Human immune response to triatomine embryo extract

Dipetalogaster maximus embryo extracts were used to stimulate peripheral blood mononuclear cells (PBMC) and in ELISA with sera either from Trypanosoma cruzi infected or non-infected individuals. The results showed that there was significant proliferative response and high antibody titers in sera of chagasic patients.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

Assembly of regularly spaced nucleosome arrays by Drosophila chromatin assembly factor 1 and a 56-kDa histone-binding protein.

To ascertain the mechanism by which nucleosomes are assembled by factors derived from Drosophila embryos, two proteins termed Drosophila chromatin assembly factors (CAFs) 1 and 4 (dCAF-1 and dCAF-4) were fractionated and purified from a Drosophila embryo extract. The assembly of chromatin by dCAF-1, dCAF-4, purified histones, ATP, and DNA is a process that generates regularly spaced nucleosomal arrays with a repeat length that resembles that of bulk native Drosophila chromatin and is not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and dCAF-4 is nearly complete within 10 min. The dCAF-1 activity copurified with the Drosophila version of chromatin assembly factor-1 (CAF-1), a factor that has been found to be required for the assembly of chromatin during large tumor (T) antigen-mediated, simian virus 40 (SV40) origin-dependent DNA replication. The dCAF-4 activity copurified with a 56-kDa core-histone-binding protein that was purified to > 90% homogeneity.

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6).Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection. (C) 2001 by Elsevier B.V.

Calbindin-immunoreactive sensory neurons in dissociated dorsal root ganglion cell cultures of chick embryo: role of culture conditions.

Immunoreactivity to calbindin D-28k, a vitamin D-dependent calcium-binding protein, is expressed by neuronal subpopulations of dorsal root ganglia (DRG) in the chick embryo. To determine whether the expression of this phenotypic characteristic is maintained in vitro and controlled by environmental factors, dissociated DRG cell cultures were performed under various conditions. Subpopulations of DRG cells cultured at embryonic day 10 displayed calbindin-immunoreactive cell bodies and neurites in both neuron-enriched or mixed DRG cell cultures. The number of calbindin-immunoreactive ganglion cells increased up to 7-10 days of culture independently of the changes occurring in the whole neuronal population. The presence of non-neuronal cells, which promotes the maturation of the sensory neurons, tended to reduce the percentage of calbindin-immunoreactive cell bodies. Addition of horse serum enhanced both the number of calbindin-positive neurons and the intensity of the immunostaining, but does not prevent the decline of the subpopulation of calbindin-immunoreactive neurons during the second week of culture; on the contrary, the addition of muscular extract to cultures at 10 days maintained the number of calbindin-expressing neurons. While calbindin-immunoreactive cell bodies grown in culture were small- or medium-sized, no correlation was found between cell size and immunostaining density. At the ultrastructural level, the calbindin immunoreaction was distributed throughout the neuroplasm. These results indicate that the expression of calbindin by sensory neurons grown in vitro may be modulated by horse serum-contained factors or interaction with non-neuronal cells. As distinct from horse serum, muscular extract is able to maintain the expression of calbindin by a subpopulation of DRG cells.

Maintenance of neuronal expression of calbindin by a muscular extract in cultures of chick dorsal root ganglion cells.

Calbindin D-28K is a calcium-binding protein which is expressed by subpopulations of dorsal root ganglion cells cultured from 10-day-old (E10) chick embryos. After 7 or 10 days of culture, more than 20% of the ganglion cells are immunostained by an anticalbindin-antiserum; however, after 14 days of culture, the proportion drops to 10%. This fall can be prevented by addition of muscle extract to cultures at 10 days. Thus the transitory expression of calbindin-immunoreactivity by responsive sensory neurons would be not only induced but also maintained by a differentiation factor of muscular origin.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Effects of Annona squamosa extract on early pregnancy in rats

Annona squamosa Linn., family Annonaceae, is said to show varied medicinal effects, including insecticide, antiovulatory and abortifacient. The purpose of present study was to investigate if A. squamosa seed aqueous extract, in doses higher than that popularly used to provoke abortion, interferes with reproductive performance, and to correlate the ingestion of this extract with possible alterations in rat embryonic implantation. Doses of 300 mg/kg (Treated Group I, n = 17) and 600 mg/kg (Treated Group II, n = 12) body wt. were administered by gavage, during days 1 to 5 of pregnancy (preimplantation period). The control group (n = 13) received water in the same manner, during the same period for comparison with experimental groups. The animals were euthanized on day 10 of pregnancy. Treatment of dams during the preimplantation period showed no signs of toxicity, and no alteration in the corpora lutea, implantations and embryo in terms of development numbers. The percentage of preimplantation and postimplantation losses in treated groups I and II did not differ from those of control. Treatment with aqueous extract of A. squamosa seeds caused no morphological change in the endometrium. The absence of morphological alterations in uterine epithelial cells in treated groups I and II permitted a viable embryonic implantation, as verified by the number of embryos in development at day 10 of pregnancy. Thus, A. squamosa seed aqueous extract did not interfere with the reproductive performance of pregnant rats.

Strategies to improve the ovarian response to equine pituitary extract in cyclic mares

Equine pituitary extract (EPE) has been reported to induce heightened follicular development in mares, but the response is inconsistent and lower than results obtained in ruminants undergoing standard superovulatory protocols. Three separate experiments were conducted to improve the ovarian response to EPE by evaluating: (1) effect of increasing the frequency or dose of EPE treatment; (2) use of a potent gonadotropin-releasing hormone agonist (GnRH-a) prior to EPE stimulation (3) administration of EPE twice daily in successively decreasing doses. In the first experiment. 50 mares were randomly assigned to one of four treatment groups. Mares received (1) 25 mg EPE once daily; (2) 50 mg EPE once daily (3) 12.5 mg EPE twice daily; or (4) 25 mg EPE twice daily. All mares began EPE treatment 5 days after detection of ovulation and received a single dose of cloprostenol sodium 7 days postovulation. EPE was discontinued once half of a cohort of follicles reached a diameter of greater than or equal to35 mm and hCG was administered. Mares receiving 50 mg of EPE once daily developed a greater number (P = 0.008) of preovulatory follicles than the remaining groups of EPE-treated mares, and more (P = 0.06) ovulations were detected for mares receiving 25 mg EPE twice daily compared to those receiving either 25 mg EPE once daily and 12.5 mg EPE twice daily. Embryo recovery per mare was greater (P = 0.05) in the mares that received 12.5 mg EPE twice daily than those that received 25 mg EPE once daily. In Experiment 2, 20 randomly selected mares received either 25 mg EPE twice daily beginning 5 days after a spontaneous ovulation. or two doses of a GnRH-a agonist upon detection of a follicle greater than or equal to35 mm and 25 mg EPE twice daily beginning 5 days after ovulation. Twenty-four hours after administration of hCG, oocytes were recovered by transvaginal aspiration from all follicles greater than or equal to35 mm. No differences were observed between groups in the numbers of preovulatory follicles generated (P = 0.54) and oocytes recovered (P = 0.40) per mare. In Experiment 3, 18 mares were randomly assigned to one of two treatment groups. Then, 6-11 days after ovulation, mares were administered a dose of PGF(2gamma) and concomitantly began twice-daily treatments with EPE given in successively declining doses, or a dose of PGF(2alpha), but no EPE treatment. Mares administered EPE developed a higher (P = 0.0004) number of follicles :35 mm, experienced more (P = 0.02) ovulations, and yielded a greater (P = 0.0006) number of embryos than untreated mares. In summary, doubling the dose of EPE generated a greater ovarian response, while increasing the frequency of treatment, but not necessarily the dose. improved embryo collection. Additionally, pretreatment with a GnRH-a prior to ovarian stimulation did not enhance the response to EPE or oocyte recovery rates. (C) 2002 Elsevier B.V. All rights reserved.

Study of the embryotoxic effects of an extract of rosemary (Rosmarinus officinalis L)

Extracts of rosemary, Rosmarinus officinalis L., have been used in folk medicine as a diuretic, an emenagogue, an antispasmodic and its aqueous extract does not present toxicity to man, presenting, however, abortive effects. In order to evaluate if this plant induces abortion and/or interferes with the normal development of the concepts, doses of 26 mg of a 30% (w/v) R. officinalis aqueous extract (13 mg solids/ml) made with leaves, flowers and stem were administered daily by gavage during two different periods of Wistar rat pregnancy. One group of animals (N = 12) received the extract from days 1 to 6 of pregnancy (preimplantation period) and another group (N = 14) received the same extract from days 6 to 15 of pregnancy (organogenic period). Control groups (N = 12) received saline in the same volume and during the same periods as their respective experimental groups. The animals were sacrificed at term. The treatment of the darns during either the preimplantation or the organogenic period did not cause significant changes in the postimplantation loss or in the number of anomalies or malformations of the term fetuses, which also showed a similar degree of development when compared with the respective controls. The percent of preimplantation loss in the group treated before embryo implantation increased, although the difference was not significant compared to the control. This result suggests that rosemary extract may present an anti-implantation effect without interfering with the normal development of the concept after implantation.

Hepatic Responses of Juvenile Fundulus heteroclitus from Pollution-adapted and Nonadapted Populations Exposed to Elizabeth River Sediment Extract.

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries region of the Elizabeth River, Virginia, have passed polycyclic aromatic hydrocarbon (PAH) resistance to their offspring as evidenced by early life stage testing of developmental toxicity after exposure to specific PAHs. Our study focused on environmentally relevant PAH mixtures in the form of Elizabeth River sediment extract (ERSE). Juvenile (5 month) F1 progeny of pollution-adapted Atlantic Wood (AW) parents and of reference site (King's Creek [KC]) parents were exposed as embryos to ERSE. Liver alterations, including nonneoplastic lesions and microvesicular vacuolation, were observed in both populations. ERSE-exposed KC fish developed significantly more alterations than unexposed KC fish. Interestingly, unexposed AW killifish developed significantly more alterations than unexposed KC individuals, suggesting that AW juveniles are not fully protected from liver disease; rapid growth of juvenile fish may also be an accelerating factor for tumorigenesis. Because recent reports show hepatic tumor formation in adult AW fish, the differing responses from the 2 populations provided a way to determine whether embryo toxicity protection extends to juveniles. Future investigations will analyze older life stages of killifish to determine differences in responses related to chronic disease.

Antioxidant And Antiproliferative Activities Of Methanolic Extract From A Neglected Agricultural Product: Corn Cobs.

Neglected agricultural products (NAPs) are defined as discarded material in agricultural production. Corn cobs are a major waste of agriculture maize. Here, a methanolic extract from corn cobs (MEC) was obtained. MEC contains phenolic compounds, protein, carbohydrates (1.4:0.001:0.001). We evaluated the in vitro and in vivo antioxidant potential of MEC. Furthermore, its antiproliferative property against tumor cells was assessed through MTT assays and proteins related to apoptosis in tumor cells were examined by western blot. MEC showed no hydroxyl radical scavenger capacity, but it showed antioxidant activity in Total Antioxidant Capacity and DPPH scavenger ability assays. MEC showed higher Reducing Power than ascorbic acid and exhibited high Superoxide Scavenging activity. In tumor cell culture, MEC increased catalase, metallothionein and superoxide dismutase expression in accordance with the antioxidant tests. In vivo antioxidant test, MEC restored SOD and CAT, decreased malondialdehyde activities and showed high Trolox Equivalent Antioxidant Capacity in animals treated with CCl4. Furthermore, MEC decreased HeLa cells viability by apoptosis due an increase of Bax/Bcl-2 ratio, caspase 3 active. Protein kinase C expression increased was also detected in treated tumor cells. Thus, our findings pointed out the biotechnological potential of corn cobs as a source of molecules with pharmacological activity.

Trypanosoma Cruzi Strains From Triatomine Collected In Bahia And Rio Grande Do Sul, Brazil.

Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains. First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice. Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%. The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship.

Presynaptic Neuromuscular Action Of A Methanolic Extract From The Venom Of Rhinella Schneideri Toad.

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording - in response to indirect stimulation - and for electrophysiological measurements. Venom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively). These results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.