Inhibition of miR-221 influences bladder cancer cell proliferation and apoptosis.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Inhibition of miR-221 influences bladder cancer cell proliferation and apoptosis, by H. Liu, J.-K. Chang, J.-Q. Hou, Z.-H. Zhao, L.-D. Zhang, published in Eur Rev Med Pharmacol Sci 2017; 21 (14): 3193-3199-PMID: 28770966" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/13140.

[The mechanism of HOTAIR regulating the proliferation and apoptosis of prostate cancer cells by targeting down-regulation of miR-152 to improve the expression of FOXR2].

Objective: To clarify the effect of FOXR2 on the proliferation and apoptosis of prostate cancer cells and to reveal the mechanism. Methods: The expression of FOXR2 in clinical samples of prostate cancer were detected by Quantitative Real-time PCR (qRT-PCR) and Western blotting. The CCK8 proliferation kit and the Annexin V-FITC apoptosis kit, flow cytometry were used to detect the proliferation and apoptosis of prostate cancer cells with or without the FOXR2 knockdown. Combined with the results of microRNA chip, we predicted the related miR-152 and detected the relationship between miR-152 and FOXR2 by luciferase reporter gene assay. The correlation between HOTAIR and miR-152 is clearly defined by software prediction and qRT-PCR. Results: FOXR2 had a relatively high expression in the prostate cancer tissue.The mRNA expression of FOXR2 is 4.9 times that of adjacent tissues, and the protein level was also significantly up-regulated. In the PC3 cell line, the specific knock-down of FOXR2 inhibits the proliferation of cells and promotes cell apoptosis. According to the microRNA chip results and luciferase reporter gene assay, we found miR-152 could regulate the expression of FOXR2; and FOXR2 3 'UTR had two miR-152 binding sites, all of which could control the expression of FOXR2. The results of LNCediting and qRT-PCR suggest that HOTAIR is negatively correlated with the expression of miR-152, and is involved in the regulation of miR-152 expression in prostate cancer. Conclusion: FOXR2 up-regulation can promote the proliferation and inhibit the apoptosis of prostate cancer cells because that HOTAIR restrains the expression of miR-152.

GENIPAC: A Genomic Information Portal for Head and Neck Cancer Cell Systems.

Head and neck cancer (HNC)-derived cell lines represent fundamental models for studying the biological mechanisms underlying cancer development and precision therapies. However, mining the genomic information of HNC cells from available databases requires knowledge on bioinformatics and computational skill sets. Here, we developed a user-friendly web resource for exploring, visualizing, and analyzing genomics information of commonly used HNC cell lines. We populated the current version of GENIPAC with 44 HNC cell lines from 3 studies: ORL Series, OPC-22, and H Series. Specifically, the mRNA expressions for all the 3 studies were derived with RNA-seq. The copy number alterations analysis of ORL Series was performed on the Genome Wide Human Cytoscan HD array, while copy number alterations for OPC-22 were derived from whole exome sequencing. Mutations from ORL Series and H Series were derived from RNA-seq information, while OPC-22 was based on whole exome sequencing. All genomic information was preprocessed with customized scripts and underwent data validation and correction through data set validator tools provided by cBioPortal. The clinical and genomic information of 44 HNC cell lines are easily assessable in GENIPAC. The functional utility of GENIPAC was demonstrated with some of the genomic alterations that are commonly reported in HNC, such as TP53, EGFR, CCND1, and PIK3CA. We showed that these genomic alterations as reported in The Cancer Genome Atlas database were recapitulated in the HNC cell lines in GENIPAC. Importantly, genomic alterations within pathways could be simultaneously visualized. We developed GENIPAC to create access to genomic information on HNC cell lines. This cancer omics initiative will help the research community to accelerate better understanding of HNC and the development of new precision therapeutic options for HNC treatment. GENIPAC is freely available at http://genipac.cancerresearch.my/ .

Inhibition of miR-221 influences bladder cancer cell proliferation and apoptosis.

OBJECTIVE: Janus kinase (JAK) - signal transducer and activator of transcription (STAT) signaling pathway participate in cell proliferation and apoptosis. Suppressors of cytokine signaling 3 (SOCS3) are negative regulators of JAK-STAT3. SOCS3 was found significantly declined, while microRNA-221 (miR-221) obviously up-regulated in bladder cancer tissue. Bioinformatics analysis revealed the complementary binding site between miR-221 and 3'-UTR of SOCS3. This study investigated the role of miR-221 in regulating SOCS3/JAK-STAT3 signaling pathway and bladder cancer cell proliferation and apoptosis. PATIENTS AND METHODS: Bladder cancer tumor tissue and para-carcinoma tissue were collected from patients to test miR-221 and SOCS3 expressions. Dual luciferase assay was used to test the targeting regulatory effect of miR-221 on SOCS3. MiR-221, SOCS3, p-JAK1, p-JAK2, and survivin expressions were compared in T24 and HBEC cells. T24 cells were divided into miR-NC, miR-221 inhibitor, pSicoR-blank, pSicoR-SOCS3, and miR-221 inhibitor + pSicoR-SOCS3 groups. Flow cytometry was applied to detect cell apoptosis. EdU staining was adopted to evaluate cell proliferation. RESULTS: MiR-221 significantly increased, while SOCS3 obviously reduced in bladder cancer tissue compared with para-carcinoma tissue. MiR-221 targeted inhibited SOCS3 expression. MiR-221, phosphorylated JAK1 (p-JAK1), phosphorylated JAK2 (p-JAK2), phosphorylated STAT3 (p-STAT3), and survivin levels markedly up-regulated, whereas SOCS3 expression apparently declined in T24 cells compared with that in HBEC cells. MiR-221 inhibitor and/or pSicoR-SOCS3 elevated SOCS3 expression, decreased p-JAK1, p-JAK2, p-STAT3, and survivin levels, enhanced cell apoptosis, and attenuated cell proliferation. CONCLUSIONS: MiR-221 elevated, while SOCS3 reduced in bladder cancer tissue. Inhibition of miR-221 suppressed T24 cell proliferation and induced apoptosis by up-regulating SOCS3 expression, lowering JAK-STAT3 signaling pathway activity, and attenuating survivin expression.

Phoenixin: A candidate pruritogen in the mouse.

Phoenixin (PNX) is a 14-amino acid amidated peptide (PNX-14) or an N-terminal extended 20-residue amidated peptide (PNX-20) recently identified in neural and non-neural tissue. Mass spectrometry analysis identified a major peak corresponding to PNX-14, with negligible PNX-20, in mouse spinal cord extracts. Using a previously characterized antiserum that recognized both PNX-14 and PNX-20, PNX-immunoreactivity (irPNX) was detected in a population of dorsal root ganglion (DRG) cells and in cell processes densely distributed to the superficial layers of the dorsal horn; irPNX cell processes were also detected in the skin. The retrograde tracer, Fluorogold, injected subcutaneously (s.c.) to the back of the cervical and thoracic spinal cord of mice, labeled a population of DRG, some of which were also irPNX. PNX-14 (2, 4 and 8 mg/kg) injected s.c.to the nape of the neck provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. The number of scratching bouts varied from 16 to 95 in 30 min, commencing within 5 min post-injection and lasted 10-15 min. Pretreatment of mice at -20 min with nalfurafine (20 µg/kg, s.c.), the kappa opioid receptor agonist, significantly reduced the number of bouts induced by PNX-14 (4 mg/kg) compared with that of saline-pretreated mice. Our results suggest that the peptide, PNX-14, serves as one of the endogenous signal molecules transducing itch sensation in the mouse.

Blunted expression of miR-199a-5p in regulatory T cells of patients with chronic obstructive pulmonary disease compared to unaffected smokers.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal regulatory T cell (T(reg)) response and increases in T helper type 1 (Th1) and Th17 cell responses. It is unclear if dysregulation of microRNAs (miRNA) within T(reg) cells contributes to the abnormal inflammatory response in COPD. In this study, we aimed to compare the miRNA profile of COPD T(reg) cells with that of healthy controls and to explore the function of differentially expressed miRNAs. We first obtained T(reg) and T effector cells (Teff ) from peripheral blood of non-smokers, unaffected current smokers and COPD current smokers. Then, we assessed their miRNA expression by microarray analysis followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) validation of particular miRNAs. Six and 96 miRNAs were expressed differentially in COPD T(reg) cells versus T(reg) cells of healthy non-smokers and healthy smokers, whereas no differences were found in miRNA expression in T(eff) cells. We found that miR-199a-5p was repressed by approximately fourfold in T(reg) cells of COPD patients compared to healthy smokers (P < 0·05). In addition, miR-199a-5p was over-expressed in T(reg) cells compared to Teff cells (P < 0·001) and had significant over-representation of its target genes in the T(reg) transcriptome, being associated with the transforming growth factor (TGF)-ß activation pathway (P < 0·01). We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan® arrays of the human TGF-ß pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favour of a Th1 and Th17 response.

Phoenixin: a novel peptide in rodent sensory ganglia.

Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 µg) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 µg) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 µg) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.

Comparison of the clinical and radiological outcomes of three minimally invasive techniques for total knee replacement at two years.

Minimally invasive total knee replacement (MIS-TKR) has been reported to have better early recovery than conventional TKR. Quadriceps-sparing (QS) TKR is the least invasive MIS procedure, but it is technically demanding with higher reported rates of complications and outliers. This study was designed to compare the early clinical and radiological outcomes of TKR performed by an experienced surgeon using the QS approach with or without navigational assistance (NA), or using a mini-medial parapatellar (MP) approach. In all, 100 patients completed a minimum two-year follow-up: 30 in the NA-QS group, 35 in the QS group, and 35 in the MP group. There were no significant differences in clinical outcome in terms of ability to perform a straight-leg raise at 24 hours (p = 0.700), knee score (p = 0.952), functional score (p = 0.229) and range of movement (p = 0.732) among the groups. The number of outliers for all three radiological parameters of mechanical axis, frontal femoral component alignment and frontal tibial component alignment was significantly lower in the NA-QS group than in the QS group (p = 0.008), but no outlier was found in the MP group. In conclusion, even after the surgeon completed a substantial number of cases before the commencement of this study, the supplementary intra-operative use of computer-assisted navigation with QS-TKR still gave inferior radiological results and longer operating time, with a similar outcome at two years when compared with a MP approach.

Irisin-immunoreactivity in neural and non-neural cells of the rodent.

Irisin is a recently identified myokine secreted from the muscle in response to exercise. In the rats and mice, immunohistochemical studies with an antiserum against irisin peptide fragment (42-112), revealed that irisin-immunoreactivity (irIRN) was detected in three types of cells; namely, skeletal muscle cells, cardiomyocytes, and Purkinje cells of the cerebellum. Tissue sections processed with irisin antiserum pre-absorbed with the irisin peptide (42-112) (1 µg/ml) showed no immunoreactivity. Cerebellar Purkinje cells were also immunolabeled with an antiserum against fibronectin type II domain containing 5 (FNDC5), the precursor protein of irisin. Double-labeling of cerebellar sections with irisin antiserum and glutamate decarboxylase (GAD) antibody showed that nearly all irIRN Purkinje cells were GAD-positive. Injection of the fluorescence tracer Fluorogold into the vestibular nucleus of the rat medulla retrogradely labeled a population of Purkinje cells, some of which were also irIRN. Our results provide the first evidence of expression of irIRN in the rodent skeletal and cardiac muscle, and in the brain where it is present in GAD-positive Purkinje cells of the cerebellum. Our findings together with reports by others led us to hypothesize a novel neural pathway, which originates from cerebellum Purkinje cells, via several intermediary synapses in the medulla and spinal cord, and regulates adipocyte metabolism.

A novel reproductive peptide, phoenixin.

Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.

Neuronostatin is co-expressed with somatostatin and mobilizes calcium in cultured rat hypothalamic neurons.

Neuronostatin (NST) is a newly identified peptide of 13-amino acids encoded by the somatostatin (SST) gene. Using a rabbit polyclonal antiserum against the human NST, neuronostatin-immunoreactive (irNST) cells comparable in number and intensity to somatostatin immunoreactive (irSST) cells were detected in the hypothalamic periventricular nucleus. Fewer and/or less intensely labeled irNST cells were noted in other regions such as the hippocampus, cortex, amygdala, and cerebellum. Double-labeling hypothalamic sections with NST- and SST-antiserum revealed an extensive overlapping of irNST and irSST cells in the periventricular nucleus. Pre-absorption of the NST-antiserum with NST (1 microg/ml) but not with SST (1 microg/ml) abrogated irNST and vice versa. The activity of NST on dissociated and cultured hypothalamic neurons was assessed by the Ca(2+) imaging method. NST (10, 100, 1000 nM) concentration-dependently elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in a population of hypothalamic neurons with two distinct profiles: (1) a fast and transitory increase in [Ca(2+)](i), and (2) an oscillatory response. Whereas, SST (100 nM) reduced the basal [Ca(2+)](i) in 21 of 61 hypothalamic neurons examined; an increase was not observed in any of the cells. Optical imaging with a slow-responding voltage sensitive dye DiBAC(4)(3) showed that NST (100 nM) depolarized or hyperpolarized; whereas, SST (100 nM) hyperpolarized a population of hypothalamic neurons. The result shows that NST and SST, though derived from the same precursor protein, exert different calcium mobilizing effects on cultured rat hypothalamic neurons, resulting in diverse cellular activities.

Amylin suppresses acetic acid-induced visceral pain and spinal c-fos expression in the mouse.

Amylin is a member of calcitonin or calcitonin gene-related peptide (CGRP) family. Immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion (DRG) and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the acetic acid-induced writhing test. Amylin (0.1, 0.5 and 1 mg/kg, intraperitoneally (i.p.) or 1-10 microg, intrathecally (i.t.)) reduced the number of writhes in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32), either by i.p. or i.t., antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 microg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were upregulated in the spinal cord of mice injected with acetic acid and the increase was attenuated by pretreatment with amylin (10 microg, i.t.). Collectively, our result demonstrates that irAMY is expressed in DRG neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the peptide by interacting with amylin receptors in the spinal cord may be antinociceptive.

C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

Simvastatin increases osteoblasts and osteogenic proteins in ovariectomized rats.

BACKGROUND: Previous reports have indicated that statins could prevent bone loss in ovariectomized (OVX) rats and increase the expressions of osteogenic genes in cultured osteoblasts. In this study, we hypothesized that simvastatin might increase osteoblast number and protein expressions of osteogenic markers localized in bones in concomitance with the prevention of bone loss in OVX rats. MATERIALS AND METHODS: Fifty-four 3-month-old OVX and sham-operated (SHAM) female Sprague-Dawley rats were used. Simvastatin (10-20 mg kg(-1) day(-1)) was administrated orally for 6 weeks. Trabecular volume, osteoblast number and osteogenic proteins including BMP2, collagen type I and osteocalcin on bone sections obtained from lumbar vertebral body, distal femur and proximal tibia were measured. RESULTS: The results showed that SHAM rats had significantly less trabecular bone volume and osteoblast number than that of OVX rats 6 weeks after operation. Oral simvastatin treatment (10-20 mg kg(-1) day(-1)) increased bone volume and osteoblast number in the distal femurs, proximal tibiae and vertebrae of OVX rats. Furthermore, the osteoblastic cells with immuno-stained BMP2, collagen type I and osteocalcin in vertebral bones were significantly increased by simvastatin treatment (20 mg kg(-1) day(-1)) in OVX rats. CONCLUSIONS: This study demonstrates that simvastatin enhances the production of osteogenic proteins in bone and this effect may contribute to the prevention of bone loss in OVX rats.

Copeptin immunoreactivity and calcium mobilisation in hypothalamic neurones of the rat.

Copeptin is cleaved from the C-terminus of vasopressin (VP) prohormone. Immunohistochemical studies have revealed intense copeptin-immunoreactivity (irCOPT) in neurones of the rat hypothalamic nuclei, including paraventricular, supraoptic, suprachiasmatic, periventricular, and accessory secretory. Varicose cell processes emanated from irCOPT neurones, some of which projected caudally and traversed the internal layer of the median eminence, and terminated in the posterior pituitary. Double-labelling hypothalamic sections with copeptin antiserum and VP or oxytocin antiserum revealed an extensive overlapping of irCOPT and irVP neurones. The biological activity of human synthetic nonglycosylated copeptin or VP was evaluated in vivo and in vitro. Copeptin (1, 10, and 20 nmol/kg) injected i.v. caused no significant changes in the mean arterial pressure (MAP) and heart rate of urethane-anaesthetised rats. VP (0.1 nmol/kg) increased MAP, which was accompanied by a small decrease of the heart rate. The ratiometric fluorescence method was employed to assess changes in intracellular Ca2+ concentrations [Ca2+](i) which served as an index of the biological activity of peptides. VP (1 microM) markedly increased [Ca2+](i) of rat hypothalamic neurones or vascular smooth muscle cells, whereas copeptin (100 nm to 1 microM) caused a low amplitude, sustained increase of [Ca2+](i) in a population of hypothalamic neurones, but not in any of the vascular smooth muscle cells tested. The results obtained demonstrate that copeptin is expressed in VP neurones and that the peptide in the concentrations tested, although causing little or no detectable changes of blood pressure and heart rate in anaesthetised rats nor changes in [Ca2+](i) of cultured aortic smooth muscle cells, increases [Ca2+](i) in a small population (< 2%) of hypothalamic neurones tested, indicating that copeptin is biologically active in mammalian neurones.

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons.

Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 microM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 microM), and were markedly reduced in a Ca(2+)-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca(2+) imaging studies showed that Tat30-86 produced three types of Ca(2+) responses: 1) a fast and transitory increase, 2) Ca(2+) oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca(2+) response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.

Portable microscopic cell counter for the determination of residual leucocytes in blood components.

BACKGROUND AND OBJECTIVES: The accurate determination of residual white blood cell (WBC) in blood components is of considerable clinical importance, and a variety of methods have been devised for the counting of low levels of residual WBC. In this study, we evaluated the performance of microscopic cell counter with microchannel plastic chip (C-reader) with regard to its ability to quantify WBC in WBC-reduced red cell concentrates. MATERIALS AND METHODS: In order to quantify residual WBC with the microscopic cell counter, WBC-reduced red cell concentrate was stained using propidium iodide. Three studies were performed: linearity, precision and correlation compared to those of manual Nageotte chamber counting and automatic flow cytometric methods. RESULTS: Dilution experiments, conducted over a range of 0.7-712 WBC/microl, showed a linearity of r(2) > 0.999, with coefficient of variation values of < or = 15.6% and accuracy of 93.8% over all tested ranges. In comparison with the Nageotte chamber counting and flow cytometric methods, the correlation coefficients were r(2) > 0.995. The detection limit of this method was 0.24 WBC/microl. Total analysis time per sample was approximately 5 min. CONCLUSION: The microscopic cell counter for residual WBC counting was determined to be efficient at the level of currently defined standards, with acceptable precision and accuracy. This method may prove useful for the quality assurance and control of WBC-depleted blood products.

Beacon immunoreactivity in the rat hypothalamus.

Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.

Tanycytomas: a newly characterized hypothalamic-suprasellar and ventricular tumor.

We report five cases of tumors occurring in three children and in two adults. The tumors had unusual histomorphology and a mixture of ependymal and piloid-like astrocytic features and a myxoid stroma similar to myxopapillary ependymomas. MR imaging in three of the cases showed aggressive, intensely enhancing partially cystic hypothalamic-suprasellar masses near midline and near the floor of the third ventricle. In the three pediatric cases, the tumor encased the circle of Willis. This newly characterized tumor, the tanycytoma, has neoplastic cells with histomorphologic and ultrastructural characteristics similar to those of the tanycyte.

Total synthesis of human and rat coupling factor-6 amide and pressor effects in the rat.

Mitochondrial coupling factor-6 (CF-6) is a component of the ATP synthase complex essential for energy transduction. CF-6, which is localized to the surface of endothelial cells (ECs) and released by shear stress, has been implicated as an endogenous vasoconstrictor. Previous methods of obtaining CF-6 through purification and recombinant methods were laborious and inefficient. Here, we describe the chemical synthesis of human CF-6, (33-108)-NH(2), its C-terminal fragment (55-108)-NH(2), which is termed pCF-6; the rat CF-6, (33-108)-NH(2), its C-terminal fragment pCF-6, (55-108)-NH(2); and two N-terminal fragments of the rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2). Biological activities of each peptide were initially screened with bioassays and verified by in vivo studies. Accordingly, intravenous administration of CF-6, pCF-6, rat CF-6, and rat pCF-6 produced a modest but statistically significant increase in blood pressure and heart rate in urethane anesthetized rats, whereas the N-terminal rat pro-coupling factor-6, (24-52)-NH(2) and (33-52)-NH(2) caused no significant pressor response. Thus, the biologically active site probably resides at the C-terminal portion of CF-6 peptides.