scholarly journals КЛЮЧОВІ ФУНКЦІОНАЛЬНІ ЗМІНИ У КЛІТИНАХ БРИЖОВИХ ЛІМФАТИЧНИХ ВУЗЛІВ НАЩАДКІВ ЩУРІВ З ЕКСПЕРИМЕНТАЛЬНИМ ГЕСТАЦІЙНИМ ДІАБЕТОМ

2020 ◽  
pp. 36-41
Author(s):  
T. M. Prozorova ◽  
K. S. Krupey ◽  
O. M. Kamyshnyi
Keyword(s):  
Sigma Chemical

Гестаційний діабет (ГД) є важливим порушенням обміну речовин, що може впливати на морфогенез органів імунної системи. Мета дослідження – з’ясувати особливості змін функціонального стану клітин брижових лімфатичних вузлів у нащадків щурів з експериментальним гестаційним діабетом (ЕГД). Матеріали і методи. Дослідження проведено на 80 нащадках щурів лінії Wistar з ЕГД віком 1 і 6 місяців. Для індукції ЕГД вводили внутрішньочеревно стрептозотоцин (SIGMA Chemical, США) у дозі 45 мг/кг. Для ідентифікації TLR2, TLR4, NOD2, RIGI, T-bet, Nlrp3, RORγt і Foxp3 у гістологічних зрізах лімфатичних вузлів застосовували імунофлюоресцентний метод. Для дослідження експресії мРНК генів Aire, Deaf1, Foxp3, IL10, Ctla4, Cxcr4, Ccr7, Madcam1, S1pr1 використовували метод полімеразної ланцюгової реакції зі зворотною транскрипцією в режимі реального часу. Результати. Встановлено комплекс ключових патофізіологічних і функціональних змін у клітинах брижових лімфатичних вузлів (БЛВ) у нащадків щурів з ЕГД: зміни експресії регуляторів рециркуляції і хоумінгу лімфоцитів; порушення формування периферичної імунологічної толерантності; активація патернрозпізнавальних рецепторів уродженої імунної системи на лімфоцитах БЛВ; зміни розподілу ефекторних Т-клітин в БЛВ. Висновки. Пренатальна гіперглікемія призводить до посилення прозапальної сигналізації та активації компонентів уродженої імунної системи більш виразно на 1 місяці життя.

scholarly journals Η ανοσολογική απόκριση ολικού ανθρώπινου αίματος σε πρόκληση με στοιχεία του τοιχώματος μυκήτων

2014 ◽  
Author(s):  
Σταύρος Αλοΐζος
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Systemic Inflammatory Response Syndrome ◽  
Inflammatory Response ◽  
Ex Vivo ◽  
Systemic Inflammatory Response ◽  
Apache Ii ◽  
Apache Ii Score ◽  
Gram Negative ◽  
Tnf Α ◽  
Sigma Chemical

Η σήψη αντιπροσωπεύει ένα σοβαρό πρόβλημα υγείας, που σχετίζεταιμε υψηλή θνητότητα και θνησιμότητα, παρατεταμένη παραμονή για νοσηλείαστο Νοσοκομείο, και αυξημένα άμεσα και έμμεσα κόστη. Ειδικά στην ΜΕΘ, ησήψη είναι το πρώτο αίτιο θανάτου. Ακολούθως με τα αποτελέσματα τηςμελέτης Sepsis Occurrence in Acutely Ill Patients (SOAP), 35% των βαρέωςπασχόντων ασθενών αναπτύσσουν σήψη σε κάποιο σημείο της νοσηλείαςτους στην ΜΕΘ, με θανατηφόρες επιπλοκές στο 27% από αυτές, έναποσοστό που πλησιάζει το 50% σε ασθενείς με σηπτικό σοκ. Τα Grampositive[gram (+)] βακτήρια έχουν αναδειχθεί ως το συχνότερο αίτιο λοίμωξηςτων βαρέως πασχόντων ασθενών, ακολουθούμενα από τα gram-negative[gram (-)] βακτήρια και από τους μύκητες, οι οποίοι εμπλέκονται σε περίπου18% των περιπτώσεων.Η ΧΝΑ συχνά επιπλέκεται από λοιμώξεις, ειδικά όταν φτάνει στο τελικότης στάδιο που απαιτεί αιμοδιάλυση. Η θνητότητα των ασθενών με τελικούσταδίου ΧΝΑ είναι περίπου 20%, με τις καρδιαγγειακές παθήσεις και τιςλοιμώξεις να ενοχοποιούνται για το 70% των θανάτων. Η αυξημένη επίπτωσητης σήψης στους ασθενείς με τελικού σταδίου ΧΝΑ είναι πολυπαραγοντική καιαποδίδεται στην ουραιμία, την υποθρεψία, και την διαδικασία αιμοδιάλυσης, ηοποία μπορεί να επηρεάσει πολλαπλώς τόσο την εγγενή όσο και την επίκτητηανοσία. Η υπεργλυκαιμία είναι ένας καλά μελετημένος παράγοντας πουευοδώνει σε λοιμώδεις επιπλοκές. Η διαταραγμένη ομοιόσταση της γλυκόζηςεπηρεάζει πολλές παραμέτρους της ανοσιακής απάντησης,συμπεριλαμβανομένης της εκκρίσεως κυτταροκινών, της λειτουργίας των κυττάρων της ανοσίας και της επιθηλιακής δυσπραγίας. Οι ασθενείς με ΣΔείναι συνήθως εξαιρετικά επιρρεπείς σε λοιμώξεις.Αμέσως μετά μια μικροβιακή προσβολή η φλεγμονώδης απάντησηξεκινάει μετά την αναγνώριση των συστατικών του μικροβιακού εισβολέα. Οικύριες προφλεγμονώδεις κυτταροκίνες είναι ο TNF-α και οι ιντερλευκίνεςIL-6,IL-1β, and IL-8, οι οποίες προωθούν την φλεγμονώδη αντίδραση καιπροκαλούν την εμφάνιση του Συνδρόμου Συστηματικής ΦλεγμονώδουςΑντίδρασης {Systemic Inflammatory Response Syndrome (SIRS)}.Ταυτόχρονα με την προφλεγμονώδη διέγερση, ξεκινάει και ηαντιφλεγμονώδης, η οποία αντιπροσωπεύεται κυρίως από την IL-10. Ηκυριαρχία του προφλεγμονώδους ή του αντιφλεγμονώδους προφίλ σχετίζεταιμε την κλινική έκφραση της λοίμωξης και την έκβαση της σήψης αμφότερα.Η παθογένεση της gram(-) σήψης έχει μελετηθεί εκτεταμένως καιαποδίδεται σχεδόν αποκλειστικά στη δράση του λιποπολυσακχαρίτη(ενδοτοξίνη, LPS) του βακτηριακού τοιχώματος. Το κυτταρικό τοίχωμα τωνμυκήτων αποτελείται από τρείς κυρίως ομάδες πολυσακχαριδών, πολυμερήτης μανόζης (μαννοπρωτεΐνες, 40% της ξηρής μάζας του κυτταρικούτοιχώματος), πολυμερή της γλυκόζης (b-γλυκάνη, 60% της ξηρής μάζας τουκυτταρικού τοιχώματος) και πολυμερή της N-acetylglucosamine (χιτίνη, 2%της ξηρής μάζας του κυτταρικού τοιχώματος). Ανάμεσα σε αυτά οι μαννάνεςέχουν ιδιαίτερη σημαντικότητα, καθώς παρέχουν αντιγονική πολυμορφία καικατέχουν ανοσοδιεγερτικές ιδιότητες.Παρότι είναι βολικό να πιστεύουμε ότι μια κοινή παθογενετική οδόςαποτελεί την βάση σε όλες τις σηπτικές προσβολές, φαίνεται να υπάρχειδιαφορετικότητα ανάμεσα σε διαφορετικά είδη βακτηρίων. Σκοπός της μελέτης: Ο κύριος σκοπός της παρούσης μελέτης είναι νααξιολογήσει την βασική ανοσιακή κατάσταση των ασθενών των ευπαθών σεμυκητιασικές λοιμώξεις, και να αξιολογήσει την ανοσολογική τους απάντησημετά διέγερση με συστατικά του κυτταρικού τοιχώματος των μυκήτων και νασυγκριθεί αυτή η αντίδραση με την αντίδραση υγειών εθελοντών ενηλίκων,χρησιμοποιώντας ένα ex vivo μοντέλο διέγερσης. Ένας δευτερεύων σκοπόςήταν να διαπιστωθεί το εάν αυτέ οι ομάδες ασθενών ευρίσκονται σεκατάσταση ανοσοπαράλυσης, κάτι που αναγνωρίζεται με ολοένα καιαυξανόμενη συχνότητα σε βαριά πάσχοντες ασθενείς.Υλικό και μέθοδος: Μετρήσαμε τα βασικά επίπεδα των κυτταροκινών τουορού καθώς και τα επίπεδα που παρήχθησαν αυτές μετά από ex vivoδιέγερση με μαννάνη σε ολικό αίμα που συνελέγη από 10 υγιείς εθελοντές, 10ασθενείς με τελικού σταδίου ΧΝΑ, 10 ασθενείς με ΣΔ και 10 ασθενείς πουβρίσκονταν στη 2η ημέρα νοσηλείας τους σε ΜΕΘ, οι οποίοι είχαν μη σηπτικόSIRS και είχαν ένα APACHE II score ≥25. Χρησιμοποιήσαμε 100 μg/mlμαννάνης για μια περίοδο επώασης 8 ωρών ώστε να διεγείρουμε 1 ml ολικούαίματος. Τα δείγματα του αίματος συνελέγησαν από περιφερική φλέβα και ηδιέγερση έλαβε χώρα κάτω από τις ίδιες συνθήκες για όλες τις ομάδες μεμαννάνη από Saccharomyces cerevisiae της εταιρείας Sigma Chemical Co.(St Louis, MO, USA). Η μέτρηση των κυτταροκινών έγινε χρησιμοποιώνταςειδικά για το ανθρώπινο είδος εμπορικά διαθέσιμα κιτ με ELISA γιακυτταροκίνες.Αποτελέσματα: Όλες οι ομάδες των ασθενών είχαν υψηλότερες βασικέςτιμές TNF-α, IL-6, IL-1β, και IL-10 συγκρινόμενες με την ομάδα ελέγχου, αλλάμόνο στους ασθενείς της ΜΕΘ οι διαφορές ήταν στατιστικά σημαντικές. Ο λόγος IL-10/IL-6 βρέθηκε 0.33, 0.22 και 0.96 αντίστοιχα στους υγιείς, τουςασθενείς με ΧΝΑ και τους ασθενείς με ΣΔ, και 1.32 για τους ασθενείς τηςΜΕΘ πριν την διέγερση και 0.22, 0.51, 1.21, 2.46 αντίστοιχα μετά τηνδιέγερση. Σε όλες τις εξετασθείσες ομάδες, τα επίπεδα των κυτταροκινώναυξήθηκαν σημαντικά μετά την διέγερση με την μαννάνη, αν και οι βαριάπάσχοντες ασθενείς εμφάνισαν σημαντικά μικρότερη μεταβολή. Δενδιαπιστώθηκε ανοσοπαράλυση σε όλες τις εξετασθείσες ομάδες, αν καιυπήρξε σχετική ανοσοπαράλυση στους ασθενείς με ΣΔ και στους ασθενείςτης ομάδας της ΜΕΘ σύμφωνα με τον λόγο IL-10/Il-6. Επιπλέον, σύμφωνα μετον λόγο IL-10/TNF-α, όλες οι ομάδες ασθενών έχουν υψηλότερη πιθανότηταθανάτου σε σύγκριση με τους υγιείς εθελοντές, με αυτούς της ομάδας τηςΜΕΘ στην κορυφή της επικινδυνότητας και ακολουθούμενους από τουςασθενείς της ομάδας του ΣΔ. Ο λόγος IL-10/TNF-α πριν και μετά από τηνδιέγερση ήταν 0.52/0.35, 0.77/0.64, 0.96/0.91 και 1.46/1.19 για τους υγιείςεθελοντές, την ομάδα της ΧΝΑ, τους ασθενείς με ΣΔ και την ομάδα της ΜΕΘαντίστοιχα.Συμπεράσματα: Οι βαρέως πάσχοντες ασθενείς και δευτερευόντως οιασθενείς με τελικού σταδίου ΧΝΑ καθώς και οι ασθενείς με ΣΔ βρίσκονται σεπροφλεγμονώδη κατάσταση. Η ανταπόκριση των ασθενών με ΧΝΑ και ΣΔστην διέγερση με μαννάνη κρίνεται ως ικανοποιητική και συγκρίνεται με τηναντίδραση των υγειών ενηλίκων, ενώ αυτή των βαρέως πασχόντωνδιαφοροποιείται, πιθανά εξαιτίας της διαφορετικής ανοσολογικής τουςκατάστασης. Οι ασθενείς της ΜΕΘ και δευτερευόντως οι ασθενείς με ΣΔ είχανσχετική ανοσοπαράλυση.

scholarly journals First Report of Cucurbit aphid-borne yellows virus in Spain

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 907-907 ◽  
Author(s):  
M. Juarez ◽  
V. Truniger ◽  
M. A. Aranda
Keyword(s):  
Nucleotide Sequence ◽  
Nucleotide Sequence Identity ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
E Coli ◽  
Sequence Identity ◽  
Tissue Print ◽  
Sigma Chemical ◽  
Beet Pseudo Yellows Virus ◽  
Cucumis Melo L

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5′-GAATACGGTCGCGGCTAGAAATC-3′ (CE9) and 5′-CTATTTCGGGTTCTGGACCTGGC-3′ (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) H. Guilley et al. Virology 202:1012, 1994. (3) H. Lecoq et al. Plant Pathol. 41:749, 1992.

scholarly journals 219EFFECTS OF GROWTH HORMONE AND GNRH ANTAGONISTS ON FOLLICULAR AND OOCYTE DEVELOPMENT IN SHEEP

2004 ◽  
Vol 16 (2) ◽  
pp. 231
Author(s):  
P. Gonzalez-Añover ◽  
T. Encinas ◽  
R.M. Garcia-Garcia ◽  
A. Veiga-Lopez ◽  
J. Santiago-Moreno ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Breeding Season ◽  
Developmental Competence ◽  
Control Group ◽  
Gh Treatment ◽  
Gnrh Antagonists ◽  
Nuclear Maturation ◽  
Final Development ◽  
Sigma Chemical ◽  
Experimental Group

Embryo output in sheep is increased when superovulatory FSH treatments are started in the presence of a high number of small follicles (2–3mm in size) and in absence of large follicles (>6mm, Gonzalez-Bulnes et al., 2002. Theriogenology, 57, 1263–1272). Administration of GnRH antagonists (GnRHa) suppresses large follicles (Cognie et al., 2003. Theriogenology, 59, 171–188), whereas the use of growth hormone (GH) would increase the number of small follicles (Campbell et al., 1995. J Reprod Fertil Suppl, 49, 335–350). Our aim was to evaluate the usefulness of pre-treatments with GH or GH plus GnRH antagonists for sheep embryo production. First, we studied the effects on follicular population by serial ultrasonographies. Thereafter, we determined whether such treatments can affect oocyte developmental competence. In a first trial, a total of 18 Manchega ewes were treated with intravaginal FGA sponges (Chronogest®, Intervet Int., H) during breeding season (beginning of April). Six animals received daily i.m. doses of 15mg of ovine GH (Tuenre, GA) for 6 days, while six females received GH plus two s.c. doses of 1.5mg of GnRHa (Antarelix™, Zentaris, G) on Days 0 and 3 of GH treatment, and six ewes acted as controls receiving saline. Number of follicles >2mm, determined by daily transrectal ultrasonography, increased to reach significant differences on Day 4 in sheep treated with GH/GnRHa (22.7±0.8 v. 16.7±0.5, P<0.001) and on Day 5 in ewes injected with GH (20.3±0.4 v. 17.0±0.6, P<0.05). The second trial involved 18 Manchega ewes treated with progestagen sponges on Day 0 and distributed in three groups at the beginning of breeding season (end of July). In the first group (n=7), sheep were treated with two doses of GnRHa on Days 0 and 3 after sponge insertion and with three doses of 15mg of GH on Days 3, 4, and 5. Thereafter, ewes from this group and from a second experimental group (n=7) were treated with 3 doses of 1.5mL of FSH (Ovagen™, ICP, NZ) every 12h, starting on the afternoon of Day 5. A third group of sheep (n=4) did not receive GH/GnRHa or FSH, acting as controls. On Day 7, follicles were aspirated and the cumulus-oocyte complexes (COC) were cultured for 24h at 38.5°C, 5% CO2, in TCM-199 supplemented with ovine FSH (Ovagen), LH, FCS, 17-βoestradiol, cysteamine, and sodium pyruvate (Sigma Chemical Co., MO, USA). Nuclear maturation was measured by Hoechst 33342 fluorescence. Mean number of COC was higher in GH/GnRHa+FSH group (8.7±0.9 v. 6.8±1.3 in FSH group, NS and 4.5±0.8 in control, P<0.05) due to higher number of follicles with similar recovery rates (45.0±4.5, 40.3±1.4, and 39.1±7.1%, respectively). There were no significant differences on the ability of COC to resume meiosis, although this was higher in FSH group (63.1±9.5% for GH/GnRHa+FSH, 79.5±6.3% for FSH and 60.0±8.8% for control group), which can indicate the necessity of a higher FSH supply to induce final development in follicles/oocytes from ewes treated with GH and GnRHa. In conclusion, the use of GH and GnRHa would help to increase the number of gonadotrophin-responsive follicles prior to gonadotrophin injections;; also, adjustment of FSH treatments improved embryo yields in superovulatory protocols.

scholarly journals First Report of Cucurbit aphid-borne yellows virus in Iran Causing Yellows on Four Cucurbit Crops

Plant Disease ◽  
2006 ◽  
Vol 90 (4) ◽  
pp. 526-526 ◽  
Author(s):  
K. Bananej ◽  
C. Desbiez ◽  
C. Wipf-Scheibel ◽  
I. Vahdat ◽  
A. Kheyr-Pour ◽  
...  
Keyword(s):  
Citrullus Lanatus ◽  
Healthy Plant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
First Report ◽  
Chain Reactions ◽  
Reference Isolate ◽  
Sigma Chemical ◽  
International Mycological Institute ◽  
Das Elisa

A survey was conducted from 2001 to 2004 in the major cucurbit-growing areas in Iran to reassess the relative incidence of cucurbit viruses. Severe yellowing symptoms were observed frequently on older leaves of cucurbit plants in various regions in outdoor crops, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) (1,2). Leaf samples (n = 1019) were collected from plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrullus lanatus L.) showing various virus-like symptoms (mosaic, leaf deformation, yellowing). All samples, collected from 15 provinces, were screened for the presence of CABYV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with IgGs and alkaline phosphatase-conjugated IgGs against a CABYV reference isolate (1). Of the 1,019 samples tested, 471 were positive for CABYV using DAS-ELISA. Some of the positive samples had typical severe yellowing symptoms while symptoms in other samples were masked by mosaic or leaf deformations caused by other viruses frequently found in mixed infections (data not shown). During the entire survey, CABYV was detected by DAS-ELISA in 201 of 503 melon samples, 72 of 129 cucumber samples, 158 of 249 squash samples, and 40 of 138 watermelon samples. These results indicate that CABYV is widely distributed on four cucurbit species in the major growing areas of Iran. In order to confirm CABYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St Louis, MO) were obtained from 25 samples that were positive using DAS-ELISA originating from Khorasan (n = 4), Esfahan (n = 6), Teheran (n = 3), Hormozgan (n = 4), Azerbaiejan-E-Sharqi (n = 4), and Kerman (n = 4). Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using forward (5′-CGCGTGGTTGTGG-TCAACCC-3′) and reverse (5′-CCYGCAACCGAGGAAGATCC-3′) primers designed in conserved regions of the coat protein gene according to the sequence of a CABYV reference isolate (3) and three other unpublished CABYV sequences. RT-PCR experiments yielded an expected 479-bp product similar to the fragment amplified with extracts from the reference isolate. No amplification of the product occurred from healthy plant extracts. To our knowledge, this is the first report of the occurrence of CABYV in Iran on various cucurbit species. The high frequency (46.2%) with which CABYV was detected in the samples assayed indicates that this virus is one of the most common virus infecting cucurbits in Iran. References: (1) H. Lecoq et al. Plant Pathol. 41:749, 1992 (2) M. A. Mayo and C. J. D'Arcy. Page 15 in: The Luteoviridae. H. G. Smith and H. Barker, eds. CAB International Mycological Institute, Wallingford, UK, 1999. (3) H. Guilley et al. Virology 202:1012, 1994.

Immunofluorescent Localization of Myosin on the Sperm Cells of Plumbago Zeylanica

1997 ◽  
Vol 3 (S2) ◽  
pp. 183-184
Author(s):  
Zhaojie Zhang ◽  
Scott D. Russell
Keyword(s):  
Immunofluorescence Microscopy ◽  
Embryo Sac ◽  
Indirect Evidence ◽  
Sperm Cells ◽  
Immunofluorescent Localization ◽  
Plumbago Zeylanica ◽  
Actomyosin Interaction ◽  
Rabbit Igg ◽  
Sigma Chemical ◽  
Phosphate Buffered Saline

Sperm cells in flowering plants are non-motile and are passive participants in their movement to the female reproductive cells. It is believed that actomyosin interaction may play a key role for sperm cell transmission in the pollen tube as well as in the embryo sac. However, indirect evidence has shown that the surface of sperm cells lacks amounts of myosin sufficient to support movement. Immunofluorescence microscopy was used in this study to further assess the presence of myosin on the surface of sperm cells of Plumbago Zeylanica.Sperm cells of Plumbago Zeylanica were isolated according to published methods. Isolated sperm cells were blocked 20 min with 1% bovine serum albumin and 2% normal goat serum in phosphate buffered saline (PBS) (pH 7.3, in 15% sucrose), incubated 2 hrs in anti-myosin antibody (M-7648, Sigma Chemical Co., St. Louis, MO) diluted 1:10 with blocking solution, washed three times (5 min) with blocking solution and incubated 1 hr in 1:30 FITC-conjugated anti-rabbit IgG as secondary antibody (EY Labs, Inc., San Mateo, CA) in blocking solution. Samples were rinsed in PBS and mounted in an anti-fading solution with 1:1 PBS:glycerol with 3% n-propyl gallate.

Evaluation of a modified alcohol dehydrogenase assay for the determination of ethanol in blood.

1982 ◽  
Vol 28 (10) ◽  
pp. 2125-2127 ◽  
Author(s):  
A Poklis ◽  
M A Mackell
Keyword(s):  
Gas Chromatography ◽  
Reaction Time ◽  
Alcohol Dehydrogenase ◽  
Correlation Coefficients ◽  
Least Square ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Sigma Chemical ◽  
Enzymic Assay

Abstract We evaluated a new alcohol dehydrogenase (EC 1.1.1.1) enzymic assay (ADH-glycine, Sigma Chemical Co.) for the determination of ethanol in blood. This assay differs from the manufacturer's previous assay (ADH-pyrophosphate) in that glycine replaces pyrophosphate as the buffer and hydrazine replaces semicarbazide as the trapping agent. The standard curve for the assay was linear over blood ethanol concentrations of 0.50-5.00 g/L. The reaction time of the assay was 10 min. At 1.00 g/L within-run and between-run CVs were 3.96% (n = 20) and 4.01% (n = 20), respectively. Mean analytical recovery of ethanol added to whole blood at 0.50-5.00 g/L was 99.7% (SD 2.6%). We performed 100 consecutive clinical and forensic determinations by the ADH-glycine assay, the ADH-pyrophosphate assay, and gas chromatography. Correlation coefficients of the results by least-square linear regression were 0.995 for ADH-pyrophosphate vs ADH-glycine, and 0.990 for gas chromatography vs ADH-glycine. The major advantage of the ADH-glycine assay over the ADH-pyrophosphate assay is the shorter reaction time, 10 min vs 30 min.

Corrections

1973 ◽  
Vol 19 (9) ◽  
pp. 1084-1084
Keyword(s):  
Aspergillus Niger ◽  
Horseradish Peroxidase ◽  
Glucose Oxidase ◽  
Gluconic Acid ◽  
Type Ii ◽  
Sigma Chemical

Abstract p 1424, Table 2, "units/liter" should read "milliunits/ liter." In reference to the article by Meites, S., and Saniel-Banrey, K., Clin. Chem. 19, 308 (1973), the authors wish to make the following clarification: The glucose oxidase used was purified from Aspergillus niger, and obtained from Sigma Chemical Co., St. Louis, Mo. 63178. It contained 1.5 EU/mg. One unit (EU) will oxidize 1 µmol of glucose to gluconic acid and H2O2 per minute at pH 5.1 at 35 °C. The peroxidase used was horseradish peroxidase and was obtained from Sigma Chemical Co. as Type II (RZ 1.0-1.5), approximately 100-150 Purpurogallin (20-s) Units/mg, or as Worthington "Peroxidase D," 626 U/mg (RZ > 1), Worthington Biochemical Corp., Freehold, N.J. 07728.

Glutamic-Oxaloacetic Transaminase Activity: A Potential End-Point-Temperature Indicator for Imported Cooked Beef†

1995 ◽  
Vol 58 (6) ◽  
pp. 686-688 ◽  
Author(s):  
G. K. SEARCY ◽  
S. D. SENTER ◽  
R. L. WILSON
Keyword(s):  
Transaminase Activity ◽  
Glutamic Oxaloacetic Transaminase ◽  
Point Temperature ◽  
End Point ◽  
Critical Range ◽  
Test Kit ◽  
Thermally Processed ◽  
Beef Products ◽  
Sigma Chemical ◽  
End Point Temperature

Glutamic-oxaloacetic transaminase (GOT) activities in thermally processed beef samples were determined with a diagnostic test kit (no. 505P, Transaminases ALT/GPT and AST/GOT, Sigma Chemical Co.) procedure for possible use as indicators of cooking end-point temperatures (EPTs) between 71.1 and 82.2°C. GOT activity in the beef samples decreased curvilinearly with increasing EPTs. Activity was 3,450, 120, and 6 Sigma-Frankel units/ml (SFUs/ml) at 71.1, 75.6, and 82.2°C, respectively; a reduction of 99.8%. GOT values at 78.9,79.4, and 80.0°C, the critical range of EPTs in evaluating beef logs imported from South America, were 31, 17, and 14 SFUs/ml, respectively. Values within this range of temperatures differed significantly (P < 0.05); we suggest that residual GOT activity may be used as an EPT indicator for imported cooked beef products.

Interactions between TGF-beta and adrenocorticotropin in growth regulation of human adrenal fetal zone cells

1994 ◽  
Vol 266 (3) ◽  
pp. E495-E500
Author(s):  
A. K. Stankovic ◽  
W. E. Grizzle ◽  
C. R. Stockard ◽  
C. R. Parker
Keyword(s):  
Cell Growth ◽  
Transforming Growth Factor Beta ◽  
Growth Regulation ◽  
Transforming Growth Factor ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Tgf Beta ◽  
Intrauterine Development ◽  
Sigma Chemical ◽  
And Function

The factors that regulate growth and function of the human adrenal gland during intrauterine development and thereafter are ill defined. Whereas others have reported that adrenocorticotropic hormone (ACTH) augments the inhibitory effect of transforming growth factor-beta (TGF-beta) on growth of fetal zone (FZ) cells of the human fetal adrenal, we recently found that ACTH interferes with TGF-beta's inhibition of growth of fetal adrenal neocortex cells. In this study we sought to assess independently the effects of TGF-beta in the absence and presence of ACTH on growth of FZ cells. TGF-beta, in a time- and dose-dependent manner, inhibited growth (i.e., [3H]thymidine incorporation) of FZ cells. ACTH (Cortrosyn), at 90 pM to 90 nM, was found to interfere with the TGF-beta inhibition of FZ growth. ACTH 1-24 and human ACTH 1-39, both from Sigma Chemical, also were found to blunt the response of FZ cells to TGF-beta. Growth inhibition due to TGF-beta action and the reversal by ACTH of TGF-beta effects on FZ cell growth were confirmed by the results of immunohistochemical analyses of 5'-bromo-2'-deoxyuridine incorporation into nuclei of FZ cells and by indirect evaluations of cell numbers. Both forskolin (10 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), but not phorbol 12-myristate 13-acetate (1 or 100 mM), were able to mimic ACTH actions in blunting the inhibitory effects of TGF-beta on DNA synthesis. We conclude that ACTH, possibly via activation of adenylate cyclase, interferes with, rather than augments, the growth-inhibitory effect of TGF-beta on FZ cell growth.

scholarly journals 89 EVALUATION OF A CUSHIONED CENTRIFUGATION TECHNIQUE FOR PROCESSING BOAR SEMEN FOR FREEZING

2005 ◽  
Vol 17 (2) ◽  
pp. 195
Author(s):  
C. Matas ◽  
J. Gadea ◽  
G. Decuadro-Hansen
Keyword(s):  
Egg Yolk ◽  
Membrane Lipid ◽  
Isotonic Solution ◽  
Circulating Water ◽  
Plasma Membrane Lipid ◽  
Penetration Ability ◽  
Thawing Process ◽  
Boar Semen ◽  
Sigma Chemical

Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.

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