Novel bio-analytical technique for estimation of gamma oryzanol in rat plasma and brain homogenate using HPLC.

Gamma oryzanol, a component of rice bran oil is used for its anticancer and antihyperlipidemic properties. Bioanalytical method for rat plasma and brain homogenate was developed by HPLC system with a PDA detector in which drug elution was performed using C-18 column (4.6mm×150cm, 5µ) with 1% acetic acid in methanol: acetonitrile (65/35, v/v) as mobile phase at 1.2ml/min flow rate and detected at 326nm wavelength. Liquid liquid extraction method was chosen for extraction of oryzanol from plasma as well as brain homogenate as it provided highest recovery (95% in plasma, 85% in brain homogenate). Various extraction solvents for each body fluid were analysed, out of which highest recovery for plasma (95%), in acetone: IPA (1/1, v/v) and for brain homogenate (85%) in isopropyl alcohol (IPA) was observed. Observed linearity was between 500ng/mL-5000ng/mL. The interday and intraday precision (i.e. %RSD) was less than 10% and accuracy was±5%. Selectivity and matrix effect was checked and found as per USFDA criteria. Plasma samples were found to be stable over the analysis period, HQC samples were stable up to third cycle in freeze and thaw stability while LQC samples were stable over fourth cycle. The method proved to be simple, useful and is appropriate, for preclinical and experimental research.

Prognosis of BRCA-associated breast cancer: a summary of evidence.

The purpose of the study is to review the clinical studies relevant to the prognosis and prognostic associations of BRCA1- and BRCA2-associated breast cancers. Reports of relevant studies obtained from a MEDLINE search and references from these articles were critically reviewed. A number of methodologic limitations have been identified in the early studies. More recent studies have failed to demonstrate, for the most part, a significant overall survival difference between BRCA-associated breast cancers and sporadic breast cancers. The risk of in-breast tumor recurrence also appears to be similar in the first 5 years following a breast cancer diagnosis with apparent increase in the risk after 5 years in one study. Many in-breast tumor recurrences are now considered to be second primary breast cancers. There is a significant increase in the risk of contralateral breast cancers in BRCA mutation carriers with an estimated 10-year risk ranging from 20-40%. The prognosis of BRCA-associated breast cancers appears to be similar to that of sporadic breast cancers based on the current literature. Future data from large prospective cohort studies will be of interest.

Does family history predict the age at onset of new breast cancers in BRCA1 and BRCA2 mutation-positive families?

Women who carry BRCA mutations are advised to begin breast cancer screening based on the age-specific risks of breast cancer development. It is not clear to what extent the family history of breast cancer influences age of onset. We evaluated the use of family history to predict the age of breast cancer onset in BRCA mutation carriers. Pedigrees from an Ontario-based registry were reviewed to identify the index case of breast cancer (most recent diagnosis) and other family cases of breast cancer. The youngest age of breast cancer diagnosis and mean age at breast cancer diagnosis in the other family cases were compared to the age of onset in the index case. The 260 BRCA1 and 213 BRCA2 pedigrees were reviewed. In BRCA2 families, the index case was diagnosed on average at 44.4 years when the youngest reported family case was less than or equal to 35 years, compared to 51.9 years when the earliest cases were diagnosed after age 50 (p = 0.04). A modest trend was seen for BRCA1 carriers, but this was not statistically significant. To a small extent, the onset of breast cancer in a BRCA2 mutation carrier can be predicted from her family history of cancer, however, the trend is modest and should not alter clinical recommendations regarding initiation of screening.

Transient receptor potential (TRP) channels: a metabolic TR(i)P to obesity prevention and therapy.

Cellular transport of ions, especially by ion channels, regulates physiological function. The transient receptor potential (TRP) channels, with 30 identified so far, are cation channels with high calcium permeability. These ion channels are present in metabolically active tissues including adipose tissue, liver, gastrointestinal tract, brain (hypothalamus), pancreas and skeletal muscle, which suggests a potential role in metabolic disorders including obesity. TRP channels have potentially important roles in adipogenesis, obesity development and its prevention and therapy because of their physiological properties including calcium permeability, thermosensation and taste perception, involvement in cell metabolic signalling and hormone release. This wide range of actions means that organ-specific actions are unlikely, thus increasing the possibility of adverse effects. Delineation of responses to TRP channels has been limited by the poor selectivity of available agonists and antagonists. Food constituents that can modulate TRP channels are of interest in controlling metabolic status. TRP vanilloid 1 channels modulated by capsaicin have been the most studied, suggesting that this may be the first target for effective pharmacological modulation in obesity. This review shows that most of the TRP channels are potential targets to reduce metabolic disorders through a range of mechanisms.

Solution structure and dynamics of GCN4 cognate DNA: NMR investigations.

A 12 bp long GCN4-binding, self-complementary duplex DNA d(CATGACGTCATG)(2) has been investigated by NMR spectroscopy to study the structure and dynamics of the molecule in aqueous solution. The NMR structure of the DNA obtained using simulated annealing and iterative relaxation matrix calculations compares quite closely with the X-ray structure of ATF/CREB DNA in complex with GCN4 protein (DNA-binding domain). The DNA is also seen to be curved in the free state and this has a significant bearing on recognition by the protein. The dynamic characteristics of the molecule have been studied by (13)C relaxation measurements at natural abundance. A correlation has been observed between sequence-dependent dynamics and recognition by GCN4 protein.

Transplant coronary artery disease: a novel model independent of cellular alloimmune response.

BACKGROUND: Allograft coronary atherosclerosis (TxCAD) is the leading cause of death after the first year after transplantation. TxCAD is believed to be a form of chronic rejection of the cardiac allografts. This study was undertaken to determine whether TxCAD could develop in the absence of a cellular alloimmune response. METHODS AND RESULTS: Inbred lean Zucker rats (>26 generations) served as donors and recipients of the cardiac grafts. Donor hearts were explanted at 60 or 90 days. Explanted hearts were processed for coronary artery histological analysis. Cytokine expression was determined by reverse transcription-polymerase chain reaction, and the presence of T cells within the explanted hearts was evaluated by immunohistochemistry. Forty-six transplantations were made, and TxCAD developed in all but one of the transplanted hearts. Overall, one third of the vessels examined were affected by TxCAD, and in roughly half of these vessels, the disease was severe. Native hearts were free of atherosclerosis. Interleukin-2 was absent from the transplanted hearts, and T cells were present in minimal amounts (<1 per low-power field). CONCLUSIONS: TxCAD developed in the absence of a cellular alloimmune response in these genetically similar donors and recipients. The observed TxCAD was significant and comparable to what is found in rat allografting models.

In vitro flow experiments for determination of optimal geometry of total cavopulmonary connection for surgical repair of children with functional single ventricle.

OBJECTIVES: This study sought to evaluate the effect of offsetting cavopulmonary connections at varying pulmonary flow ratios to determine the optimal geometry of the connection. BACKGROUND: Previous investigators have demonstrated energy conservation within the streamlined contours of the total cavopulmonary connection compared with that of the atriopulmonary connection. However, their surgical design of connecting the two cavae directly opposite each other may result in high energy losses. Others have introduced a unidirectional connection with some advantages but with concerns about the formation of arteriovenous malformation in the lung excluded from hepatic venous return. Thus, an optimal surgical design has not been determined. METHODS: In the present models, the caval connections were offset through a range of 0.0 to 2.0 diameters by 0.5 superior cava diameter increments. Flow ratios were fixed for superior and inferior cavae and varied for right and left pulmonary arteries as 70:30, 60:40, 50:50, 40:60 and 30:70 to stimulate varying lung resistance. Pressure measurements and flow visualization were done at steady flows of 2, 4 and 6 liters/min to stimulate rest and exercise. RESULTS: Our data show that the energy losses at the 0.0-diameter offset were double the losses of the 1.0 and 1.5 diameters, which had minimal energy losses. This result was attributable to chaotic patterns seen on flow visualization in the 0.0-diameters offset. Energy savings were more evident at the 50:50 right/left pulmonary artery ratio. Energy losses increased with increased total flow rates. CONCLUSIONS: The results strongly suggest the incorporation of caval offsets in future total cavopulmonary connections.

Functional foods as potential therapeutic options for metabolic syndrome.

Obesity as part of metabolic syndrome is a major lifestyle disorder throughout the world. Current drug treatments for obesity produce small and usually unsustainable decreases in body weight with the risk of major adverse effects. Surgery has been the only treatment producing successful long-term weight loss. As a different but complementary approach, lifestyle modification including the use of functional foods could produce a reliable decrease in obesity with decreased comorbidities. Functional foods may include fruits such as berries, vegetables, fibre-enriched grains and beverages such as tea and coffee. Although health improvements continue to be reported for these functional foods in rodent studies, further evidence showing the translation of these results into humans is required. Thus, the concept that these fruits and vegetables will act as functional foods in humans to reduce obesity and thereby improve health remains intuitive and possible rather than proven.

NMR identification of local structural preferences in HIV-1 protease tethered heterodimer in 6 M guanidine hydrochloride.

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus-1 protease in the 'unfolded state'. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native-like, and there are also some non-native structural elements. Three-bond H(N)-H(alpha) coupling constants that could be measured for some of the N-terminal and C-terminal residues are consistent with the native-like beta-structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.

Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven by autolysis.

Structural studies in proteases have been hampered because of their inherent autolytic function. However, since autolysis is known to be mediated via protein unfolding, careful monitoring of the autolytic reaction has the potential to throw light on the folding-unfolding equilibria. In this paper we describe real time nuclear magnetic resonance investigations on the tethered dimer construct of the human immunodeficiency virus-1 protease, which have yielded insights into the relative stabilities of several residues in the protein. The residues lying along the active site (bottom, side and top of the active site) and those in helix have lower unfolding free energy values than the other parts of the protein. The residue level stability differences suggest that the protein is well suited to adjust itself in almost all the regions of its structure, as and when perturbations occur, either due to ligand binding or due to mutations.

The passage of trimethylamine across rat and human skin.

Trimethylamine is a volatile low molecular weight tertiary aliphatic amine that has known toxicity and the potential for human exposure from industrial and environmental sources is considerable. It is generally believed that absorption across the skin is an unimportant route of entry but there is little, if any, supporting evidence for this assumption. Passage across rat and human skin has been investigated employing excised skin circles in an in vitro diffusion cell apparatus. Trimethylamine was found to penetrate readily when applied to the epidermal surface of skin at three different dose levels (0.1, 1.0 and 10 mg per skin membrane 0.32 cm2). The apparent dermal flux was calculated as 3.40 +/- 1.60, 58.3 +/- 30.6 and 265.0 +/- 155.0 microg/cm2/h for rat and 0.98 +/- 0.75, 9.21 +/- 3.06 and 92.7 +/- 31.9 microg/cm2/h for human at the three dose levels, respectively. Both rat and human skin was able to act as a reservoir, with the trimethylamine not remaining in the stratum corneum but passing through. When presented to the underneath of rat and human skin circles, both [U-14C]-trimethylamine and [U-14C]-trimethylamine N-oxide were able to pass from the dermis to the epidermis. Small but detectable amounts of trimethylamine were oxidised to its N-oxide during passage through the skin.

Cloning, expression and purification of the DNA binding domain of RFX protein.

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimer DNA binding proteins which have diverse regulatory functions in eukaryotic organisms. To characterize this novel motif, a 78mer polypeptide corresponding to the DBD of human hRFX (hrfX1/DBD), a prototypical member of the RFX family has been cloned and overproduced in Escherichia coli. A purification procedure using cation exchange chromatography has also been developed.

Painless electrodiagnostic current perception threshold and pain tolerance threshold values in CRPS subjects and healthy controls: a multicenter study.

The purpose of this study is to evaluate both painless and painful sensory transmission in patients with Complex Regional Pain Syndrome (CRPS) using the automated electrodiagnostic sensory Nerve Conduction Threshold (sNCT) test. This test generates reliable, painless Current Perception Threshold (CPT) and atraumatic Pain Tolerance Threshold (PTT) measures. Standardized CPT and PTT measures using constant alternating current sinusoid waveform stimulus at 3 different frequencies 5 Hz, 250 Hz, and 2 kHz (Neurometer CPT/C Neurotron, Inc. Baltimore, MD) were obtained from CRPS subjects at a distal phalange of the affected extremity and at an ipsilateral asymptomatic control site. Matched sites were tested on healthy subjects. Detection sensitivities for an abnormal PTT and CPT test were calculated based on specificity of 90% as determined from data obtained from healthy controls. A Spearman rank correlation was used to test for a significant association between presence of allodynia and an abnormal PTT or CPT at any frequency tested. Thirty-six CRPS subjects and 57 healthy controls were tested. The highest detection sensitivity of the PTT test from symptomatic test sites was 63% for the finger and 71% for the toe. PTT abnormalities were also detected, to a lesser degree, at the asymptomatic control site (41% finger control site, 16% toe control site). The highest CPT detection sensitivity at the symptomatic site was 37% for the finger site and 53% for the toe site. CPT abnormalities were also detected at the asymptomatic control site (29% finger control site, 37% toe control site). Eighty-six percent of the CRPS subjects had either a PTT or CPT abnormality at any frequency at the symptomatic site. There was a significant correlation between presence of allodynia and presence of an abnormal CPT and PTT, respectively (P < .01). The correlation coefficient was lower for CPT than for PTT, ie, 0.34 versus 0.6 for the finger and 0.48 versus 0.67 for the toe, respectively. In studied CRPS patients an abnormal PTT was detected with higher sensitivity than an abnormal CPT. Assessing PTT may become a useful electrodiagnostic quantitative sensory test for diagnosing and following the course of neuropathic pain conditions.

Prebiotics in obesity.

Obesity was probably rare in ancient times, with the current increase starting in the Industrial Revolution of the eighteenth century, and becoming much more widespread from about 1950, so concurrent with the increased consumption of carbohydrates from cereals in the Green Revolution. However, dietary components such as oligosaccharides from plants including cereals may improve health following fermentation to short-chain carboxylic acids in the intestine by bacteria which constitute of the microbiome. Such non-digestible and fermentable components of diet, called prebiotics, have been part of the human diet since at least Palaeolithic times, and include components of the cereals domesticated in the Neolithic Revolution. If consumption of these cereals has now increased, why is obesity increasing? One reason could be lowered prebiotic intake combined with increased intake of simple sugars, thus changing the bacteria in the microbiome. Processing of food has played an important role in this change of diet composition. Since obesity is a low-grade inflammation, changing the microbiome by increased consumption of simple carbohydrates and saturated fats may lead to obesity via increased systemic inflammation. Conversely, there is now reasonable evidence that increased dietary prebiotic intake decreases inflammation, improves glucose metabolism and decreases obesity. Would widespread increases in prebiotics in the modern diet, so mimicking Palaeolithic or Neolithic nutrition, decrease the incidence and morbidity of obesity in our communities?

Molecular battles between plant and pathogenic bacteria in the phyllosphere.

The phyllosphere, i.e., the aerial parts of the plant, provides one of the most important niches for microbial colonization. This niche supports the survival and, often, proliferation of microbes such as fungi and bacteria with diverse lifestyles including epiphytes, saprophytes, and pathogens. Although most microbes may complete the life cycle on the leaf surface, pathogens must enter the leaf and multiply aggressively in the leaf interior. Natural surface openings, such as stomata, are important entry sites for bacteria. Stomata are known for their vital role in water transpiration and gas exchange between the plant and the environment that is essential for plant growth. Recent studies have shown that stomata can also play an active role in limiting bacterial invasion of both human and plant pathogenic bacteria as part of the plant innate immune system. As counter-defense, plant pathogens such as Pseudomonas syringae pv tomato (Pst) DC3000 use the virulence factor coronatine to suppress stomate-based defense. A novel and crucial early battleground in host-pathogen interaction in the phyllosphere has been discovered with broad implications in the study of bacterial pathogenesis, host immunity, and molecular ecology of bacterial diseases.

Opioid-induced bowel dysfunction: prevalence, pathophysiology and burden.

As a result of the undesired action of opioids on the gastrointestinal (GI) tract, patients receiving opioid medication for chronic pain often experience opioid-induced bowel dysfunction (OBD), the most common and debilitating symptom of which is constipation. Based on clinical experience and a comprehensive MEDLINE literature review, this paper provides the primary care physician with an overview of the prevalence, pathophysiology and burden of OBD. Patients with OBD suffer from a wide range of symptoms including constipation, decreased gastric emptying, abdominal cramping, spasm, bloating, delayed GI transit and the formation of hard dry stools. OBD can have a serious negative impact on quality of life (QoL) and the daily activities that patients feel able to perform. To relieve constipation associated with OBD, patients often use laxatives chronically (associated with risks) or alter/abandon their opioid medication, potentially sacrificing analgesia. Physicians should have greater appreciation of the prevalence, symptoms and burden of OBD. In light of the serious negative impact OBD can have on QoL, physicians should encourage dialogue with patients to facilitate optimal symptomatic management of the condition. There is a pressing need for new therapies that act upon the underlying mechanisms of OBD.

Molecular battles between plant and pathogenic bacteria in the phyllosphere

The phyllosphere, i.e., the aerial parts of the plant, provides one of the most important niches for microbial colonization. This niche supports the survival and, often, proliferation of microbes such as fungi and bacteria with diverse lifestyles including epiphytes, saprophytes, and pathogens. Although most microbes may complete the life cycle on the leaf surface, pathogens must enter the leaf and multiply aggressively in the leaf interior. Natural surface openings, such as stomata, are important entry sites for bacteria. Stomata are known for their vital role in water transpiration and gas exchange between the plant and the environment that is essential for plant growth. Recent studies have shown that stomata can also play an active role in limiting bacterial invasion of both human and plant pathogenic bacteria as part of the plant innate immune system. As counter-defense, plant pathogens such as Pseudomonas syringae pv tomato (Pst) DC3000 use the virulence factor coronatine to suppress stomate-based defense. A novel and crucial early battleground in host-pathogen interaction in the phyllosphere has been discovered with broad implications in the study of bacterial pathogenesis, host immunity, and molecular ecology of bacterial diseases.

Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of HIV-I protease tethered dimer by real time NMR spectroscopy.

HIV I protease has been the target of extensive and variety of investigations in recent years because of its importance in the AIDS viral life cycle. We describe here real time NMR studies on the unfolding kinetics of two tryptophans, W6 and W42, which are located in the dimerization and hinge domains of the protein, respectively. Unfolding seems to get initiated in the dimerization domain. The kinetic data at two temperatures, 32 and 42 degrees C, can both be described by two-state models for both the tryptophans, and the final state reached at 42 degrees C does not depend on the path of unfolding. Unfolding free energy changes derived from the kinetic fitting parameters are less than 3 kJ/mol, indicating that the energy landscape is very shallow. The free energy values and the rates for the two tryptophans are different at 32 degrees C, but are nearly the same at 42 degrees C. These are interpreted in the light of the "new view" of protein folding and the relative behaviors of the two tryptophans suggest the existence of cooperative pathways in the unfolding reaction of the protein. These observations would provide valuable insights into protein function, stability, and effects of nonactive site mutations conferring drug resistance.