Examining the mediational role of psychological flexibility, pain catastrophizing, and visceral sensitivity in the relationship between psychological distress, irritable bowel symptom frequency, and quality of life.

The aim of the current study was to use Structural Equation Modelling (SEM) to examine whether psychological flexibility (i.e. mindfulness, acceptance, valued-living) mediates the relationship between distress, irritable bowel syndrome (IBS) symptom frequency, and quality of life (QoL). Ninety-two individuals participated in the study (12 male, 80 female, Mage = 36.24) by completing an online survey including measures of visceral sensitivity, distress, IBS-related QoL, mindfulness, bowel symptoms, pain catastrophizing, acceptance, and valued-living. A final model with excellent fit was identified. Psychological distress significantly and directly predicted pain catastrophizing, valued-living, and IBS symptom frequency. Pain catastrophizing directly predicted visceral sensitivity and acceptance, while visceral sensitivity significantly and directly predicted IBS symptom frequency and QoL. Symptom frequency also had a direct and significant relationship with QoL. The current findings suggest that interventions designed to address unhelpful cognitive processes related to visceral sensitivity, pain catastrophizing, and psychological distress may be of most benefit to IBS-related QoL.

The effect of benzalkonium chloride additions to AH Plus sealer. Antimicrobial, physical and chemical properties.

OBJECTIVES: The aim of this study was to determine the antimicrobial and antibiofilm activities and physicochemical properties of AH Plus sealer mixed with different concentrations of benzalkonium chloride (BC). METHODS: AH Plus was tested alone and mixed with 1%, 2% and 3% of BC. The antimicrobial and antibiofilm activities of the sealers against Enterococcus faecalis were evaluated by the direct contact test (DCT) and by confocal laser scanning microscopy, respectively. Setting time, flow and solubility were assessed according to ANSI/ADA specifications. Microhardness and contact angle tests were also performed. The chemical changes of the sealers were evaluated by X-ray diffraction analysis, and both Fourier transform infrared spectroscopy (FT-IR) and attenuated total reflectance Fourier transform infrared (ATR FT-IR). RESULTS: AH Plus+3% BC was the only sealer to promote total elimination of E. faecalis and the biovolume in this group was significantly lower than in the rest of the sealers (p>0.05). The physical properties of the sealers were according to the ANSI/ADA specifications. The microhardness decreased significantly when BC was added and a significant reduction in contact angle was obtained when incorporating 2% and 3% BC (p<0.05). No phase changes were observed with the modified sealers. CONCLUSIONS: The addition of 2% or higher concentrations BC to AH Plus showed antimicrobial and antibiofilm activities without affecting the properties specified in ANSI/ADA standards. However, additives to the root canal sealer altered other physical and chemical properties that are not commonly found in the literature to evaluate filling materials. CLINICAL SIGNIFICANCE: The present study highlights that the antimicrobial properties of AH Plus can be significantly improved with the addition of BC. Testing beyond what is specified in standards may be indicated.

Effect of hCG treatment on the oestrous and ovulation responses to FSH in prepubertal gilts.

To ensure sufficient numbers of pregnant females, particularly at hotter times of the year, hormonal induction of gilt oestrus may be necessary. However, the gilt oestrus and ovulation responses to gonadotrophin treatment have often proven unpredictable. The objective of this study was to examine possible reasons for this unpredictability. Prepubertal gilts (approximately 150 days of age, n = 63) were assigned to one of three treatments: injection of 300 IU hCG (n = 15); pre-treatment with 100 mg FSH in polyvinylpyrrolidinone administered as 2 x 50 mg injections 24 h apart, followed by 600 IU eCG at 24 h after the second FSH injection (n = 23); or FSH pre-treatment as above followed by 300 IU hCG at 24 h after the second FSH injection (n = 25). To facilitate oestrus detection, gilts were exposed to a mature boar for 15 min daily for 7 days. Blood samples were obtained on the day of eCG or hCG injection and again 10 days later and gilt ovulation responses determined based on elevated progesterone concentrations. The oestrus responses by 7 days were 6.7%, 17.5% and 64.0% for gilts treated with hCG, FSH + eCG and FSH + hCG, respectively (p < 0.001). The oestrous gilt receiving hCG alone and one oestrous FSH + hCG gilt did not ovulate, all other oestrous gilts ovulated. A further two anoestrous FSH + eCG-treated gilts ovulated. These data suggest that FSH pre-treatment facilitated the development of ovarian follicles to the point where they became responsive to hCG, but had little effect on the response to eCG.

Effect of prior FSH treatment on the estrus and ovulation responses to eCG in prepubertal gilts.

The objective of this study was to determine the effect of pre-treatment of prepubertal gilts with FSH on the estrus and ovulatory responses to eCG injection at two ages. A total of 149 prepubertal Hypor gilts were selected at 150 days (n=76) or 180 days (n=73) of age and assigned to injection of 400 IU eCG plus 200 IU hCG (PG600), 600IU eCG alone (Folligon), pre-treatment with 72 mg FSH (Folltropin) administered as 6 x 12 mg injections at 12 h intervals with 600 IU Folligon 12h after last FSH injection, or non-injected controls. To facilitate detection of estrus, gilts were exposed to a mature boar for 15 min daily for 7 days. To determine ovulatory responses, blood samples were obtained on the day of injection and 10 days later and assayed for progesterone content. Following treatment at 150 days, one control gilt (5.3%) was deemed estrus but ovulation did not occur. Compared to treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus response (52.6% compared with. 26.3%) and increased (P<0.01) the ovulatory response (89.5% compared with. 47.4%). The estrous response in gilts pretreated with Folltropin was intermediate (42.1%) but the ovulatory response (47.4%) was the same as for Folligon alone. Following treatment at 180 days, two control gilts (10.5%) were deemed estrus and ovulation did occur in these gilts. There was no difference between hormone-treated groups for estrus or ovulatory responses, although the ovulatory response of PG600-treated gilts tended (P=0.1) to be greater than for the Folligon-treated group (89.5% compared with 66.7%), with Folltropin-pretreated gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory responses of gilts were greater for PG600 than for Folligon and that while responses to PG600 were not affected by gilt age, for the combined Folligon groups, estrous response (P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.

Effect of eCG or eCG Plus hCG on oestrus expression and ovulation in prepubertal gilts.

To meet weekly breeding targets, it is occasionally necessary to inject exogenous gonadotrophins to induce oestrus in prepubertal gilts. However, the gilt oestrus response to equine chorionic gonadotrophin (eCG) either alone or in combination with human chorionic gonadotrophin (hCG) can be unpredictable. The objective of the present study was to examine possible reasons for this unpredictability. Prepubertal gilts (90 kg and 153 days of age, n = 109) received an injection of either 600 IU eCG or a combination of 400 IU eCG and 200 IU hCG (PG600), or were non-injected controls, and were then exposed to a mature boar for 15 min daily for 7 days for oestrus detection. At the time of injection, real-time ultrasound revealed that the gilt ovaries had primarily 1-2 mm follicles. Blood samples were obtained at time of hormone injection (day 0) and at days 3, 7 and 10 for assay of serum progesterone concentrations. The oestrus responses by 7 days were 15.5%, 73.3% and 0%, for eCG, PG600, and control gilts, respectively (p < 0.001). The oestrus response improved (p < 0.05) with increasing body weight. Based on circulating progesterone levels, all oestrous gilts ovulated except for four of the PG600 gilts. Failure to express oestrus in PG600 gilts was not associated with a premature rise in progesterone.

Effect of insemination-ovulation interval and addition of seminal plasma on sow fertility to insemination of cryopreserved sperm.

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.

Weak TCR stimulation induces a calcium signal that triggers IL-4 synthesis, stronger TCR stimulation induces MAP kinases that control IFN-gamma production.

Th1 and Th2 cells produce different cytokines and have distinct functions. Th1/Th2 cell differentiation is influenced, among other factors, by the nature of TCR-MHC interactions. However, how the TCR transduces a signal resulting in IFN-gamma or IL-4 production is a matter of debate. For example, some authors reported a loss of calcium signaling pathway in Th2 cells. We used a T cell hybridoma producing IL-4 upon weak TCR stimulation and both IL-4 and IFN-gamma for strong TCR engagement as a model to study how TCR signaling pathways are differentially activated in both conditions of stimulation and how this influences the production of cytokines. We show that: (1) the calcium response is identical following weak and strong TCR stimulation; (2) mitogen-activated protein kinase(MAPK) activation is a gradual phenomenon depending upon the strength of TCR activation; (3) a calcium response, even weak, triggers IL-4 expression; (4) IFN-gamma synthesis requires not only a calcium response but also MAPK activation. The MAPK pathway is dispensable for IL-4 production, although it amplifies IL-4 synthesis upon strong TCR stimulation; (5) TCR-induced IL-4 production also depends on calcium signaling in Th2 cells, while IFN-gamma synthesis is dependent, in addition, on MAPK activation in Th1 cells.

Gold is a T cell polyclonal activator in BN and LEW rats but favors IL-4 expression only in autoimmune prone BN rats.

Gold salts are beneficial in the treatment of rheumatoid arthritis but may induce immune-mediated disorders in predisposed patients. Gold salts induce Th2-dependent autoimmunity in Brown-Norway (BN) rats but not in Lewis (LEW) rats. The aim of this study was to define molecular targets of gold salts and to approach why LEW rats are resistant. Gold salts act on early steps of transduction in T cells from BN and LEW rats since they trigger tyrosine phosphorylation of numerous proteins including p56(lck) and a calcium signal which results in IL-4 and IFN-gamma expression by BN and LEW T cells. However, the IL-4 response was favored in BN spleen cells in vitro and in vivo. IFN-gamma, produced in part by CD8(+) cells, contributes to the resistance of LEW rats since gold salt-injected LEW rats receiving anti-CD8 or anti-IFN-gamma mAb displayed the parameters characteristics of gold salt-induced Th2 autoimmunity although to a lesser extent than in BN rats. Gold salts transduce a signal in BN and LEW spleen cells resulting in IL-4 and IFN-gamma gene transcription with a preferential IL-4 response in BN rats, a Th2-prone strain, while IFN-gamma contributes to the resistance of LEW rats.

Evaluation of environmental levels and biological impact of TBT in Malta (central Mediterranean).

Chemical monitoring for butyltins in bulk seawater, surface microlayer and superficial sediments determined that the two main local sources of marine contamination by tributyltin (TBT) compounds in Malta are ship-repairing dockyards and marinas. In bulk seawater, TBT values ranged from below the detection limit of 5 ng Sn l(-1) to 300 ng Sn l(-1); in sediments derived from the most polluted areas, TBT concentrations as high as 1500 ng Sn g(-1) were measured. At TBT levels found in local harbours, several sublethal biological responses are possible and were observed, including a significant reduction in MFO enzyme system activities of fish; digestive cell atrophy in the oyster Ostrea edulis; and induction of imposex in the snail Hexaplex trunculus. The latter two responses are evident at TBT concentrations below the environmental quality standard (20 ng TBT l(-1)). The ecological implications of these results are discussed and it is concluded that exposure of marine organisms to TBT in local harbours may be expected to lead to a reduction in the long-term survival of a number of such organisms. For these reasons, both the use of TBT-based antifouling paints for pleasure boats as well as drydock practices need to be controlled by appropriate regulations and their enforcement.

Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells.

Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.

Thymocyte development in Ah-receptor-deficient mice is refractory to TCDD-inducible changes.

The arylhydrocarbon receptor (AhR), a ligand-activated transcription factor, is differentially distributed in tissues and abundant in the thymus epithelium. The activated AhR can induce the transcription of an array of genes, including genes of cell growth and differentiation. Neither the physiological function of the AhR nor its putative natural ligand is known. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a xenobiotic high-affinity activator of the AhR, and appears to be essential for most of the multifold toxic effects of TCDD. Activation of the AhR by even low doses of TCDD results in general immunosuppression and thymus hypoplasia. TCDD exposure interferes with thymocyte development; for instance, it reduces the proliferation rate of the very immature (CD4- CD8- and CD4- CD8+ HSA+) thymocytes, leads to preferential emigration of very immature cells, and drastically skews the differentiation of thymocyte subpopulations towards mature CD4- CD8+ alphabeta TCRhigh thymocytes. As shown here, in fetal thymi of AhR-deficient mice, thymocyte differentiation kinetics as defined by CD4 and CD8 surface markers, was comparable to AhR+/+ C57BL/6 mice. Also, the cell emigration characteristics were similar to AhR+/+ mice. These parameters were refractory to TCDD exposure in the AhR-/- mice, but not in the C57BL/6 mice. However, in AhR deficient mice at gestation day 15 more CD4- CD8- immature cells bore high amounts of the (alphabeta-T-cell receptor. Also, fetal thymocyte numbers were significantly lower, as compared to strain C57BL/6. Thus, the AhR is the mediator of thymotoxic effects of TCDD.

Observations on ploidy of cells and on reproductive performance in parthenogenetic turkeys.

Using flow cytometry, the ploidy levels of parthenogenetic turkeys were quantified from blastodisc stage to adulthood. Eggs were collected from noninseminated hens of the Beltsville Small White flock, known for their high degree of parthenogenesis, and the blastodermal cells from developing embryos were compared with those of embryos produced by hens inseminated with semen from males of the same flock. Erythrocytes of parthenogens from Day 10 of incubation to 27 mo of age were also used for ploidy determination. Sperm and erythrocyte preparations from normal males of the above flock served as haploid and diploid standards, respectively. In parthenogenetically developing blastoderms, 40.3 +/- 14.5% of the cells were haploid and 48.9 +/- 11.9% diploid; blastoderms from fertilized eggs had no haploid cells. The haploid cell content of parthenogens declined from the blastodermal stage to adult life, with 1.9 +/- 2.3% at 10 to 20 d of embryonic development, 1.5 +/- 1.4% at 21 to 29 d of development, 1.4 +/- 2.6% at 4 wk posthatch, and 1.3 +/- 1.9% in adulthood, although changes between the 1st mo after hatch and adult stage were not significant. It is possible, therefore, that parthenogenetic embryos with a low proportion of haploid cells could be the ones that survive to Day 10 of development and beyond, whereas those with a higher proportion of haploid cells fail to develop. The semen volume of male parthenogens was significantly lower than that of normal males, although the concentration of spermatozoa and their fertilizing capacity did not vary significantly between groups, suggesting that the germ cells of these parthenogens are capable of normal meiosis and sperm maturation leading to a normal fertility.

Differentiating between parthenogenetic and "positive development" embryos in turkeys by molecular sexing.

In mated or inseminated turkeys, 5 to 15% of eggs set for incubation show only rudimentary development. Most of these embryos die during the first 24 to 48 h of incubation and contain only unorganized sheets of tissue. This abnormal development is termed "positive development" (PD). Turkey eggs also show incidence of parthenogenesis and the resulting progeny is believed to be always male. As both types of embryos are morphologically similar at the early stage of incubation, it has been speculated that PD embryos may in fact be parthenogens. By identifying the sex at the blastodermal stage with the help of DNA markers, we have differentiated between the PD embryos and parthenogens. Parthenogenetic embryos were obtained from eggs laid by uninseminated or virgin Beltsville Small White (BSW) hens, and the PD embryos were obtained from eggs of inseminated Nicholas and British United Turkeys of America (BUTA) hens. DNA was extracted from blastoderms of parthenogenetic and PD embryos. Turkey W-chromosome specific DNA probe and primers were used to detect females in all samples by Southern blot and polymerase chain reaction (PCR), respectively. No female was detected among the 35 parthenogens examined, whereas there were 3 females among the 11 PD embryos. The presence of both males and females among PD embryos suggests that they are products of fertilization, and that at least these 3 female embryos, if not all the 11 PD embryos, are not of parthenogenetic origin. It is concluded, therefore, that PD embryos result from errors in fertilization or from early embryonic mortality following successful fertilization, and that they are unlikely to be of parthenogenetic origin.

Acute myeloid leukemia cells with low P-glycoprotein expression and high rhodamine 123 efflux capacity display high clonogenicity.

This study was designed to correlate the clonogenic capacity of acute myeloid leukemia (AML) cells with P-glycoprotein (P-gp) expression level and P-gp-mediated efflux capacity. Fifty AML cell samples were tested for P-gp expression using MRK16 monoclonal antibody and flow cytometry. Among them, 12 samples were selected for sorting experiments according to the following two criteria: their clonogenic capacity in methylcellulose in the presence of 5637 conditioned medium, and the heterogeneity of P-gp distribution in leukemic cells. For each of these 12 samples, leukemic cells which displayed the highest P-gp expression level (P-gp++) and P-gp- leukemic cells were sorted after MRK16 staining and seeded into methylcellulose for primary clonogenic assay. In each case, the number of CFU-L in the P-gp fraction was significantly higher than that of the P-gp++ fraction (P < 0.01); the median number of CFU-L for 10(5) seeded cells being 147 (range 3-1855) and 495 (range 60-4100) for P-gp++ and P-gp- populations, respectively. Furthermore, in order to correlate clonogenic capacity and P-gp function, AML cells were stained with rhodamine 123 (Rh 123), washed and then sorted after 4 h incubation at 37 degrees C in Rh 123-free media on the basis of their residual fluorescence intensity before plating. For each of six samples, we found that the number of CFU-L in the AML cell fraction which displayed the most efficient Rh 123 efflux capacity (Rh 123dull) was significantly higher compared to that of the AML cell fraction which displayed high residual fluorescence signal (Rh 123bright) (P = 0.05); the median number of CFU-L for 10(5) seeded cells being 1025 (range 250-2240) and 296 (range 11-838) for Rh 123dull and Rh 123bright populations, respectively. Altogether this study suggests that, for an individual AML cell population, the clonogenic fraction is preferentially recruited in AML cells which display low P-gp expression and high P-gp-mediated efflux capacity.

Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro.

Glucosinolates hydrolysis products are attracting increasing attention since many studies have suggested that they may be involved in the anticarcinogenic property of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin, respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma HT29 cells. Moreover, these products are able to inhibit quiescent cells to re-enter the cell cycle. Interestingly, our results clearly show that low doses of DIM and sulforaphane, although very effective on undifferentiated intestinal HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The reversibility of their effects has also been tested and is discussed.

Effect of 5637-conditioned medium and recombinant cytokines on P-glycoprotein expression in a human GM-CSF-dependent leukemic myeloid cell line.

This study was aimed at evaluating the influence of 5637-conditioned medium (5637-CM) and human recombinant cytokines on both expression and function of P-glycoprotein (P-gp) in TF-1, a GM-CSF/IL-3-dependent acute myeloid leukemia cell line which constitutively expresses functional P-gp. P-gp expression was measured by flow cytometry using MRK16 monoclonal antibody. P-gp function was measured by rhodamine 123 (Rh 123) efflux kinetics. When TF-1 cells were cultured with 5637-CM (50% v/v), both P-gp expression and P-gp efflux capacity were increased in a time-dependent manner with a 4-fold increase in P-gp expression level at day 6 whereas TF-1 cell differentiation status remained unchanged as assessed by morphological studies, phenotypical and cytochemistry analysis. Recombinant cytokines including GM-CSF, G-CSF, IL-1 beta, IL-6, stem cell factor, LIF, erythropoietin, and IL-3 had no effect on P-gp expression whereas TNF alpha induced dose- and time-dependent P-gp and mdr-1 gene overexpression. However, TNF alpha-induced P-gp overexpression had no influence on P-gp efflux capacity. Furthermore, when TF-1 cells were exposed to IL-3 for periods longer than 1 month, we found that P-gp efflux capacity was increased as compared to cells cultured with GM-CSF whereas P-gp expression was unchanged. Both TNF alpha and IL-3 did not induce TF-1 differentiation. Collectively, these results suggest that cytokines may influence both expression and function of P-gp in TF-1 cells without interfering with their differentiation status. In contrast to cytokines, phorbol esters enhanced expression and efflux capacity of P-gp in parallel with TF-1 cell monocytic differentiation. Finally, our study suggests that paracrine and/or autocrine secretion of cytokines may interfere with P-gp activity in some acute myeloid leukemia cells.

Sex chromosome complement and developmental diversity in pre-and post-hatching porcine embryos.

To examine sex and development relationships in porcine embryos in early gestation, 10 gilts were killed on Day 4, 5, or 6 post mating (first day of standing estrus = Day 0). Embryos recovered immediately after slaughter were cultured in Medium 199 with colcemid (0.05mug/ml), fixed on slides, and stained with 4% Giemsa. The number of cells in each specimen was counted from the slides, and, whenever cell dispersion allowed, sex was determined by presence or absence of the Y-chromosome in at least 2 spreads from each embryo. Three gilts slaughtered on Day 4 yielded 2- and 4-cell stage embryos (n = 38), but no data on sex could be obtained due to lack of mitosis or readable metaphase spreads. Three Day 5 litters had individual specimens ranging from 8 to 14 cells (n = 8), 32 to 64 cells (n = 10), and 13 to 31 cells (n = 11), with the sex determined in 15 of these. Cell numbers ranged from 18 to 165 (n = 14), 16 to 32 (n = 9), 36 to 82 (n = 12), and 16 to 30 (n = 9) in the 4 gilts slaughtered on Day 6, with the sex determined in 26 of these. Embryos within each litter were divided into low, medium and high cell numbers by 3 equal divisions of the range of cell numbers. Three Day-5 embryos and 1 Day-6 embryo were lost during preparation; neither the cell numbers nor the sex could be determined in 4 Day-5 and in 3 Day-6 embryos. The overall sex ratio approximated 1:1, but on Day 5, the ratios for males to females were 0:5, 1:3 and 6:0 for the low, medium and high cell number groups, respectively. Embryos of undetermined sex in these same groups numbered 3, 1 and 3, respectively. On Day 6 the distribution was 1:11, 4:2 and 8:0 in favor of the males, while embryos of undetermined sex in the low, medium and high cell number groups numbered 5, 7 and 2, respectively. Chi-square analysis of the combined Day-5 and Day-6 results indicated the presence of significantly more females among embryos with low cell numbers and more males in the high cell number group (P < 0.01).

Optimization of immunohistochemical detection of P-glycoprotein in chronic lymphoid disorders.

To optimize the immunohistochemical detection of the multidrug resistance (MDR)-associated P-glycoprotein (P-gp) in chronic lymphoid disorders, the authors compared the sensitivity of three different monoclonal antibodies (MoAb) directed against P-gp (C219, JSB-1, and MRK 16) by using the APAAP technique on four tissue preparations obtained from lymphoid tumors: Cryostat sections, ModAMEX processed sections, frozen cytospin preparations, and fresh cytospin preparations. Tumor samples were obtained from patients with previously treated chronic lymphocytic leukemia (6 cases) or non-Hodgkin's malignant lymphoma (4 cases). Lymph nodes (n = 9), spleen (n = 3), and blood (n = 5) were analyzed. JSB-1 MoAb detected P-gp in 4 of 12 cases (33.3%) on either frozen sections or ModAMEX processed sections, and in 6 of 17 cases (35.3%) on frozen cytospin preparations. The sensitivity of JSB-1 was significantly improved when fresh cytospin preparations were used with an incidence of P-gp positive samples as high as 70.6% (P < .05). C219 MoAb was unreactive with lymphoid cells whatever the technique used, whereas this antibody stained stromal cells. MRK 16 MoAb was equally reactive to JSB-1 on fresh cytospin preparations, but unreactive when the other preparations were used. The specificity of JSB1 MoAb was confirmed by both Western blot analysis and Rhodamine 123 efflux assay. The authors used JSB-1 MoAb on fresh cytospin smears prepared from 28 CLL patients. Overall incidence of P-gp positive cases was 39.2%. Univariate analysis showed that P-gp expression was correlated with prior therapy, refractoriness to treatment, Rai stratification, and time of tissue storage after diagnosis. The authors recommend the use of JSB-1 on fresh cytospin preparations for the immunocytochemical detection of P-gp in chronic lymphoid disorders.

Effect of the protein kinase C inhibitor staurosporine on chemosensitivity to daunorubicin of normal and leukemic fresh myeloid cells.

The effect of the protein kinase C (PKC) inhibitor staurosporine (ST) on the chemosensitivity of normal (colony-forming unit granulocyte-macrophage [CFU-GM]) and leukemic (acute myeloid leukemia-CFU [AML-CFU]) myeloid progenitors to daunorubicin (DNR) was evaluated. Primary colony inhibition assays allowed us to characterize two distinct groups of AML, a DNR-resistant group (patients no. 1 through 6), which displayed significantly lower DNR sensitivity than normal CFU-GM (D50 = 11.3 +/- 1.4 ng/mL v 1.8 +/- 0.5 ng/mL, after 7 days of exposure, respectively; P < 0.01) and a DNR-sensitive group (patients no. 7 through 12) with D50 = 2.7 +/- 0.4 ng/mL. This classification remained unaltered when assessed by secondary colony inhibition assay (evaluating the self-renewal fraction of AML-CFU) or by viability assay (evaluating the ultimately differentiated blast cell population), suggesting that the DNR sensitivity profile in maintained throughout AML-CFU differentiation. DNR resistance of the differentiated blast cell population was not correlated with the level of P-glycoprotein (P-gp) expression but rather with the ability to extrude rhodamine 123 (Rh123). ST used at subtoxic concentrations induced a twofold to threefold enhancement of DNR cytotoxicity, increased Rh123 accumulation, and decreased Rh123 efflux kinetics in resistant AML cells. These effects were observed for ST concentrations much lower than those required to displace the P-gp-binding probe azidoprazosin, suggesting that ST might act through its PKC inhibitory effect and not through P-gp binding. Finally, this study provides evidence that DNR resistance in AML cells is, at least in part, related to the multidrug-resistance (MDR) phenotype. Because P-gp function can be downregulated by ST, it seems likely that the MDR pheno-type can be functionally regulated by cellular signalization in AML cells.

Influence of sex on early growth of pig conceptuses.

Nineteen gilts were used in an experiment to examine the relationship between rate of development and embryonic sex on day 10 of pregnancy. All gilts were mated to the same boar approximately 24 h after detection of second oestrus. They were individually housed and fed similar diets until slaughter on day 10 of gestation (day 0 = day of insemination) for subsequent recovery of the conceptus. All conceptuses were photographed and their surface areas (mm2) measured by tracing outlines on a digitized tablet interfaced with a computer program. Within each litter, individuals were categorized as small, medium or large by three equal divisions of the size range between the smallest and largest member. Conceptuses were individually cultured in Medium 199 with 1% colcemid and stained with 4% Giemsa. Metaphase spreads were located and sex was determined by presence or absence of the Y chromosome in at least two spreads from each specimen. A total of 214 conceptuses were recovered but only 125 (58%) were successfully karyotyped. The overall sex ratio was not significantly different from 1:1 (57 males and 68 females; P > 0.25). Sex was determined in 51 of 88 small embryos, 22 of 44 medium embryos and 52 of 82 large embryos and males represented 9 (17.6%), 10 (45.5%) and 38 (73%), respectively. Logistic analysis indicated significantly more females in the small and significantly more males in the large groups (P < 0.001). Results demonstrate that most male conceptuses grow faster than females before commencement of attachment to the uterine lining.