Normalization is essential to the in-cell western (ICW) assay, a near-infrared immunocyto-blot for protein analysis. Here we report that cells reacted with glutaraldehyde fluoresced in the near-infrared region of the spectrum, and the intensity of fluorescence was directly proportional to cell number over a range of 3,125 to 100,000 per well. We took advantage of this property to develop a method for quantification of cells, and applied it to ICW assay for normalization. The application of glutaraldehyde may make the ICW assay more popular due to the reduced cost and simplified procedure.

Recent studies have demonstrated that immunization with nontoxic mutant staphylococcal enterotoxin C (mSEC) provides protection against Staphylococcus aureus infection in mouse models. In the present study, we investigated whether vaccination with a glutathione S-transferase-fused SEC (GST-mSEC) can protect against S. aureus-induced bovine mastitis. Cows were immunized with the GST-mSEC plus alum adjuvant and then challenged with viable S. aureus by an intramammary route. The results showed that immunization with GST-mSEC-induced production of SEC-specific antibodies in sera and the high titers of antibodies could persist for over 12 weeks. Importantly, immunization with GST-mSEC also induced production of SEC-specific antibodies in milk. The somatic cell counts in the milk from S. aureus challenged quarters of vaccinated lactating cows were significantly lower than those of the non-vaccinated control animals. Furthermore, the sera from GST-mSEC-immunized cows significantly inhibited interferon-gamma and tumor necrosis factor-alpha production from mouse spleen cells induced by wild-type SEC. These results suggest that vaccination with GST-mSEC provides protection against S. aureus-induced bovine mastitis and that the protection might be mediated by SEC-neutralizing antibodies.

The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has been widely applied in many fields. In this study, a special designed superconducting magnet, which can produce three apparent gravity levels (0, 1, and 2 g), namely high magneto-gravitational environment (HMGE), was used to simulate space gravity environment. The effects of HMGE on osteoblast gene expression profile were investigated by microarray. Genes sensitive to diamagnetic levitation environment (0 g), gravity changes, and high magnetic field changes were sorted on the basis of typical cell functions. Cytoskeleton, as an intracellular load-bearing structure, plays an important role in gravity perception. Therefore, 13 cytoskeleton-related genes were chosen according to the results of microarray analysis, and the expressions of these genes were found to be altered under HMGE by real-time PCR. Based on the PCR results, the expressions of WASF2 (WAS protein family, member 2), WIPF1 (WAS/WASL interacting protein family, member 1), paxillin, and talin 1 were further identified by western blot assay. Results indicated that WASF2 and WIPF1 were more sensitive to altered gravity levels, and talin 1 and paxillin were sensitive to both magnetic field and gravity changes. Our findings demonstrated that HMGE can affect osteoblast gene expression profile and cytoskeleton-related genes expression. The identification of mechanosensitive genes may enhance our understandings to the mechanism of bone loss induced by microgravity and may provide some potential targets for preventing and treating bone loss or osteoporosis.

OBJECTIVES:Irritable bowel syndrome (IBS) is associated with a state of chronic visceral hypersensitivity, but the underlying molecular mechanisms of visceral hyperalgesia remain elusive. This study was designed to examine changes in the excitability and alterations of voltage-gated K(+) currents in subpopulations of colonic dorsal root ganglion (DRG) neurons in a rat model of IBS-like visceral hypersensitivity.METHODS:The model of IBS-like visceral hypersensitivity was induced by intracolonic infusion of 0.5% acetic acid (AA) in saline from postnatal days 8 -21. Experiments were conducted when rats became adults. DRG neurons innervating the colon were identified by 1,1'-dioleoyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate (DiI) fluorescence labeling and were immunostained for isolectin B4 (IB4) binding to classify these colonic neurons. Patch-clamp recordings were made from acutely dissociated DiI-labeled DRG neurons, and the expression of K(+) channel in L6-S2 DRG was examined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot.RESULTS:(1) Neonatal AA treatment induced long-lasting visceral hypersensitivity without significant inflammation but with mast cell hyperplasia.(2) Colonic DRG neurons contained IB4-positive and negative neurons with different electrophysiological properties. IB4-positive colonic neurons have longer action potentials (APs) and larger A-type K(+) currents (I(A)) than the IB4-negative neurons, and IB4 phenotypic changes of colonic neurons were not involved in the chronic visceral hypersensitivity.(3) Neonatal AA treatment decreased I(A) density and changed the electrophysiological properties of I(A) and I(K) by shifting the steady-state inactivation toward a negative direction in IB4-positive colonic neurons. The excitability of these cells increased.(4) Kv4.3 was downregulated in neonatal AA-treated rats compared with control rats, which suggests a possible mechanism regarding the changes in electrical activity of DRG neurons in these rats.CONCLUSIONS:A new model for chronic visceral hypersensitivity following a diluted AA stimulus in the neonatal period is described. The hypersensitivity may be associated with mast cell hyperplasia in the colon and increased excitability of IB4-positive colonic neurons as a result of suppression of I(A) density and a shift in the inactivation curves of I(A) and I(K) in a hyperpolarizing direction in these cells. This study identifies for the first time a specific molecular mechanism in subpopulations of colonic DRG neurons that underlies chronic visceral hypersensitivity.Am J Gastroenterol advance online publication, 2 June 2009; doi:10.1038/ajg.2009.227.

Angiogenesis, the formation of new blood vessels, is critical in many normal and pathological processes such as development, reproduction, tumor growth, and metastasis. Recently, exposure to moderate-intensity static magnetic fields (1 mT to 1 T) has attracted much attention for its potential therapeutic value as a noninvasive intervening method. Nevertheless, the effects of moderate-intensity and spatial gradient static magnetic fields (GSMF) on angiogenesis have not received enough attention. In this study, the effects of GSMF (0.2-0.4 T, 2.09 T/m, 1-11 days) on angiogenesis were investigated both in vitro and in vivo. An MTT assay was used as an in vitro method to detect the proliferation ability of human umbilical veins endothelial cells (HUVECs). Two kinds of in vivo models, a chick chorioallantoic membrane (CAM) and a matrigel plug, were used to detect the effects of GSMF on angiogenesis. The results showed that the proliferation ability of HUVECs was significantly inhibited 24 h after the onset of exposure. With regard to the CAM model, vascular numbers in the CAM that was continuously exposed to the GSMF were all less than those in normal condition. In accordance with the gross appearance, the contents of hemoglobin in the models exposed to GSMF for 7-9 days were also less. In addition, similar to the CAM model, the results of vascular density and hemoglobin contents in the matrigel plug also demonstrated that the GSMF exposure for 7 or 11 days inhibited vascularization. These findings indicate that GSMF might inhibit or prevent new blood vessels formation and could be helpful for the treatment of some diseases relevant to pathological angiogenesis. Bioelectromagnetics, 2009 (c) 2009 Wiley-Liss, Inc.

OBJECTIVE: To analyze the expression of genes from chromosomal region 22q11.2 and assess the association between mutation(s) of particular gene(s) from this region and malformations of the urinary system. METHODS: Expression of rat homologs of 33 genes from above region was determined in kidney tissues derived from rats of different fetal development ages (E13, E15, E19) and adulthood with reverse transcriptase-PCR. Potential mutation(s) in candidate gene SNAP29, whose expression pattern appeared to be unique, was screened in 44 patients and 220 normal controls with PCR-single strand conformation polymorphism (SSCP). Suspected positive regions were sequenced to verify the mutations. RESULTS: Nine genes showed no expression throughout the whole development process; 18 genes with various expression levels showed continuous expression from the beginning of development; 6 genes only expressed for a short time, among which SNAP29 was selected for mutation screening. Upon sequencing, three mutations were identified from the 44 patients, including a G to A transition (GAG to AAG) in exon 2, and two A to G transitions (AGC to GGC) in exon 3. CONCLUSION: Through systematic analysis of the expression of genes from chromosomal region 22q11.2, the SNAP29 gene was found to have a potential role in the development of genitourinary system. Two missense mutations were identified in three patients. These included one in exon 2 (featuring cryptorchidism), and the other in exon 3 (featuring cryptorchidism and hypospadia). Neither of the mutations was found in the normal controls. The results suggested that mutation(s) of gene(s) from chromosomal region 22q11.2 may play an important role in the genesis of genitourinary malformations.

The full length cDNA of silkworm hibadh gene was cloned by RT-PCR and RACE (Rapid amplification of cDNA ends) technique. The hibadh gene and its deduced amino acid sequences were analyzed. The tissue distribution of hibadh gene in 5th instar silkworm larvae was tested by RT-PCR. The expression patterns of hibadh gene in simulated weightless environment were analyzed by real time RT-PCR. The results showed that the full length hibadh cDNA sequence was 1074 bp in lenth, including an open read frame of 969 bp encoding the entire coding region of Hibadh (GenBank accession No. EU719652). The deduced amino acid sequence similarities of hibadh between silkworm and Burkholderia ambifaria, Drosophila melanogaster, Apis mellifera, Xenopus tropicalis, Mus musculus, Homo sapiens were 46%, 43%, 48%, 44%, 45%, 45%, respectively. Signal peptide analysis showed that Hibadh was a secretory protein. There wasn't glycosyl-phosphatidyl inositol anchor site in Hibadh amino acid sequence. Molecular weight and isoelectric point of Hibadh were 34.1 kD and 9.14 respectively. The RT-PCR tests indicated that the hibadh gene expressed in head, silk gland, midgut, cuticle, blood, fat body, tuba malpighii of the 5th instar silkworm larvae. There were different expression patterns of hibadh gene during different silkworm embryo period in simulated weightless environment. Simulated weightlessness resulted in the expression of silkworm hibadh gene up regulated 2.3-fold (P < 0.05), up regulated 4.6-fold (P<0.01), down regulated 7.6-fold (P < 0.01), down regulated 2.6-fold (P < 0.05) during apophysis formation period, inverse period, trachea formation period, and whole embryo period, respectively. There was no significant change of hibadh gene expression during other period of silkworm embryo between simulated weightless and control groups. There were different response patterns to simulated weightless environment between hibadh gene and whole body of silkworm. Gene showed much higher sensitivity compared to whole body in response to environment. This study is useful for the further research on the gravity biological mechanism of hibadh gene.

Proper synaptic function requires the seamless integration of the transport, assembly, and regulation of synaptic components and structures. Inasmuch as the synapse is often distant from the neuronal cell body, newly synthesized synaptic proteins, the precursors of synaptic vesicles, active zone compartments, channels and receptors, and mitochondria, must be transported along lengthy neuronal processes to participate in synaptogenesis. Neuronal transport is mediated by motor proteins that associate with their cargoes via adaptors (or receptors) and that travel along the cytoskeleton network within the neuronal processes. Thus, the identity of membranous protein cargoes and the specificity of motor-cargo interactions are critical for correctly targeting cargoes and properly assembling synapses in developing neurons and in remodeling synapses of mature neurons in response to neuronal activity. In this article, the authors review recent progress in characterizing microtubule- and actin-based motor proteins that are involved in delivering synaptic components and discuss potential mechanisms underlying the formation of motor-receptor-cargo complexes that contribute to synaptogenesis and activity-induced synaptic plasticity.

Angiotensin II (ANG II), the biologically active peptide of the renin-angiotensin system (RAS), is generated by angiotensin-converting enzyme (ACE) and is a regulator of cardiovascular homeostasis. Recently, there has been increasing evidence that ANG II is involved in the regulation of cell proliferation and migration, as well as angiogenesis via the ANG II-type 1 receptor (AT1R). These findings suggest that the ACE-ANG II-AT1R pathway is related to cancer biology. Previous reports have shown that ACE is preferentially expressed in pancreatic ductal adenocarcinoma (PDAC) tissues. Recently a homologue of ACE, angiotensin-converting enzyme 2 (ACE2), was reported to counterbalance the function of ACE, but the expression and role of ACE2 in PDAC are still unclear. In the present study, we analyzed the expression of ACE2 in invasive human PDAC and surrounding non-malignant tissues by Western blot analysis and immunohistochemistry. The ANG II concentration in homogenates of pancreatic tissues was measured with ELISA, and ACE2 protein was detected by Western blot analysis in BxPC3 and SW1990 human pancreatic ductal cancer cells. We have shown for the first time that the expression of ACE2 is decreased in PDAC tissues, in which ANG II was accumulated. Treatment of BxPC3 and SW1990 cells with ANG II decreased the expression of ACE2. Therefore, ANG II may contribute to the down-regulation of ACE2. Moreover, reduction of ACE2 expression by RNA interference promoted the proliferation of cultured pancreatic cancer cells. These findings suggest that ACE2 may have clinical potential as a novel molecular target for the treatment of PDAC.

INTRODUCTION: Skeletal unloading during spaceflight leads to bone loss. Following long-duration flight, such loss may be very slow or impossible to fully recover. Therefore, it is important to seek countermeasures to prevent the loss. METHODS: We studied the effects on bone of whole-body vibration (WBV) with variable parameters (10-60 Hz and 0.1-1 g) using hind-limb unloading (HLU) in the rat as a model for microgravity. The bone mineral densities (BMD) of the femur, tibia, and lumbar spines were measured and the mechanical properties of the femur were determined by mechanical testing. Serum alkaline phosphatase (ALP) and pyridinoline (PYD) levels were analyzed. RESULTS: After 28 d of HLU, WBV reduced the losses of BMD in the femur and tibia from 18.8 to 10.1% and from 16.7 to 7.1%, respectively, and prevented the loss in stiffness of the femur after HLU. However, WBV had no effect on the lumbar spine. ALP levels were suppressed by HLU and maintained by WBV (28.3% increase). There was no significant difference in PYD among groups. In the recovery period following HLU, there were no significant effects of WBV on BMD, and the elastic modulus of the femur returned to normal. DISCUSSION AND CONCLUSIONS: Mechanical stimulation in the form of WBV limited reduction of bone density when it was applied during the unloading, but not afterward. It is of interest that the mechanical properties of the femur were influenced by WBV in the control femurs as well as in the unloaded bones of this rat model.