Development of a progestin-based estrus synchronization program: I. Reproductive response of cows fed melengestrol acetate for 20 days with an injection of progesterone

We designed two experiments to determine the efficacy of an estrus control system in cows that combined long-term progestin exposure (20 d) with an acute increase in progesterone concentration. In Exp. 1, cows (n = 30) were fed either melengestrol acetate (MGA; .5 mg x cow(-1) x d(-1)) or ground ear corn (MGA carrier) for 20 d. On d -15 (last day of MGA feeding = d 0), cows were administered 25 mg of PGF2alpha to regress the corpus luteum (CL) and establish an environment conducive to the development of persistent follicles. To synchronously regress persistent follicles, cows fed MGA (n = 15) were injected with 200 mg of progesterone on d -2 (MGA-P), and the cows fed the MGA carrier were not treated (CONT; n = 15). Cows in the CONT group were artificially inseminated 12 h after detection of spontaneous estrus from d -20 to d 8. Estrus was observed, and all cows in the MGA-P group were artificially inseminated during the period of estrus synchronization (SYNC; d 1 to 8). No difference in conception rate was observed between treatments. In Exp. 2, postpartum cows (n = 113) received either the MGA-P (n = 56) or CONT (n = 57) treatment. More (P < .05) cows were observed in estrus during SYNC in the MGA-P (50%) than in the CONT (28%) group. Of the cows in the MGA-P group that were not observed in estrus during SYNC, 50% were in estrus for the first time 23 to 29 d after MGA withdrawal (SYNC2), suggesting that these cows ovulated without observable estrus during SYNC. Estrus was observed for the first time during SYNC2 in more (P < .05) cows in the MGA-P (25%) than in the CONT (7%) group. Conception rate at the synchronized estrus, pregnancy rate, and interval to first service and pregnancy were similar between treatments. We conclude that administration of MGA-P results in the synchronization and(or) induction of a fertile estrus in cows.     

Extension of corpus luteum lifespan and reduction of uterine secretion of prostaglandin F2 alpha of cows in response to recombinant interferon-tau

Two experiments tested the effect of recombinant ovine and bovine interferon-tau on corpus luteum lifespan, interestrous interval, and oxytocin-induced uterine secretion of prostaglandin F2 alpha. Cows received intrauterine injections of 100 micrograms of recombinant ovine interferon-tau plus 1.4 mg of BSA or of 1.5 mg of BSA alone in Experiment 1 and 200 micrograms of recombinant bovine interferon-tau plus 1.3 mg of BSA or 1.5 mg of BSA alone in Experiment 2. Twice daily injections (0700 and 1900 h) were split evenly between the uterine horns from d 14 to 24 of the experimental estrous cycle via an AI pipette in Experiment 1 and via intrauterine catheters in Experiment 2. On d 17, cows were injected with 100 IU of oxytocin, and plasma was collected for analysis of 13,14-dihydro-15-keto-prostaglandinF2 alpha. Recombinant ovine interferon-tau extended the lifespan of the corpus luteum (27.5 vs. 19.2 d) and interestrous interval (30.5 vs. 20.6 d) and abolished the oxytocin-induced increase in 13,14-dihydro-15-keto-prostaglandinF2 alpha, which peaked at 30 min for the BSA control group (210.8 pg/ml). Recombinant bovine interferon-tau also extended the lifespan of the corpus luteum (29.0 vs. 21.4 d) and interestrous interval (31.5 vs. 22.6 d) and abolished the oxytocin-induced increase in 13,14-dihydro-15-keto-prostaglandin F2 alpha, which peaked at 30 min for the BSA control group (205.6 pg/ml). In conclusion, recombinant ovine interferon-tau and recombinant bovine interferon-tau were effective antiluteolytic agents in cattle.     

Induced and synchronized estrus in cattle: dose titration of estradiol benzoate in peripubertal heifers and postpartum cows after treatment with an intravaginal progesterone-releasing insert and prostaglandin F2alpha

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.     

A comparative study of plasma 17beta-oestradiol, progesterone, placental lactogen and chorionic gonadotrophin in abortion induced with intra-amniotic prostaglandin F2alpha

Serial estimations were made in plasma of 17beta-oestradiol (E2), progesterone and human placental lactogen (HPL) in 43 patients and of human chorionic gonadotrophin (HCG) in 34 patients during mid-trimester abortions induced with intra-amniotic prostaglandin F2alpha (PGF2alpha. Mean plasma concentrations of all the hormones showed a progressive fall after PGF2alpha. There was no relationship between the fall in levels of progesterone, HPL and HCG and the induction-abortion interval, signs of fetal distress or of intrauterine fetal death. Both the control level and the rate of fall of E2 were related to the induction-abortion interval and a rapid decline preceded intrauterine fetal death. The relationships of the progesterone/E2 ratio and the amniotic fluid volume/progesterone ratio to the induction-abortion interval were examined. The variation in the time at which significant falls in the concentration of individual hormones occurred was probably related to their respective half-lives in plasma.     

Evidence that Fe-NTA-induced renal prostaglandin F2 alpha is responsible for hyperplastic response in kidney: implications for the role of cyclooxygenase-dependent arachidonic acid metabolism in renal tumor promotion

Oxidative stress in a tissue activates phospholipase A2 which releases free arachidonic acid. In addition, a low grade oxidative tone also stimulates the tissue cyclooxygenase activity. Cyclooxygenase-dependent arachidonic acid metabolites such as PGF 2 alpha are known to play an important role in the development and maintenance of hyperplasia in skin in response to the application of tumor promoters. In this study we show that Fe-NTA, an oxidant renal tumor promoter induces PGF 2 alpha which was maximum at 12 hours after Fe-NTA treatment. However, at all time points studied, the elevated levels of PGF 2 alpha have been observed. As a result of the induction of PGF 2 alpha, the hyperplastic response can also be observed in the histopathology of the tissue. Additionally, an increased incorporation of [3H]thymidine in renal DNA has also been observed. Pretreatment of animals with indomethacin suppresses Fe-NTA-mediated hyperproliferation suggesting a role of cyclooxygenase in Fe-NTA-mediated stimulation of hyperplastic activity. The pretreatment of animals with the chain breaking antioxidants, Vit. E, BHA and BHT were only partially effective in inhibiting Fe-NTA-mediated PGF2 alpha production, further suggesting a role of non-free radical-dependent mechanism in its production. Our data suggest that Fe-NTA-induced PGF2 alpha through the activation of cyclooxygenase is responsible for the development and maintenance of hyperplasia in kidney.     

A comparative study of uterine activity in labour induced with prostaglandin F2alpha or oxytocin and in spontaneous labour. I. Pattern of the uterine contractions

The uterine contraction patterns and the changes in fetal heart rate (FHR) were studied in cardiotocographic recordings from 26 women in oxytocin-induced labour, 26 women in PGF2alpha-induced labour and 24 women during the later part of spontaneous labour. The contraction patterns and their effect on the FHR did not differ between the three groups. During the course of labour an increasing steepness of the upward slope of the contraction wave with increasing intensity of the contraction was found. High frequency of atypical contraction patterns, suggesting some degree of uterine incoordination was found during the active phase of labour in 10 patients, 8 of whom were primiparae. This incoordination could not be related to the effect of induction with either drug. Incoordinated contractions were associated with longer duration of labour and a tendency to more pronounced acidosis in the infant at birth, although mean values still fell in the normal range. Ominous FHR patterns were only seen in 2 cases of uterine hyperactivity during induction of labour.     

Fate of the dominant follicle, embryonal survival, and pregnancy rates in dairy cattle treated with prostaglandin F2 alpha and progestins in the absence or presence of a functional corpus luteum

Our objective was to examine the role of progestin type on serum concentrations of progesterone (p4) and estradiol-17 beta (E2), ovarian follicular dynamics, and fertility in cattle in the presence or absence of a corpus luteum (CL) in an estrus synchronization scheme using progestin and PGF2 alpha. In Exp. 1, 325 cows and heifers were given one injection of PGF2 alpha (d 0) and then assigned randomly within parity to five treatments: to receive a second PGF2 alpha injection 14 d later (control); to receive norgestomet (NORG) for 7 d beginning on d 8, with a second PGF2 alpha injection given either 1 d (NORG + no CL) or 6 d (NORG + CL) after insertion; or to receive a P4-releasing intravaginal device (PRID) in lieu of norgestomet at comparable times. Presence or absence of a CL was based on concentrations of serum P4 on d 14. Pregnancy rates after insemination were greater (P < .01) with luteal treatments than with nonluteal treatments. Embryonal survival between two stages of pregnancy was 87.6%. In Exp. 2, ovarian structures in 50 cows were examined daily using ultrasonography and the same five treatments. Diameter of the ovulatory follicle was greater (P < .05) with the nonluteal treatments (NORG and PRID + no CL) than with the control and luteal treatments (PRID and NORG + CL). Replacement of the dominant follicle during progestin treatment was altered by treatment (luteal status) and stage of the estrous cycle. Fertility was not enhanced by exogenous progestins when a CL was present. In the absence of a CL, progestin (P4 less than NORG at the doses used) reduced fertility by increasing E2 and the diameter of the ovulatory follicle and decreasing turnover of dominant follicles.     

Development of a single-shot subunit vaccine for HIV-1. 2. Defining optimal autoboost characteristics to maximize the humoral immune response

The design of a single-shot subunit vaccine for HIV-1 with polylactic-coglycolic acid (PLGA) sustained-release technology to effect an autoboost of antigen (MN gp120) at a given time after the primary immunization requires in-depth knowledge about the timing, the duration, and the need for coadjuvant in the autoboost. These questions cannot be answered unambiguously with PLGA microspheres, so we have conducted studies using Alzet minipumps to release antigen at prescribed times to mimic a PLGA autoboost. The results show that a discrete autoboost is preferred over continuous release of antigen, that the time profile of the autoboost (whether pulsatile or a 2-week continuous release) does not affect the booster immune response, and that only antigen is required in the booster immunization (a coadjuvant in the boost does not give higher titers).     

Hemoglobin Sydney--alpha beta 2 67 (E11) Val-Ala and hemoglobin Olomouc alpha 2 beta 2 86 (F 2) Ala-Asp in Czech families. DNA sequence analysis for a more accurate diagnosis of hemoglobinopathies

The paper demonstrates the importance of sequence analysis of DNA for the identification of Hb-Sydney [alpha2-beta2 67 (E11) Val-Ala]. The latter was considered erroneously, based on results of biochemical analyses to be Hb-M-Milwaukee [alpha2 beta2 67 (E 11) Val-Glu]. With the unstable Hb-Sydney correspond also phenotypical manifestations of disease (haemolytic anaemia with Heinz bodies in red blood cells). Sequence analysis of DNA of patients with Hb-Olomouc [alpha 2 beta 2 (F 2) Ala-Asp] revealed that mutation of Ala-Asp in position 86 (F 2) of the beta globin chain is coded by mutation C-->A (GCC-GAC).     

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. II. Evidence for direct binding

The present study was designed to obtain evidence for direct interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by Gbeta gamma was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by Galpha was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the Galpha-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N- and P/Q-type channels revealed direct interaction of Galpha with C-terminal fusion proteins as well as direct interaction of Gbeta gamma with loop 1 fusion proteins. These findings define loop 1 of N- and P/Q-type Ca2+ channels as an interaction site for Gbeta gamma and the C termini for Galpha.     

Immunological status of septic and trauma patients. II. Proliferative response and production of interleukin 6 and tumor necrosis factor alpha by peripheral blood mononuclear cells from septic survivor, nonsurvivor and trauma patients: a correlation with the survival rate

OBJECTIVE: The reasons for conflicting prognostic results as to DNA ploidy and cell cycle variables (DNA index, percent S (%S) phase) may be found mainly at different levels of the flow cytometric methodology used. The present study concentrated on how many nuclei have to be measured with flow cytometry for reliable DNA histogram interpretation. STUDY DESIGN: Twenty-three samples of fresh frozen and 22 samples of paraffin-embedded material from different sites were used. For each sample, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 (x1,000) events were cumulatively measured. The resulting DNA histograms were analyzed with the MultiCycle computer program using a highly reproducible interpretation protocol. RESULTS: No disagreements about DNA ploidy classification were found in samples with 10,000 or more events for the fresh frozen and 40,000 or more events for the paraffin-embedded material. Excluding DNA ploidy disagreements, the DNA index was stable in all cases. To obtain a %S-phase cell measurement within 20% of the reference value, at least 20,000 and 40,000 events were needed for, respectively, DNA diploid and DNA nondiploid cases for the fresh frozen material and, respectively, 40,000 and 50,000 events for the paraffin-embedded material. CONCLUSION: For reliable combined determination of DNA ploidy, DNA index and %S-phase cells, at least 40,000 and 50,000 events are necessary for fresh frozen and paraffin-embedded material, respectively.     

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.     

Development of a two-stage procedure for the automatic recognition of dysfluencies in the speech of children who stutter: II. ANN recognition of repetitions and prolongations with supplied word segment markers

This program of work is intended to develop automatic recognition procedures to locate and assess stuttered dysfluencies. This and the following article together, develop and test recognizers for repetitions and prolongations. The automatic recognizers classify the speech in two stages: In the first, the speech is segmented, and, in the second, the segments are categorized. The units that are segmented are words. Here assessments by human judges on the speech of 12 children who stutter are described using a corresponding procedure. The accuracy of word boundary placement across judges, categorization of the words as fluent, repetition or prolongation, and duration of the different fluency categories are reported. These measures allow reliable instances of repetitions and prolongations to be selected for training and assessing the recognizers in the subsequent paper.     

The murine (H-2k) T-cell epitopes of bee venom phospholipase A2 Lie outside the active site of the enzyme. Implications with respect to a paracrine activation of Th2 cells for an IgE antibody response

A 51-year-old drunken male was carried to a hospital with acute abdominal pain and was suspected of acute pancreatitis. The patient was treated with fasting, electrolyte transfusion, and anodyne, but took a sudden turn for the worse and died in 16 hours. In the judicial autopsy, rupture of a small intestine was detected. As the police investigated, he had been kicked in the abdomen by an assailant before coming to the hospital. The cause of death was diagnosed to be acute peritonitis due to the rupture of a small intestine. Several problems were pointed out on medical examinations and treatments of this case.     

Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L. A candidate gene for lysinuric protein intolerance

We have identified a new human cDNA (y+L amino acid transporter-1 (y+LAT-1)) that induces system y+L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes. Human y+LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1. Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y+L, respectively. y+LAT-1 protein forms a approximately 135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of approximately 85 kDa (i.e. 4F2hc) and approximately 40 kDa (y+LAT-1). Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the co-expressed transport activity. These data suggest that y+LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters. Human y+LAT-1 mRNA is expressed in kidney > peripheral blood leukocytes > lung > placenta = spleen > small intestine. The human y+LAT-1 gene localizes at chromosome 14q11.2 (17cR approximately 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P. , Savontaus, M. L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P. (1997) Am. J. Hum. Genet. 60, 1479-1486). LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues. The pattern of expression of human y+LAT-1, its co-expressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y+LAT-1 as a candidate gene for LPI.