Angiogenic Effect of Intercellular Adhesion Molecule-1

In order to investigate the angiogenic effect of intercellular adhesion molecule-1 （ICAM-1）, two parts of experiment were performed. Chick embryo chorioallantoic membrane （CAM） assay was used for in vivo angiogenic research. The chick embryos were divided into 4 groups： ICAM-1 group （divided into 3 subgroups, Ⅰ, Ⅱ and Ⅲ） for screening the angiogenic effect of ICAM-1 by adding different concentrations of ICAM-1 （0.1, 0.2 and 0.3 μg/μL） 5 μL into the chick embryo CAMs on the day 10 after incubation for every subgroup; Anti-ICAM-1 group A （divided into 2 subgroups, Ⅰ and Ⅱ） by adding different concentrations of Anti-ICAM-1 （1：100, 1：50） 5 μL into the chick embryo CAMs on the day 10 after incubation for every subgroup to evaluate the effect of ICAM-1 on the survival of microvessels through observing whether Anti-ICAM-1 could induce involution of the microvessels on CAMs; Anti-ICAM-1 group B （divided into 2 subgroups, Ⅰ and Ⅱ ） by adding different concentrations of Anti-ICAM-1 （1：100, 1：50） 5 μL into the chick embryo CAMs on the day 6 after incubation for every subgroup to evaluate whether ICAM-1 involved in embryonic angiogenesis through observing the growth of microvessels on CAMs; Control group： ICAM-1 or Anti-ICAM-1 was substituted by PBS 5 μL on the day 10 or day 6 after incubation. Three days later, the CAMs were photographed in vivo, excised, sectioned and the number of microvessels was counted. In ICAM-1 group, there was increased number of microvessels arranged radially with ＂spoked-wheel＂ pattern around the gelatin sponges. The new microvessels growing perpendicularly to gelatin sponges were observed. The number of the microvessels growing in the CAM mesenchymes around the sponges in 3 subgroups was higher than that in control group （P〈0.01）, however, there was no significant difference among the 3 subgroups （P〉0.05）. In anti-ICAM-1 group A, the radially arranged microvessels were very unclear around the sponges contrast to that of ICAM-1 group. Few new microvessels were detected in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in subgroup Ⅱ was lower than that in control group （P〈0.01）. There was no significant difference in the number of the microvessels around the sponges between subgroup I and control group （P〉0.05）. In anti-ICAM-1 group B, the radially arranged microvessels were very unclear around the sponges contrast to that of control grouμ New microvessels were very scarce in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in the 2 subgroups were less than that in control group （P〈0.01）, and there was significant difference between the 2 subgroups （P〈0.05）. It was suggested that ICAM-1 could induce angiogenesis and support the survival of microvessels, and ICAM-1 was involved in embryonic angiogenesis.

Degenerative alterations in noradrenergic neurons of the locus coeruleus in Alzheimer's disease

Mice carrying mutant amyloid precursor protein and presenilin-1 genes （APP/PS1 double trans- genic mice） have frequently been used in studies of Alzheimer＇s disease; however, such studies have focused mainly on hippocampal and cortical changes. The severity of Alzheimer＇s disease is known to correlate with the amount of amyloid-13 protein deposition and the number of dead neurons in the locus coeruleus. In the present study, we assigned APP/PS1 double transgenic mice to two groups according to age： young mice （5-6 months old） and aged mice （16-17 months old）. Age-matched wild-type mice were used as controls. Immunohistochemistry for tyrosine hydroxylase （a marker of catecholaminergic neurons in the locus coeruleus） revealed that APP/PS1 mice had 23% fewer cells in the locus coeruleus compared with aged wild-type mice. APP/PS1 mice also had increased numbers of cell bodies of neurons positive for tyrosine hydroxylase, but fewer tyrosine hydroxylase-positive fibers, which were also short, thick and broken. Quantitative analysis using unbiased stereology showed a significant age-related increase in the mean volume of tyrosine hy- droxylase-positive neurons in aged APP/PS1 mice compared with young APP/PS1 mice. Moreover, the mean volume of tyrosine hydroxylase-positive neurons was positively correlated with the total volume of the locus coeruleus. These findings indicate that noradrenergic neurons and fibers in the locus coeruleus are predisposed to degenerative alterations in APP/PS1 double transgenic mice.

Comparison of scanning electron microscopy findings regarding biofilm colonization with microbiological results in nasolacrimal stents for external, endoscopic and transcanalicular dacryocystorhinostomy

AIM:To compare bacterial biofilm colonization in lacrimal stents following external dacryocystorhinostomy(EX-DCR),endoscopic dacryocystorhinostomy(EN-DCR),and transcanalicular dacryocystorhinostomy(TC-DCR)with multidiode laser.METHODS:This prospective study included 30consecutive patients with nasolacrimal duct obstruction who underwent EXT-,EN-,or TC-DCR.Thirty removed lacrimal stent fragments and conjunctival samples were cultured.The lacrimal stent biofilms were examined by scanning electron microscopy(SEM).RESULTS:Eleven(36.7%)of the 30 lacrimal stent cultures were positive for aerobic bacteria(most commonly Staphylococcus epidermidis and Pseudomonas aeruginosa).However anaerobic bacteria and fungi were not identified in the lacrimal stent cultures.Twenty-seven(90%)patients had biofilmpositive lacrimal stents.The conjunctival culture positivity after the DCR,biofilm positivity on stents,the grade of biofilm colonization,and the presence of mucus and coccoid and rod-shaped organisms did not significantly differ between any of the groups(P】0.05).However,a significant difference was found when the SEM results were compared to the results of the lacrimal stent and conjunctival cultures(P【0.001).CONCLUSION:Type of dacryocystorhinostomy(DCR)surgery did not affect the biofilm colonization of the lacrimal stents.SEM also appears to be more precise than microbiological culture for evaluating the presence of biofilms on lacrimal stents.

Expression of vimentin and glial fibrillary acidic protein in central nervous system development of rats

Objective: To investigate the distribution and contents of vimentin(Vim) and glial fibrillary acidic protein(GFAP) immunoreactivities in the central nervous system(CNS)of normal newborn, adult and aged rats.Methods: In this study, thirty healthy and normal Sprague–Dawley rats were simply classified into three groups: Newborn(7 days aged), adult(5 months aged) and aged(24 months aged) rats. Brains and spinal cord were dissected and cut into frozen sections. The expression of Vim and GFAP in CNS were detected by confocal immunofluorescence.Results: In each group, Vim was expressed in all the regions of CNS including the hippocampal, cerebral cortex, the third ventricle and spinal cord, and the expression was highest in neuron-like cell of newborn rats, while Vim was mainly expressed in cell bodies in adult and aged rats. GFAP was expressed in all the regions of CNS including the hippocampal, cerebral cortex, the third ventricle and spinal cord, and the expression was in astrocytes of aged rats. In the third ventricle, Vim was detected in all groups, and only observed in neuron-like cells of newborn. Meanwhile, the GFAP expression showed no significant differences between adult and aged rats in this region. The co-localization of Vim and GFAP were mainly observed in hippocampus and cerebral cortex of newborn,but this co-localization was found in the third ventricle of the rats in all groups.Conclusion: Our data demonstrate for the first time that the expression of Vim and GFAP in the rat's CNS during development. This data may provide a foundation for the further mechanistic studies of these two main intermediate filaments during development of CNS.

Cloning and bioinformatical analysis of the N-terminus of the sonic hedgehog gene

The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.

The observation on the levels of VIP in the cerebral cortex of rats with bilateral hemispheric ischemia

The rat vertebral and common carotid arteries were obstructed to cause bilateralhemispheric ischemia.Vasoactive intestinal peptide(VIP)in the rat cerebral cortex following 10～60min of ischemia was analysed by radioimmunoassay(RIA).The results showed that the levels ofVIP were significantly lower in the ischemic animals than controls(P【0.01).The distribution andmetabolism of VIP in the cerebral cortex and the probable mechanism during ischemia are dis-cussed.

siRNA Specific to Pdx-1 Disturbed the Formation of the Islet in Early Zebrafish Embryos

Pdx-1, an important transcription factor highlighting in the early pancreatic development, islet functions and pancreatic disorders, needs to be more investigated in zebrafish, and siRNA is still seldom applied in zebrafish embryo-related research.Our aim was to explore the role of pdx-l in pancreatic development of zebrafish embryos by using siRNA approach. Microinjection, reverse transcriptase-PCR （RT-PCR）, in situ hybridization and immunofluorescent staining were used in this research, and the morphology of the islet in normal zebrafish embryos, and in those treated with the siRNA specific to pdx-1 （siPDX-l） or siGFP was observed and compared. The expression of pdx-1 was detected in the stages of 1-cell, 2-cell, 4-cell, 8-cell, 16-cell, 16-hour by RT-PCT. The in situ hybridization and immunofluorescent staining results showed that siPDX-1 disturbed the formation of the islet in zebrafish embryos. Pdx-1 played multiple roles in maintaining the phenotype of the islet during embryogenesis in zebrafish.

EFFECT OF MIFEPRISTONE (RU486)ON MARKERS OF ENDOMETRIAL RECEPTIVITY

Objective To study the effect of single and low dose of RU486 on endometrial receptivity of healthy women. Methods A total of 5 healthy women were followed for one control and one treatment cycle. In the treatment cycle, a dose of 10 mg RU486 was administered on day luteinizing hormone (LH)-2. In both the control and treatment cycle, an endometrial biopsy was obtained on LH+7. These biopsies were assessed by immunohistochemical analysis to find the difference in expression of integrins and progesterone receptor (PR) between the control and treatment cycle. Results The treatment with RU486 increased the expression of α 1 and α 4 subunits of integrin in glandular epithelial cells, but did not influence β 3 subunit. Moreover, the normal down regulation of PR in epitherial cell nuclei was inhibited by 10 mg RU486. Conclusion Single dose of 10 mg RU486 impairs the establishment of endometrial receptivity on time.

Improvement of mount preparations in showing myenteric nerve plexus from intestines of mice

Objective: The whole mount preparations of digestive tract is an effective experimental way to study the appearance and distribution of nerve plexus in digestive tract. Although myentric nerve plexus preparations technique was reported very early. But we have done experiment over and over during our research work in order to improve this traditional method and to meet the needs of our research work, we made some progresses in regular mount preparations after many experiments, which helped offer better situation in observing myentric nerve plexus. Methods: Five healthy male adult Kunming mice (20-30 g in weight) were used in this study. After intraperitoneal injection of muscle relaxant, with dislocation of cervical vertebra method, the abdominal cavity was exposed through abdominal median incision. After several steps of mount preparations the mucous layer and longitudinal muscle layer mount preparations with myentric nerve plexus were stripped under anatomical microscope. Immunohistochemical staining was also used in our study. Results: The mount preparation samples with myentric nerve plexus from intestines of mice showed positive SP immunoreaction. The positive cells were dark brown. Many of the cytons appeared circular and oval, while some appeared triangular or irregular. Conclusion: Our improved method is really a good method to show enteric nerve plexus. The method has many advantages and is particularly applied to small animals such as Kunming mice and BALB/c mice, weighing from 20 g to 30 g.

Involvement of Shp-2 phosphatase in IL-1α mediated mouse embryo fi- broblast MMP-1 secretion and migration

Objective: To investigate the relation between Shp-2 (a cytoplasmic tyrosine phosphotase) and matrix metalloproteinase-1 (MMP-1) in the migration of mouse embryo fibroblast cell. Methods: Shp-2 -/-embryo fibroblast was separated from Shp-2 knockout mouse on E10. 5, and Shp-2 cDNA was transfected into Shp-2 -/- cell. The cell migration was observed with the wound healing, the MMP-1 expression and secretion was analyzed by immunoblotting and immunoprecipitation. The activation of MMP-1 in the supernatant was detected with type I collagenase activity assay after the stimulation of IL-1α compared with E10. 5 mouse embryo fibroblast cell(Shp-2 +/+). Results: There was obvious migration in Shp-2 +/+ cells and Shp-2 -/-R cells, but not in Shp-2 -/- cells. The shape of Shp-2 -/- cell was epithelial-like. The expression and secretion was increased in Shp-2 +/+ cells and Shp-2 -/-R cells, and it had not changed in Shp-2 -/- cell. The activation of MMP-1 was lower in Shp-2 -/- cells compared to the other cells. Conclusion: IL-1 induces the expression and secretion of MMP-1α at the physical dose, and the cell migration is involved in MMP-1 by way of Shp-2 signal transduction.

<i>In vivo</i>effect of 17-β-estradiol, progesterone, hCG and expression of P53 and P21 in endometrial Ishikawa cells

The following article has been retracted due to the investigation of complaints received against it. The Editorial Board found that the first author published the paper without other authors’ consent and approval. The scientific community takes a very strong view on this matter, and the OJOG treats such behavior seriously. This paper published in Vol. 3 No. 1, 105-110 (pages), 2013, has been removed from this site.

Fabella Syndrome: A Typical Case of Misdiagnosis and Discussion

Background: Fabella is a natural occurring sesamoid bone, cartilage or a mixture of both that is usually located at the posterolateral corner (PLC) of the knee [1]. Recently the PLC of the knee has been extensively investigated because it is a common site of injuries and diseases [2] [3]. The complexity of PLC anatomy needs to be fully understood because the fabella could be missed diagnosis as an osteochondral defect, osteochondritis dissecans (OCD), calcific tendinitis, or foreign body. We present a case report of fabella syndrome triggered by trauma and also performed a review of literature for the various diagnoses that might be confused with fabella syndrome. Case presentation: A 29-year-old, Sudanese male presented to the trauma center in King Khalid Hospital in KSA, complaining of pain and partial swelling in his left knee joint due to trauma. Fabella was detected in the posterior lateral corner (PLC) of the knee joint embedded in the lateral head of the gastrocnemius muscle. All other pathological conditions were excluded. Conclusion: Fabella is sesamoid bone with variable size, found in the PLC. Commonly if present it causes periodic pain especially in the fully extended knee or it remains asymptomatic. Trauma, surgery, sports or heavy extreme activities may trigger the pain of asymptomatic fabella. Clinicians should consider that pain in the PLC of the knee can result from the presence of the fabella in a condition called fabella syndrome.

Critical Amino Acid Residues for Nicotine 5'-Hydroxylation in Human CYP2A Enzymes

Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.