcium-independent basal PKC activity in the VMN but not the POA. These rapid changes were not the result of changes in PKC protein levels or phosphorylation status. These data indicate that progesterone- and SKF-recruit distinct signalling molecules within the same regions of the brain to activate region-specific signal transduction pathways.     

Cholinesterases exhibiting aryl acylamidase activity in human amniotic fluid.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid.     

Determining compliance with a dietary fiber supplement

The purpose of this pilot study was to evaluate possible ways to determine compliance with a dietary fiber supplement. A wheat bran supplement (30 g daily) significantly increased mean daily wet and dry stool weights (SW) in 7 adults, when compared to SW during an ad libitum low-fiber diet (paired t-test, P less than .01). Because unpaired data would be used during a clinical trial, distribution of the 7 ad libitum low-fiber mean SW observations was used to establish a reference distribution and an upper confidence limit against which the bran supplement SW could be compared. Only one of the seven bran supplement mean SW was above the confidence limit of the low-fiber period, independent of the number of days of collection (2-10) used to calculate the individual mean daily SW. Total fecal output over varying periods of time (2-10 days) suffered the same intersubject and intrasubject variability. Most (5-6) of the bran mean daily SW were above the group mean SW of the low-fiber period. However, this dose of bran was large enough to significantly decrease calcium absorption, and differences in SW produced by lower doses of wheat bran would probably not be as great. The bulk (greater than or equal to 80%) of a single dose of a fecal marker, chromium sesquioxide, which could be incorporated into a specific day's fiber supplement, was recovered in 5 days of excretion during the control period and in 4 days during the bran period. However, the blue color of the chromium before ingestion is clearly a negative feature. Another marker, polyethylene glycol, could not be recovered in excreta when transient time was 4 days or more. In a separate study, demonstration of very little overlap in the concentration of fecal neutral detergent fiber between the control and bran periods suggests that fecal fiber may be a marker of compliance with a fiber supplement.