Attochemistry Regulation of Charge Migration.

Charge migration (CM) is a coherent attosecond process that involves the movement of localized holes across a molecule. To determine the relationship between a molecule's structure and the CM dynamics it exhibits, we perform systematic studies of para-functionalized bromobenzene molecules (X-C6H4-R) using real-time time-dependent density functional theory. We initiate valence-electron dynamics by emulating rapid strong-field ionization leading to a localized hole on the bromine atom. The resulting CM, which takes on the order of 1 fs, occurs via an X localized → C6H4 delocalized → R localized mechanism. Interestingly, the hole contrast on the acceptor functional group increases with increasing electron-donating strength. This trend is well-described by the Hammett σ value of the group, which is a commonly used metric for quantifying the effect of functionalization on the chemical reactivity of benzene derivatives. These results suggest that simple attochemistry principles and a density-based picture can be used to predict and understand CM.

Patient quality of life and short-term surgical outcomes between robotic and laparoscopic anterior resection for adenocarcinoma of the rectum.

PURPOSE: To compare patient quality of life (QoL) and short-term surgical outcomes between robotic anterior resection (r-AR) and laparoscopic (l-AR) approach. METHODS: Consecutive patients having undergone either robotic or laparoscopic AR for adenocarcinoma were studied. All operations were performed by two surgeons experienced in laparoscopic and recently introduced robotic surgery. Surgical outcomes were determined by post-operative histology and short-term complications. QoL was prospectively assessed using the EORTC QLC-CR30 and QLC-CR29 questionnaires. RESULTS: In total, 36 patients (18 r-AR) with a median follow-up of 12 months following surgery (9-month robotic and 20-month laparoscopic) were studied. The two groups were similarly matched for age and gender. Laparoscopic patients had a lower ASA grade (p = 0.02). There was no significant difference in surgical outcomes between groups. r-AR patients reported lower pain scales (2 ± 6 vs. 11 ± 13) (p = 0.04), lower levels of insomnia 0 vs. 8 ± 15 (p = 0.04) and a lower abdominal pain scale (2 ± 9 vs. 17 ± 27) (p = 0.04). Male impotence scores were higher in l-AR 33 ± 35 compared to r-AR 7 ± 21 (p = 0.03). CONCLUSION: Despite its recent introduction to our centre, the quality of oncological resection using the robotic surgery is comparable to laparoscopy. Lower impotence and QoL scores in patients after robotic procedure may be explained on the basis of better visualisation and precise tissue handling.

Magnetic Resonance Cholangiopancreatography (MRCP) Evaluation of Obstructive Jaundice in Comparison with Endoscopic Retrograde Cholangiopancreatography (ERCP).

This cross sectional study was carried out in the department of Radiology and Imaging in collaboration with Department of Gastroenterology of Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh from July 2013 to June 2014 to evaluate the efficacy of Magnetic resonance cholangiopancreatography (MRCP) and ERCP in the management of obstructive jaundice and also to determine diagnostic validity accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of MRCP in evaluation of obstructive jaundice. For this purpose, a total of 60 patients with obstructive jaundice who underwent MRCP and Endoscopic retrograde cholangiopancreatography (ERCP) in the above mentioned hospital were enrolled. More than one third (35.0%) patients were in 5th decade and the mean age was 46.2±12.9 years. Male female ratio was 1.1:1. Most (45.0%) of the patients had filling defect, 28.3% had concentric stenosis and 26.7% eccentric stenosis. In MRCP findings more than one third (35.0%) patients had choledocholithiasis followed by 26.7% had cholangiocarcinoma, 10.0% benign CBD stricture and 8.3% had ascariasis. In ERCP findings 31.7% patients had choledocholithiasis followed by 16.7% had cholangiocarcinoma, 13.3% benign CBD stricture and 10.0% ascariasis. All patients had increased serum bilirubin.

Protective effect of sterubin against neurochemical and behavioral impairments in rotenone-induced Parkinson's disease.

This study was conducted to evaluate how sterubin affects rotenone-induced Parkinson's disease (PD) in rats. A total of 24 rats were distributed into 4 equal groups: normal saline control and rotenone control were administered saline or rotenone (ROT), respectively, orally; sterubin 10 received ROT + sterubin 10 mg/kg po; and sterubin alone was administered to the test group (10 mg/kg). Rats of the normal saline and sterubin alone groups received sunflower oil injection (sc) daily, 1 h after receiving the treatments cited above, while rats of the other groups received rotenone injection (0.5 mg/kg, sc). The treatment was continued over the course of 28 days daily. On the 29th day, catalepsy and akinesia were assessed. The rats were then euthanized, and the brain was extracted for estimation of endogenous antioxidants (MDA: malondialdehyde, GSH: reduced glutathione, CAT: catalase, SOD: superoxide dismutase), nitrative (nitrite) stress markers, neuroinflammatory cytokines, and neurotransmitter levels and their metabolites (3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA), norepinephrine (NE), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA)). Akinesia and catatonia caused by ROT reduced the levels of endogenous antioxidants (GSH, CAT, and SOD), elevated the MDA level, and altered the levels of nitrites, neurotransmitters, and their metabolites. Sterubin restored the neurobehavioral deficits, oxidative stress, and metabolites of altered neurotransmitters caused by ROT. Results demonstrated the anti-Parkinson's activities of sterubin in ROT-treated rats.

GSTPi-positive tumour microenvironment-associated fibroblasts are significantly associated with GSTPi-negative cancer cells in paired cases of primary invasive breast cancer and axillary lymph node metastases.

BACKGROUND: Glutathione S-transferase Pi (GSTPi) expression is one of the factors, which is known to be associated with development of resistance to chemotherapeutics in cancer patients, including those with breast cancer. Yet, its expression has been reported to be undetectable in cancer cells in high percent of patients with primary breast cancer. However, GSTPi expression in stromal cells in breast tumour microenvironment, namely cancer-associated fibroblast (CAF), which is recognised to have major roles in cancer progression, remains poorly reported. METHODS: The aim of the study was to determine the expression of GSTPi; vimetin, a fibroblast-associated cytoskeleton protein; and α-smooth muscle actin (α-SMA), a known marker of CAF in breast cancer tissue, by immunohistochemical staining method in consecutive histologic sections of formalin-fixed and paraffin-embedded tissue biopsy specimens from a cohort of 39 paired cases of patients with invasive breast cancer and the corresponding axillary lymph nodes metastases. RESULTS: Ductal and acinar luminal epithelial cells, myoepithelial cells and surrounding fibroblasts exhibited a homogeneous cytoplasmic reactivity with anti-GSTPi antibody in 11 of 11 cases of benign breast tissue biopsies. The vimentin-positive fibroblasts were unreactive with anti-α-SMA antibody. Loss of GSTPi expression was observed in breast cancer cells, at both the primary and metastatic sites, in 31 of 39 paired cases, as compared with benign breast epithelial cells (Fisher's exact test P<0.001). A significant association was observed between GSTPi-positive, vimentin-positive and α-SMA-positive fibroblast in tumour microenvironment at both sites. CONCLUSION: This is an original report of demonstration of a significance association between tumour microenvironment-associated GSTPi-positive CAF (vimentin/α-SMA-positive) and the GSTPi-negative cancer cells in paired cases of primary invasive breast cancer and the corresponding axillary lymph nodes metastases.

Evaluation of matrix effects in analysis of estrogen using liquid chromatography-tandem mass spectrometry.

Matrix effects of different biological samples, including phosphate-buffered saline-bovine serum albumin (PBS-BSA), gelded horse serum, mouse serum, and mouse brain, were investigated for the determination of 17α- and ß-estradiol using derivatization with dansyl chloride prior to LC-MS/MS. Matrix effects were evaluated based on the slopes of regression lines plotted from results obtained in biological matrices versus pure standard solutions. Such plots indicate the enhancement or suppression of signal based on the presence of a particular biological fluid for a particular method. The matrix effects from PBS-BSA were similar to those of mouse serum. In contrast, analyses performed from horse serum and mouse brain yielded significant ion suppression, especially for 17ß-estradiol. Precipitation during derivatization was observed when pre-concentrated samples were processed with ethyl acetate as an extraction solvent. This was overcome with the use of methyl tert-butyl ether; however, matrix effects from this preparation were still present, evidenced by signal suppression and poor linearity in the standard curve. This work affirms that caution should be taken in the transfer of methods for use with different biological matrices, especially in the case where surrogate matrices are necessary for calibration purposes.

Luteal blood flow and concentrations of circulating progesterone and other hormones associated with a simulated pulse of 13,14-dihydro-15-keto-prostaglandin F2alpha in heifers.

Progesterone and luteal blood flow effects of an i.u. 2-h infusion of 0.25 mg/h of prostaglandin F(2)(alpha) (PGF) that simulated a natural pulse of 13,14-dihydro-15-keto-PGF (PGFM) were compared to the effects of a single bolus i.u. injection of PGF (4 mg) that induced complete luteolysis in heifers. Blood sampling and an estimate of the percentage of luteal area with colour-Doppler signals of blood flow were performed every 2 min for 20 min and less frequently thereafter for 6 h. After the beginning of PGF infusion or a bolus injection, progesterone increased to a peak at 14 and 10 min respectively, and was accompanied by an increase in blood flow in the bolus group but not in the infusion group. Progesterone then decreased for 1 or 2 h and was accompanied by a continued elevation in blood flow in the PGF bolus group and by a slight increase in the PGF infusion group. Progesterone then rebounded in both groups, but the rebound was greater in the infusion group. Blood flow decreased during the descending arm of the progesterone rebound. Cortisol and prolactin began to increase 6 min after the bolus PGF injection but did not increase during or after PGF infusion. The increases in cortisol, prolactin and blood flow after a PGF bolus treatment but not during a simulated PGFM pulse indicated that the bolus treatment was pharmacologic, and its use may lead to faulty conclusions on the nature of physiologic luteolysis. The comparisons between progesterone and blood flow are novel.

Supervised and unsupervised methods for prostate cancer segmentation with multispectral MRI.

PURPOSE: Magnetic resonance imaging (MRI) has been proposed as a promising alternative to transrectal ultrasound for the detection and localization of prostate cancer and fusing the information from multispectral MR images is currently an active research area. In this study, the goal is to develop automated methods that combine the pharmacokinetic parameters derived from dynamic contrast enhanced (DCE) MRI with quantitative T2 MRI and diffusion weighted imaging (DWI) in contrast to most of the studies which were performed with human readers. The main advantages of the automated methods are that the observer variability is removed and easily reproducible results can be efficiently obtained when the methods are applied to a test data. The goal is also to compare the performance of automated supervised and unsupervised methods for prostate cancer localization with multispectral MRI. METHODS: The authors use multispectral MRI data from 20 patients with biopsy-confirmed prostate cancer patients, and the image set consists of parameters derived from T2, DWI, and DCE-MRI. The authors utilize large margin classifiers for prostate cancer segmentation and compare them to an unsupervised method the authors have previously developed. The authors also develop thresholding schemes to tune support vector machines (SVMs) and their probabilistic counterparts, relevance vector machines (RVMs), for an improved performance with respect to a selected criterion. Moreover, the authors apply a thresholding method to make the unsupervised fuzzy Markov random fields method fully automatic. RESULTS: The authors have developed a supervised machine learning method that performs better than the previously developed unsupervised method and, additionally, have found that there is no significant difference between the SVM and RVM segmentation results. The results also show that the proposed methods for threshold selection can be used to tune the automated segmentation methods to optimize results for certain criteria such as accuracy or sensitivity. The test results of the automated algorithms indicate that using multispectral MRI improves prostate cancer segmentation performance when compared to single MR images, a result similar to the human reader studies that were performed before. CONCLUSIONS: The automated methods presented here can help diagnose and detect prostate cancer, and improve segmentation results. For that purpose, multispectral MRI provides better information about cancer and normal regions in the prostate when compared to methods that use single MRI techniques; thus, the different MRI measurements provide complementary information in the automated methods. Moreover, the use of supervised algorithms in such automated methods remain a good alternative to the use of unsupervised algorithms.

Effect of oviductal proteins on structural and functional characteristics of cryopreserved sperm in Murrah buffaloes.

The present study was undertaken to elucidate the effect of non-luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non-luteal based on ovarian morphology. Non-luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10,000 g at 4 degrees C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at -20 degrees C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris-citrate-egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non-luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5 degrees C, filled in 0.5 ml French straws, exposed to LN(2) vapour, plunged into LN(2) and then stored at -196 degrees C. The equilibrated and frozen-thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo-osmotic sperm swelling test (HOST). In frozen-thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non-luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze-thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen-thawed semen. It was inferred that incorporation of non-luteal whole oviductal fluid proteins improved the sperm quality in frozen-thawed semen in Murrah buffaloes.

Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARbeta2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens.

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted "gold standard" for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARbeta2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARbeta2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher's exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARbeta2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARbeta2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.

The Chlamydomonas cell wall degrading enzyme, lysin, acts on two substrates within the framework of the wall.

The Chlamydomonas cell wall is a multilayered, extracellular matrix containing 20-25 proteins and glycoproteins, many of which are highly enriched in hydroxyproline. 80-90% of the wall protein is located in a crystalline portion of the wall that is soluble in sarkosyl-urea solutions as well as in chaotropic salts. Although the wall has no cellulose it contains a noncrystalline, highly insoluble framework portion that is responsible for the integrity and overall shape of the wall. In the present report we show that the framework of the wall is composed of two components that are acted upon by lysin, a wall degrading enzyme released by mating gametes. One, which makes up the major portion of the framework, is insoluble upon boiling in SDS-PAGE sample buffer. Lysin treatment of this portion leads to its physical degradation and the concomitant appearance of several SDS-dithiothreitol-soluble polypeptides ranging in relative molecular mass from greater than 400,000 to less than 60,000. The second component is the flagellar collar. This hollow cylinder composed of striated fibers aligned in parallel array serves as the tunnel in the wall through which the flagella protrude. Our evidence indicates that the primary collar polypeptide is a 225,000-Mr molecule that itself has at least two functional domains. One domain, contained in a 185,000-Mr fragment, permits the self-association of the molecules to form the main body of the collar. The second part of the molecule anchors the collar to the wall framework via sarkosyl-urea-insensitive, SDS-dithiothreitol-sensitive linkages.

A monoclonal antibody that blocks adhesion of Chlamydomonas mt+ gametes.

During the mating reaction (fertilization) in the biflagellated alga, Chlamydomonas reinhardtii, mt+ and mt- gametes adhere to each other via their flagella and subsequently fuse to form quadriflagellated zygotes. In the studies reported here, we describe a monoclonal antibody directed against an mt+ flagellar surface molecule. The antibody blocks the adhesiveness of mt+ gametes, isolated mt+ flagella, and detergent extracts thereof. It has no effect on mt- gametes. Cyanogen bromide-activated Sepharose beads derivatized with the antibody bind only mt+ gametes; mt- gametes and mt+ and mt- vegetative cells are unreactive with the derivatized beads. The interaction of mt+ gametes with the beads is dynamic and cells continuously bind, detach, and rebind to the beads. Surprisingly, antibody-derivatized beads that have been incubated with mt+ gametes acquire the ability to bind mt- gametes. Moreover, extraction of the preincubated beads with detergents releases active mt+ adhesion molecules. The evidence suggests that binding of the antibody to the flagellar surface adhesion molecules causes their release from the flagellar surface, possibly mimicking the normal mechanism of flagellar de-adhesion.

The Chlamydomonas cell wall: characterization of the wall framework.

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.

Activation of the cell wall degrading protease, lysin, during sexual signalling in Chlamydomonas: the enzyme is stored as an inactive, higher relative molecular mass precursor in the periplasm.

During the mating reaction in Chlamydomonas reinhardtii mating type plus and mating type minus gametes adhere to each other via adhesion molecules on their flagellar surfaces. This adhesive interaction induces a sexual signal leading to release of a cell wall degrading enzyme, lysin, that causes wall release and degradation. In this article, we describe the preparation of a polyclonal antibody against the 60,000-Mr lysin polypeptide excised from SDS-PAGE gels. After absorption of the IgG with cell walls to remove antibodies against a carbohydrate epitope common to several Chlamydomonas glycoproteins, the immune IgG reacted with the 60,000-Mr polypeptide, and a 47,000-Mr species that we show here was immunologically cross-reactive with the 60,000-Mr molecule. By use of several fractionation methods including ion exchange and molecular sieve chromatography, sucrose gradient centrifugation, and affinity chromatography, we showed that the 60,000-Mr antigen copurified with lysin activity, thereby demonstrating that the antibody was indeed directed against the enzyme. Immunoblot experiments on suspensions of nonmating and mating gametes showed that the 60,000-Mr antigen was missing in the nonmating gametes. Instead, they contained a 62,000-Mr antigen that was not present in suspensions of mating gametes that had undergone sexual signalling. Furthermore, nonmating gametes whose walls were removed with exogenously added lysin did not contain either form of the antigen. We also found that the 62,000-Mr form of the antigen, which could be released from gametes by freeze-thawing, did not have wall degrading activity. These results indicate that lysin in gametes is stored in the periplasm as a higher relative molecular mass, inactive precursor and also that sexual signalling induces conversion of this molecule to a lower relative molecular mass, active enzyme. This may be a novel example of processing of an extracellular protease induced by cell contact.

Diabetic myopathy differs between Ins2Akita+/- and streptozotocin-induced Type 1 diabetic models.

Mechanistic studies examining the effects of Type 1 diabetes mellitus (T1DM) on skeletal muscle have largely relied on streptozotocin-induced diabetic (STZ) rodents. Unfortunately, characterization of diabetic myopathy in this model is confounded by the effects of streptozotocin on skeletal muscle independent of the diabetic phenotype. Here we define adolescent diabetic myopathy in a novel, genetic model of T1DM, Ins2(Akita+/-) mice, and contrast these findings with STZ mice. Eight weeks of diabetes resulted in significantly reduced gastrocnemius-plantaris-soleus mass (control: 0.16 +/- 0.005 g; Ins2(Akita+/-): 0.12 +/- 0.003 g; STZ: 0.12 +/- 0.01g) and IIB/D fiber area in Ins2(Akita+/-) (1,294 +/- 94 microm(2)) and STZ (1,768 +/- 163 microm(2)) compared with control (2,241 +/- 144 microm(2)). Conversely, STZ type I fibers (1,535 +/- 165 microm(2)) were significantly larger than Ins2(Akita+/-) (915 +/- 76 microm(2)) but not control (1,152 +/- 86 microm(2)). Intramyocellular lipid increased in STZ (122.9 +/- 3.6% of control) but not Ins2(Akita+/-) likely resultant from depressed citrate synthase (control: 6.2 +/- 1.2 micromol.s(-1).mg(-1); Ins2(Akita+/-): 5.2 +/- 0.8 micromol.s(-1).mg(-1); STZ: 2.8 +/- 0.5 micromol.s(-1).mg(-1)) and 3-beta-hydroxyacyl coenzyme-A dehydrogenase (control: 4.2 +/- 0.6 nmol.s(-1).mg(-1); Ins2(Akita+/-): 5.0 +/- 0.6 nmol.s(-1).mg(-1); STZ: 2.7 +/- 0.6 nmol.s(-1).mg(-1)) enzyme activity in STZ muscle. In situ muscle stimulation revealed lower absolute peak tetanic force in Ins2(Akita+/-) (70.2 +/- 8.2% of control) while STZ exhibited an insignificant decrease (87.6 +/- 7.9% of control). Corrected for muscle mass, no force loss was observed in Ins2(Akita+/-), while STZ was significantly elevated vs. control and Ins2(Akita+/-). These results demonstrate that atrophy and specific fiber-type loss in Ins2(Akita+/-) muscle did not affect contractile properties (relative to muscle mass). Furthermore, we demonstrate distinctive contractile, metabolic, and phenotypic properties in STZ vs. Ins2(Akita+/-) diabetic muscle despite similarity in hyperglycemia/hypoinsulinemia, raising concerns of our current state of knowledge regarding the effects of T1DM on skeletal muscle.

J1-31 protein expression in astrocytes and astrocytomas.

J1-31 is one of the astrocytic proteins, the expression of which has not been evaluated in astrocytomas. In the present study, we studied the expression of J1-31 protein in astrocytes and astrocytomas in comparison with GFAP, p53 and Ki-67. Materials consisted of formalin-fixed paraffin-embedded tissue specimens that included five cases of normal brain, 17 of gliosis, 15 of pilocytic astrocytoma (WHO grade I), 26 of low-grade diffuse astrocytoma (WHO grade II), four of anaplastic astrocytoma (WHO grade III), and eight of glioblastoma (WHO grade IV). GFAP was highly expressed in all specimens examined. The anti-J1-31 antibody exhibited strong cytoplasmic staining of reactive gliosis in 17/17 (100%) cases with a higher intensity of staining than that observed in the adjacent normal astrocytes. The antibody showed reactivity with tumor cells in 12/15 (80%) cases of pilocytic astrocytoma, although intensity of staining was generally weaker and more focal than observed in reactive gliosis. J1-31-positive tumor cells were detected in only 9/26 (35%) cases of the low-grade diffuse astrocytoma and none of the cases of anaplastic astrocytoma and glioblastoma. Increasing Ki-67 indices paralleled advancing tumor grades. p53 protein was expressed more commonly in infiltrating astrocytomas compared to pilocytic astrocytoma. In conclusion, down-regulation of J1-31 expression correlates with advancing grade of astrocytomas. The result suggests this protein plays some role in astrocytes that is progressively lost in malignant progression. The anti-J1-31 antibody may help further our understanding of astrocytes in disease and may be useful as an aid in the pathologic diagnosis of astrocytic lesions.

Protective effect of sterubin against neurochemical and behavioral impairments in rotenone-induced Parkinson's disease

This study was conducted to evaluate how sterubin affects rotenone-induced Parkinson's disease (PD) in rats. A total of 24 rats were distributed into 4 equal groups: normal saline control and rotenone control were administered saline or rotenone (ROT), respectively, orally; sterubin 10 received ROT + sterubin 10 mg/kg po; and sterubin alone was administered to the test group (10 mg/kg). Rats of the normal saline and sterubin alone groups received sunflower oil injection (sc) daily, 1 h after receiving the treatments cited above, while rats of the other groups received rotenone injection (0.5 mg/kg, sc). The treatment was continued over the course of 28 days daily. On the 29th day, catalepsy and akinesia were assessed. The rats were then euthanized, and the brain was extracted for estimation of endogenous antioxidants (MDA: malondialdehyde, GSH: reduced glutathione, CAT: catalase, SOD: superoxide dismutase), nitrative (nitrite) stress markers, neuroinflammatory cytokines, and neurotransmitter levels and their metabolites (3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA), norepinephrine (NE), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA)). Akinesia and catatonia caused by ROT reduced the levels of endogenous antioxidants (GSH, CAT, and SOD), elevated the MDA level, and altered the levels of nitrites, neurotransmitters, and their metabolites. Sterubin restored the neurobehavioral deficits, oxidative stress, and metabolites of altered neurotransmitters caused by ROT. Results demonstrated the anti-Parkinson's activities of sterubin in ROT-treated rats.

Unmethylated E-cadherin gene expression is significantly associated with metastatic human prostate cancer cells in bone.

BACKGROUND: The concurrent determination of methylation status of E-cadherin gene and E-cadherin protein expression remains scant in metastatic prostate cancer cells in bone, the most prevalent site for metastatic growth. Therefore, the study was undertaken to ascertain the methylation status of E-cadherin gene, a most frequent and known epigenetic mechanism of its regulation, and the protein expression in prostate tissue biopsy specimen. METHODS: The methylation of E-cadherin gene was determined by methylation specific-PCR and the protein expression by immunohistochemical method in the consecutive sections of each prostate tissue biopsy specimen. RESULTS: The unmethylated E-cadherin gene and homogeneous E-cadherin protein expression was significantly associated with BPH as compared to the primary prostate carcinoma (Fisher's Exact P < 0.001). A significant association was observed between the concurrent methylated gene and markedly reduced expression of the protein in the primary prostate cancer cells as compared to the BPH cells, suggesting methylation-dependent regulation of the gene expression in these cases. In contrast to the primary cancer, a highly significant increase in the frequency of metastatic prostate cancer cells in bone exhibited the concurrent expression of unmethylated gene and homogeneous protein (Fisher's Exact P < 0.001). CONCLUSIONS: The study clearly demonstrated a significant association of the concurrent expression of unmethylated E-cadherin gene and E-cadherin protein with metastatic prostate cancer cells in bone, and that its expression may have a role in the intercellular adhesion in the formation of metastatic lesions in bone.

Effect of oviductal fluid proteins on buffalo sperm characteristics during cryopreservation.

The objective was to determine the effects of oviductal proteins on sperm function. Abbatoir-derived buffalo oviducts were flushed with PBS; the fluid recovered (protein concentration, 2.3 mg/mL; average of 3.5 mg protein/oviduct) was centrifuged, dialyzed, and clarified, and the supernatant applied to a Heparin-Sepharose affinity column. Unbound fractions were collected and bound proteins were separately eluted (with elution buffer). Eight distinct protein bands (from 12 to 177 kDa) in the H-unbound fraction and 15 distinct protein bands (from 12 to 165 kDa) in the H-bound fraction were detected in SDS-PAGE. Semen from four buffalo bulls was divided into three parts: Parts 1 and 2 were treated with the heparin binding (H-bound) and non-heparin binding (H-unbound) oviductal proteins, respectively, whereas Part 3 remained as an untreated control. Equilibrated and frozen-thawed semen was assessed for motility, viability, intact acrosome percentage, mucus penetration distance, and hypo-osmotic swelling test. The H-bound oviductal fluid proteins enhanced (P<0.05) the proportion of sperm that were progressively motile, alive, had an intact acrosome and functional plasma membrane (hypo-osmotic swelling test), as well as the distance covered in the cervical mucus sperm penetration test during cryopreservation. Addition of the H-unbound oviductal protein fraction did not increase sperm motility and penetration distance but increased (P<0.05) the proportion of sperm that were live, had an intact acrosome, and functional plasma membrane (hypo-osmotic swelling test). We concluded that the H-bound fraction of buffalo oviductal fluid protein(s) maintained sperm motility, viability and membrane integrity during cryopreservation, whereas the H-unbound proteins maintained sperm viability and membrane integrity.

Epistaxis: when are coagulation studies justified?

BACKGROUND: Epistaxis is a common emergency in otolaryngology. There is some evidence questioning the use of routine coagulation studies (prothrombin time and activated partial thromboplastin time (APTT)) in these patients, but this practice continues in most centres. AIM: To identify groups of patients likely to present with coagulation abnormalities. METHODS: Charts of all patients aged >14 years with epistaxis, requiring admission to Aga Khan University Hospital, Karachi, Pakistan, through the emergency department between January 2002 and December 2005, were retrospectively reviewed for the presence of comorbid conditions and coagulation abnormalities. Deranged coagulation was defined as an APTT of >7 s above control or an international normalised ratio >1.5. Analysis was carried out using SPSS V.13.0. RESULTS: All 108 patients were included in the study (male patients, 71.3%; female patients, 28.7%; mean age 40.4 years). Only 49 patients had an associated condition that could potentially cause epistaxis. Of these, the coagulation profiles of 10 patients were deranged, which included 6 patients receiving anticoagulant treatment, 2 with chronic active hepatitis, 1 with liver cancer and 1 with haemophilia. CONCLUSION: Routine coagulation screening of all patients with epistaxis is of little value and only adds to treatment costs and emergency room occupancy times. Comorbid conditions such as hypertension or cases with thrombocytopenia do not merit coagulation screening. However, coagulation studies are justified in patients receiving anticoagulant treatment and those with known coagulopathy or chronic liver disease.