Localization of vasoactive intestinal polypeptide in penile erectile tissue and in the major pelvic ganglion of the rat

Vasoactive intestinal polypeptide was localized by immunocytochemical techniques in the major pelvic ganglion and penile erectile tissue of the rat. Vasoactive intestinal polypeptide fibers were concentrated in penile crura with the density of innervation decreasing distally. The helicine arteries were very densely innervated while fewer fibers surrounded the deep artery of the penis. Intrinsic smooth muscle of the cavernous bodies received a moderate supply of vasoactive intestinal polypeptide immunoreactive fibers. Dorsal vascular structures, including the deep dorsal vein were innervated by vasoactive intestinal polypeptide fibers. Vasoactive intestinal polypeptide immunoreactive cell bodies were found in the major pelvic ganglion, concentrated on one end of the ganglion. Rectrograde studies with a dye injected into the penile crura indicated that neurons in major pelvic ganglion projected to the penis. Combined dye and immunofluorescent studies showed that all the dye-labeled neurons were immunoreactive for vasoactive intestinal polypeptide.

It is concluded that all vascular beds in the penis of the rat are innervated by vasoactive intestinal polypeptide fibers and that the extent of the innervation is related to the occurrence of smooth muscle. Neurons in the major pelvic ganglion probably are the main source of vasoactive intestinal polypeptide fibers to the penis.     

Antiproliferative activity and standardization of Tecomella undulata bark extract on K562 cells

Ethnopharmacological relevance

The bark of Tecomella undulata is primarily used in the treatment of syphilis, painful swellings and cancer by traditional healers. Also, it is claimed to be useful in treating urinary discharges, enlargement of spleen, leucorrhoea, leukoderma, tumors, liver disorders, gonorrhea, gout and promotes wound healing in Indian traditional system of medicine.

Aim

To establish a scientific validation for the antitumor effects of Tecomella undulata bark and explore the mechanistic pathway in chronic myeloid leukemia cell line, K562. The study was further extended to standardize the extract using quercetin as biomarker.

Methods

Induction of apoptosis by chloroform extract of Tecomella undulata bark (CTUB) was determined by MTT, Annexin V and caspase activation assays. The cell cycle analysis was done by flow cytometer and nuclear staining by DAPI. The standardization of the extract was performed through reverse phase-HPLC method under PDA detection.

Result

Results clearly showed the induction of apoptosis by CTUB in K562 cells. The effect was found to be dose dependent, having IC₅₀ of 30 μg/ml with activation of FAS, FADD, caspase 8, caspase 3/7 and fragmentation of DNA. The bioactive CTUB was determined to possess 0.03% (w/w) of quercetin.

Conclusion

The investigation clearly demonstrated the potential antitumor effect of CTUB, thereby validating the traditional claim. Quercetin, known to have anticancer activity is being reported and quantified for the first time from the bark of Tecomella undulata.     

Ablation of the microglial protein DOCK2 reduces amyloid burden in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) neuropathology is characterized by innate immune activation primarily through prostaglandin E2 (PGE₂) signaling. Dedicator of cytokinesis 2 (DOCK2) is a guanyl nucleotide exchange factor expressed exclusively in microglia in the brain and is regulated by PGE₂ receptor EP2. DOCK2 modulates microglia cytokine secretion, phagocytosis, and paracrine neurotoxicity. EP2 ablation in experimental AD results in reduced oxidative damage and amyloid beta (Aβ) burden. This discovery led us to hypothesize that genetic ablation of DOCK2 would replicate the anti-Aβ effects of loss of EP2 in experimental AD. To test this hypothesis, we crossed mice that lacked DOCK2 (DOCK2 −/−), were hemizygous for DOCK2 (DOCK2 +/−), or that expressed two DOCK2 genes (DOCK2 +/+) with APPswe-PS1Δe9 mice (a model of AD). While we found no DOCK2-dependent differences in cortex or in hippocampal microglia density or morphology in APPswe-PS1Δe9 mice, cerebral cortical and hippocampal Aβ plaque area and size were significantly reduced in 10-month-old APPswe-PS1Δe9/DOCK2 −/− mice compared with APPswe-PS1Δe9/DOCK2 +/+ controls. DOCK2 hemizygous APPswe-PS1Δe9 mice had intermediate Aβ plaque levels. Interestingly, soluble Aβ₄₂ was not significantly different among the three genotypes, suggesting the effects were mediated specifically in fibrillar Aβ. In combination with earlier cell culture results, our in vivo results presented here suggest DOCK2 contributes to Aβ plaque burden via regulation of microglial innate immune function and may represent a novel therapeutic target for AD.     

Iron-Oxide Nanocluster Labeling of Clostridium novyi-NT Spores for MR Imaging–Monitored Locoregional Delivery to Liver Tumors in Rat and Rabbit Models

PurposeTo label Clostridium novyi-NT spores (C. novyi-NT) with iron oxide nanoclusters and track distribution of bacteria during magnetic resonance (MR) imaging-monitored locoregional delivery to liver tumors using intratumoral injection or intra-arterial transcatheter infusion.Materials and MethodsVegetative state C. novyi-NT were labeled with iron oxide particles followed by induction of sporulation. Labeling was confirmed with fluorescence microscopy and transmission electron microscopy (TEM). T2 and T2* relaxation times for magnetic clusters and magnetic microspheres were determined using 7T and 1.5T MR imaging scanners. In vitro assays compared labeled bacteria viability and oncolytic potential to unlabeled controls. Labeled spores were either directly injected into N1-S1 rodent liver tumors (n = 24) or selectively infused via the hepatic artery in rabbits with VX2 liver tumors (n = 3). Hematoxylin-eosin, Prussian blue, and gram staining were performed. Statistical comparison methods included paired t-test and ANOVA.ResultsBoth fluorescence microscopy and TEM studies confirmed presence of iron oxide labels within the bacterial spores. Phantom studies demonstrated that the synthesized nanoclusters produce R2 relaxivities comparable to clinical agents. Labeling had no significant impact on overall growth or oncolytic properties (P >.05). Tumor signal-to-noise ratio (SNR) decreased significantly following intratumoral injection and intra-arterial infusion of labeled spores (P <.05). Prussian blue and gram staining confirmed spore delivery.ConclusionsC. novyi-NT spores can be internally labeled with iron oxide nanoparticles to visualize distribution with MR imaging during locoregional bacteriolytic therapy involving direct injection or intra-arterial transcatheter infusion.     

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.     

Detection of human chromosomal abnormalities using a new technique combining 4',6-diamidino-2-phenyl-indole staining and image analysis

Chromosomal abnormalities are associated with a variety of diseases. We have developed a new technique for detecting chromosomal abnormalities, and the technique combines conventional 4',6-diamidino-2-phenyl-indole staining (DAPI) with image analysis. The image analysis consists of two simple steps: deconvolution and three-dimensional reconstruction. The technique has been reported for analyzing plant chromosomes but has not been applied to analyze human chromosomes yet. To test the technique, we analyzed five translocations: 46,XX,t(3;21)(12;18), 46,XX,t(11;22), 46,XY,t(7;22), 46,XY,t(11;18), and 46,XY,t(3;7). The results showed that the karyotype of the 46,XX,t(3;21)(12;18) was 46,XX,t(3;21)(q11.1;p13),t(12;18) (q21.2;q23), and the karyotypes of the 46,XX,t(11;22), 46,XY,t(7;22), 46,XY,t(11;18), and 46,XY,t(3;7) were 46,XX,t(11;22)(q23;q12.1); 46,XY,t(7;22)(q32;q13.2); 46,XY,t(11;18)(q13.3;q23), and 46,XY,t(3;7)(q22.1;p13), respectively. The identity of derivative chromosomes involved in the translocations was verified by chromosome painting as well as FISH analyses with centromere probes. The new technique has two advantages: the procedure is simple and convenient, and the results are accurate. The technique has the potential to be used in cytogenetic studies and clinical diagnosis of human diseases in the future.     

Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation.     

Serial culture of murine primary airway epithelial cells and ex vivo replication of human rhinoviruses

Human rhinoviruses (HRV) are the primary etiological agents in cold infections, and represent a serious risk to individuals with chronic respiratory disease such as asthma. In order to develop treatment options for HRV infections, murine models are a crucial component in the study of infection mechanisms due to the wide array of reagents and techniques available to study murine immunology. We present here a cell culture system for studying isolated murine epithelial cell responses to HRV. Monolayers of primary mouse airway epithelial cells were maintained in a serial culture system, and the identity and purity of the cell population was confirmed via immunostaining (positive for cytokeratin, negative for vimentin). Infection of these cells with a minor group rhinovirus (HRV-1A) was evidenced by increases in viral RNA, de novo synthesis of viral proteins, and production of infectious virus. This model will be useful in experiments to define mechanisms of viral replication and host/virus interactions within airway epithelial cells.     

High levels of histidine decarboxylase in the striatum of mice and rats

Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a novel fibrinolytic agent with anti-inflammatory effect. Previous study demonstrated that SMTP-7 further ameliorated infarction volume in a mouse embolic stroke model compared with tissue type plasminogen activator (tPA), but the reason SMTP-7 has more beneficial effect than tPA has not yet been determined. In the present study, we investigated whether SMTP-7 has an intrinsic neuroprotective effect against transient focal cerebral ischemia (tFCI). Sprague-Dawley rats were subjected to tFCI by intraluminal middle cerebral artery occlusion for 2 h. Following induction of tFCI, rats were randomized into two groups based on the agent administered: SMTP-7 group and vehicle group. We examined cerebral infarction volume 24 h after reperfusion, and evaluated superoxide production, the expressions of nitrotyrosine and matrix metalloproteinase-9 (MMP-9), which play major roles in secondary brain injury and hemorrhagic transformation. The findings showed that SMTP-7 significantly suppressed superoxide production, the expression of nitrotyrosine and MMP-9 after tFCI, and consequently attenuated ischemic neuronal damage. These results suggest that SMTP-7 has an intrinsic neuroprotective effect on ischemia/reperfusion injury through the suppression of oxidative stress and MMP-9 activation.