bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Protease Inhibitors on the Acrosome Reaction and Sperm‐Zona Pellucida Binding in Bovine Sperm
Authors: Deppe M , Morales P , Sánchez R 
Published: July 2008

ContentsAcrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm‐zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin‐like, chymotrypsin‐like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim‐up method (4 × 106 cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N‐α‐p‐tosyl‐l‐lysine‐chloromethyl‐ketone (trypsin inhibitor); Trypsin inhibitor (I‐S Type from soybean); N‐tosyl‐l‐phenylalanine‐chloromethyl‐ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm‐zona binding capacity was evaluated using cumulus cell‐free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome‐reacted sperm was observed only in cells incubated with trypsin (10.2 ± 1%) and chymotrypsin inhibitors (18.5 ± 1%) in relation to the control (52.2 ± 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 ± 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.
Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen
Authors: Selvaraju S , Ghosh J , Ravindra JP 
Published: July 2008

ContentsTwenty‐four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4–8 years were used to assess various attributes of spermatozoa influencing the zona‐binding and zona‐penetration tests. Ejaculates from each bulls were subjected to in vitro sperm‐‐zona binding and sperm‐‐zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 ± 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 ± 0.64. The average number of zona‐bound and ‐penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin‐‐nigrosin stain and hypo‐osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 ± 2.32) was significantly (p < 0.05) higher than hypo‐osmotic swelling‐Giemsa (HOS‐G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 ± 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin‐‐nigrosin (r = 0.85, p < 0.05). The HOS‐G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin‐‐nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS‐G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.
Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares
Authors: Araujo GHM , Rocha Filho AN , Lopes EP , Moya CF , Alvarenga MA 
Published: July 2008

ContentsThe effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19°45′45′S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post‐ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35‐mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35‐ to 40‐mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively – one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low‐dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.
Porcine β1,4‐Galactosyltransferase‐I Sequence and Expression
Authors: Landers EA , Burkin HR , Bleck GT , Howell‐Skalla L , Miller DJ 
Published: July 2008

Contents β1,4‐Galactosyltransferase‐I (B4GALT1), one of seven β1,4‐galactosyltransferases, is an enzyme commonly found in the trans‐Golgi complex that adds galactose to oligosaccharides. In the three mammals studied to date, the B4GALT1 gene directs production of B4GALT1 protein using either of two transcription start sites. The product of the smaller transcript serves the traditional biosynthetic role in the Golgi. This form also complexes with α‐lactalbumin, a mammary‐specific protein, to form lactose synthase. In addition to a biosynthetic role, the protein translated from the longer transcript appears on the plasma membranes of some cells where it serves as a signalling receptor in cell‐matrix interactions such as sperm‐egg binding. The objective of this study was to sequence the protein‐coding region of porcine B4GALT1 and examine the sequence for relationships to the bovine, human, murine and chicken B4GALT1 genes. The sequence for the 1203 base pair protein‐coding region of porcine B4GALT1 was obtained. Analysis of the deduced protein sequences revealed that the transmembrane region displayed the highest identity between the four mammals. The catalytic domain was 84–88% identical between the porcine sequence and those of the bovine, human and mouse. The porcine protein had the lowest overall homology to the chicken amino acid sequence, 58% identity. Conservation of both transcription start sites in the porcine gene supports the existence of two isoforms. When compared to the other mammalian B4GALT1 genes, the porcine coding sequence contained a single threonine codon inserted into the region encoding the cytoplasmic domain. Two putative phosphorylation sites in the mouse cytoplasmic domain were conserved in the porcine sequence. Northern blots revealed a widely expressed 4.4 kb transcript that was more abundant in the mammary gland during lactation. These results are important for studies of the function of this unusual and important glycosyltransferase during glycoprotein biosynthesis, lactation and fertilization.
Effect of GnRH Dose on Occurrence of Short Oestrous Cycles and LH Response in Cyclic Dairy Heifers
Authors: Rantala MH , Peltoniemi OAT , Katila T , Taponen J 
Published: July 2008

ContentsProstaglandin F2α (PGF2α) and GnRH treatments given 24 h apart have been shown to result in short oestrous cycles (8–12 days) in some cows and heifers. The differences in responses may depend on the dose of GnRH. Therefore, the effect of the dose of GnRH on occurrence of short cycles and LH response was studied here. Oestrus was induced with dexcloprostenol (0.15 mg) in two groups of Ayrshire heifers. A second luteolysis was induced similarly on day 7 after ovulation; 24 h after PGF2α treatment, the heifers were administered either a high (0.5 mg, n = 15, group T500) or low (0.1 mg, n = 10, group T100) dose of gonadorelin. Blood samples for progesterone analyses were collected daily from the second PGF2α administration to the second ovulation after the PGF2α injection. Beginning 24 h after the GnRH treatment, ovaries were examined by transrectal ultrasonography every 6 h until ovulation, and daily between day 4 and the next ovulation. Five heifers from both groups were sampled for LH analyses via a jugular catheter every 30 min from 1 h before to 6 h after the GnRH administration. Short oestrous cycles were detected in 7 of 10 cases in group T100 and in 12 of 15 cases in group T500. No significant differences in LH responses were detected between the groups. In group T500, the rise in LH concentration tended to be somewhat slower than in group T100. The dose of GnRH (0.1 vs 0.5 mg) did not affect the occurrence of short oestrous cycles and LH response.
DECREASED AMPA GLuR2, BUT NOT GLuR3, mRNA EXPRESSION IN RAT AMYGDALA AND DORSAL HIPPOCAMPUS FOLLOWING MORPHINE‐INDUCED BEHAVIOURAL SENSITIZATION
Authors: Sepehrizadeh Zargham , Bahrololoumi Shapourabadi Mina , Ahmadi Shamseddin , Hashemi Bozchlou Saeed , Zarrindast Mohammad‐Reza , Sahebgharani Mousa 
Published: July 2008

SUMMARYRepeated administration of psychostimulants and µ‐opioid receptor agonists elicits a progressive enhancement of drug‐induced behavioural responses, a phenomenon termed behavioural sensitization. These changes in behaviour may reflect plastic changes requiring regulation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionic acid (AMPA) receptor function.In the present study, rats were treated for 7 days with saline or morphine (10 mg/kg). After a washout period of either 24 h or 7 days, locomotion, oral stereotypy and state‐dependent memory in a passive avoidance test were measured in the presence or absence of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione disodium salt (CNQX; 3 mg/kg), an AMPA receptor antagonist. In order to evaluate the mechanism underlying the behavioural responses, quantitative real‐time reverse transcription–polymerase chain reaction was used to evaluate mRNA expression of the AMPA receptor subunits GluR2 and GluR3 in the striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala of animals treated repeatedly with morphine.The results indicate that repeated morphine treatment followed by 7 days (but not 24 h) washout produces behavioural sensitization, as determined by locomotion, oral stereotypy and state‐dependent memory. Blockade of AMPA receptors with CNQX on the test day did not alter these behavioural responses. In addition, repeated morphine treatment followed by 7 days (but not 24 h) washout decreased GluR2 mRNA expression in both the amygdala (by 50%) and hippocampus (by 35%). Repeated morphine treatment did not alter GluR3 mRNA expression in any brain area assessed.These data imply that AMPA receptors are involved in the development (but not expression) phase of behavioural sensitization. The decreases in GluR2 mRNA expression in the amygdala and hippocampus may result in the formation of calcium‐permeable AMPA receptors, which are believed to play an important role in behavioural sensitization.
NEGATIVE REGULATION OF QUINONE REDUCTASE 2 BY RESVERATROL IN CULTURED VASCULAR SMOOTH MUSCLE CELLS
Authors: Cai Jing‐Bo , Zhang Zhi‐Hua , Xu Dong‐Jie , Qian Zhi‐Yong , Wang Zhi‐Rong , Huang Yuan‐Zhu , Zou Jian‐Gang , Cao Ke‐Jiang 
Published: July 2008

SUMMARY

Resveratrol, a polyphenol in red wine, has a cardioprotective effect. Resveratrol‐targeting protein (RTP) has been purified using a resveratrol affinity column (RAC) and has been identified as quinone reductase type 2 (NQO2). We hypothesize that NQO2 is the target protein of resveratrol in vascular smooth muscle cells (VSMC) and that resveratrol inhibits proliferation of VSMC through its action on NQO2. In the present study, we investigated the correlation between NQO2 regulation and cell proliferation in VSMC in response to resveratrol treatment.
The RTP was purified using RAC and was detected with a NQO2 polyclonal antibody. The VSMC were incubated with resveratrol (1, 10 and 50 µmol/L) for 24, 48 and 72 h. Cell proliferation was detected by cell counting and bromodeoxyuridine (BrdU) assay. A lentiviral vector incorporating NQO2 short interference (si) RNA of short hairpin design was constructed and transduced into VSMC. Real‐time quantitative polymerase chain reactionwas used to measure NQO2 mRNA levels; NQO2 expression was determined by western blot analysis.
Using RAC, we extracted a 26 kDa protein from aortic smooth muscle, which was referred to as RTP‐26. Proliferation of VSMC was inhibited by resveratrol in a concentration‐ and time‐dependent manner. The mRNA and protein expression of NQO2 was also repressed by resveratrol in a concentration‐ and time‐dependent manner. A similar pattern of inhibition was observed for cells treated with resveratrol (25 µmol/L) as for cells transduced with a lentiviral vector containing siRNA sequences against NQO2.
Collectively, these data indicate that the suppression of VSMC proliferation mediated by resveratrol correlates with NQO2 downregulation.
NORSOLORINIC ACID INHIBITS PROLIFERATION OF T24 HUMAN BLADDER CANCER CELLS BY ARRESTING THE CELL CYCLE AT THE G0/G1 PHASE AND INDUCING A FAS/MEMBRANE‐BOUND FAS LIGAND‐MEDIATED APOPTOTIC PATHWAY
Authors: Wang Clay CC , Chiang Yi‐Ming , Kuo Po‐Lin , Chang Jiunn‐Kae , Hsu Ya‐Ling 
Published: July 2008

SUMMARY

Norsolorinic acid, isolated from Aspergillus nidulans, has been shown to have antiproliferative activity in T24 human bladder cancer cells by arresting the cell cycle at the G0/G1 phase and inducing apoptosis. The aim of the present study was to investigate the antiproliferative activity of norsolorinic acid in T24 human bladder cancer cells.
The effects of norsolorinic acid (1, 5, 10 and 20 µmol/L) on the proliferation of T24 cells and on the distribution of cells within different phases of the cell cycle were investigated indirectly using an XTT assay and a flow cytometer, respectively. Factors affecting the cell cycle and apoptosis, including p53, p21, Fas receptor, Fas ligand (FasL) and caspase 8 activity, were examined using ELISA.
The results showed that norsolorinic acid inhibited proliferation of T24 cells in a dose‐dependent manner, with an IC50 of 10.5 µmol/L. The effect involved the induction of cell cycle arrest at the G0/G1 phase and apoptosis.
These results demonstrate that G0/G1 phase arrest is due to increased expression of p21 in cells treated with norsolorinic acid (10 and 20 µmol/L) for 24 h. Moreover, enhanced Fas and membrane‐bound Fas ligand (mFasL) may be responsible for the apoptotic effect of norsolorinic acid. Thus, the present study reports, for the first time, that induction of p21 and the Fas/mFas ligand apoptotic system may participate in the antiproliferative action of norsolorinic acid in T24 human bladder cancer cells.
ROLE OF NAOFEN, A NOVEL WD REPEAT‐CONTAINING PROTEIN, IN REDUCING NITRIC OXIDE‐INDUCED RELAXATION
Authors: Feng Guo‐Gang , Yamada Mitsuru , Wongsawatkul Orapin , Li Chang , Huang Lei , An Jun , Komatsu Toru , Fujiwara Yoshihiro , Naohisa Ishikawa 
Published: July 2008

SUMMARYNaofen, a novel WD40 repeat domain‐containing protein, has recently been found in the intracellular compartment. The aim of the present study was to determine whether naofen affects thoracic aortic vascular reactivity in normotensive and hypertensive rats and whether naofen is present in the thoracic aorta. In addition, we examined whether naofen modulates acetylcholine (ACh)‐stimulated nitric oxide (NO) release from the endothelium.Immunohistochemistry showed greater naofen expression in endothelial cells in the DOCA‐salt group compared with controls. There was increased naofen mRNA expression in deoxycorticosterone acetate (DOCA)‐salt hypertensive rats compared with normotensive rats.Acetylcholine‐induced relaxation of rat aortic strips was decreased in DOCA‐salt hypertensive rats compared with normotensive rats. Naofen‐N‐ but not naofen‐C‐terminal protein caused a significant decrease in ACh‐induced relaxation of aortic strips from normotensive rats.Using a nitrite assay in a murine aortic endothelial cell line demonstrated that naofen‐N‐terminal protein, but not naofen‐C‐terminal protein, significantly reduced ACh‐induced NO production, suggesting that naofen interferes with NO production.Administration of naofen‐N‐terminal protein, but not naofen‐C‐terminal protein, significantly inhibited cyclohydrolase (GCH) I mRNA expression in a murine aortic endothelial cell line, suggesting that naofen‐N‐terminal protein interferes with NO synthesis by inhibiting GCH I mRNA expression.The results of the present study suggest that naofen is present in vascular endothelial cells and has an inhibitory effect on ACh‐induced relaxation under normotensive conditions. The findings reinforce the functional significance of naofen‐N‐terminal protein on rat vascular reactivity.
ACCELERATED DOSING FREQUENCY OF A PULMONARY FORMULATION OF TISSUE PLASMINOGEN ACTIVATOR IS WELL‐TOLERATED IN MICE
Authors: Stringer Kathleen A , Tobias Meghan , Dunn John S , Campos Jackie , Van Rheen Zachary , Mosharraf Mitra , Nayar Rajiv 
Published: July 2008

SUMMARY

Tissue plasminogen activator (tPA) has both fibrinolytic and anti‐inflammatory activity. These properties may be useful in treating inflammatory lung diseases, such as acute respiratory distress syndrome (ARDS).
We have previously demonstrated the feasibility of targeted pulmonary delivery of tPA. As part of our research to develop a clinically viable pulmonary formulation of tPA, we assessed the tolerability and incidence of haemorrhage associated with the administration of a pulmonary formulation of mouse tPA (pf‐mtPA).
Intratracheal doses of nebulized pf‐mtPA or sterile saline were administered with increasing frequency to male and female B6C3F1 mice. After dosing, the mice entered a recovery period, after which they were killed and their lungs were lavaged and harvested. Post‐mortem gross necropsy was performed and all major organs were assessed histologically for haemorrhage. The bronchoalveolar lavage fluid was assessed for markers of lung injury.
Mouse tPA that was formulated to mimic a previously characterized human pf‐tPA was well tolerated when given intratracheally with increasing dosing frequency. The administration of pf‐mtPA did not result in any detectable haemorrhagic‐related events or signs of lung injury.
The results of the present longitudinal study demonstrate that a maximally feasible dose of pf‐mtPA (3 mg/kg) can be given frequently over a short period of time (12 h) without haemorrhagic complications. Although these data were generated in a healthy mouse model, they provide support for the continued evaluation of pf‐tPA for the treatment of pulmonary diseases, such as ARDS.
DIFFERENTIAL EFFECTS OF THE ADENOSINE A1 RECEPTOR AGONIST ADENOSINE AMINE CONGENER ON RENAL, FEMORAL AND CAROTID VASCULAR CONDUCTANCE IN PRETERM FETAL SHEEP
Authors: Booth Lindsea C , Tummers Leonie , Jensen Ellen C , Barrett Carolyn J , Malpas Simon C , Gunn Alistair J , Bennet Laura 
Published: July 2008

SUMMARY

Adenosine A1 receptor activation is critical for endogenous neuroprotection from hypoxia–ischaemia, raising the possibility that treatment with A1 receptor agonists may be an effective physiological protection strategy for vulnerable preterm infants. However, the A1 receptor can mediate unwanted systemic effects, including vasoconstriction of the afferent glomerular arteriole. There is limited information on whether this occurs at doses that improve cerebral perfusion in the immature brain.
Therefore, in the present study, we examined whether infusion of the selective A1 receptor agonist adenosine amine congener (ADAC) is associated with reduced renal perfusion in chronically instrumented preterm (0.7 gestation) fetal sheep. In the present study, ADAC was given in successive doses of 2.5, 5.0 and 15.0 µg, 45 min apart.
Treatment with ADAC was associated with a marked reduction in renal vascular conductance (and blood flow), whereas carotid conductance was increased and there was no significant effect on femoral conductance. In contrast with the stable effects of increasing ADAC dose on vascular conductance, there was a significant dose‐related fall in fetal heart rate and blood pressure.
In conclusion, these short‐term data support the concern that A1 receptor agonist infusion can selectively impair renal perfusion, even at low doses.
GLUCOSE REGULATION OF DIPEPTIDYL PEPTIDASE IV GENE EXPRESSION IS MEDIATED BY HEPATOCYTE NUCLEAR FACTOR‐1α IN EPITHELIAL INTESTINAL CELLS
Authors: Gu Ning , Tsuda Mariko , Matsunaga Tetsuro , Adachi Tetsuya , Yasuda Koichiro , Ishihara Akihiko , Tsuda Kinsuke 
Published: July 2008

SUMMARY

Dipeptidyl peptidase IV (DPP‐IV) is a new drug target in the treatment of Type 2 diabetes. Dipeptidyl peptidase IV enzyme activity is significantly altered in Type 2 diabetic patients with hyperglycaemia, but the underlying molecular mechanisms remain unclear.
The first aim of the present study was to clarify whether glucose regulates DPP‐IV enzyme activity. To address this, DPP‐IV gene expression and enzyme activity were measured in Caco2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) concentrations of glucose. We observed that high glucose inhibited DPP‐IV gene expression and enzyme activity.
The second aim of the present study was to investigate whether hepatocyte nuclear factor (HNF)‐1α contributes to glucose regulation of DPP‐IV gene expression. To explore this question, associations between the gene expression of DPP‐IV and HNF‐1α were examined in Caco‐2 cells cultured in the presence of low (2.5 mmol/L) or high (16.7 mmol/L) glucose. We found that the pattern of glucose‐regulated DPP‐IV gene expression is similar to that of HNF‐1α. Moreover, to elucidate whether glucose regulation of DPP‐IV gene expression is affected when HNF‐1α is inhibited, we produced two stable cell lines in which a dominant‐negative mutant HNF‐1αR271G or basic vectors were stably expressed. We found that glucose regulation of DPP‐IV gene expression was compromised in HNF‐1αR271G cells, but was well maintained in basic vector cells.
These results suggest that glucose regulation of DPP‐IV gene expression is mediated by HNF‐1α.