Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 x 10(-4) M) had effects similar to those of oxytocin pretreatment (1 x 10(-5) M). PKC depletion with phorbol 12-myristate 13-acetate (1 x 10(-6) M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 x 10(-5) M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 microg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.     

Effects of the ovarian cycle on sympathetic neural outflow during static exercise

We compared reflex responses to static handgrip at 30% maximal voluntary contraction (MVC) in 10 women (mean age 24.1 +/- 1.7 yr) during two phases of their ovarian cycle: the menstrual phase (days 1-4) and the follicular phase (days 10-12). Changes in muscle sympathetic nerve activity (MSNA; microneurography) in response to static exercise were greater during the menstrual compared with follicular phase (phase effect P = 0.01). Levels of estrogen were less during the menstrual phase (75 +/- 5.5 vs. 116 +/- 9.6 pg/ml, days 1-4 vs. days 10-12; P = 0.002). Generated tension did not explain differences in MSNA responses (MVC: 29.3 +/- 1.3 vs. 28.2 +/- 1.5 kg, days 1-4 vs. days 10-12; P = 0.13). In a group of experiments with the use of 31P-NMR spectroscopy, no phase effect was observed for H+ and H2PO-4 concentrations (n = 5). During an ischemic rhythmic handgrip paradigm (20% MVC), a phase effect was not observed for MSNA or H+ or H2PO-4 concentrations, suggesting that blood flow was necessary for the expression of the cycle-related effect. The present studies suggest that, during static handgrip exercise, MSNA is increased during the menstrual compared with the follicular phase of the ovarian cycle.     

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.     

The p11 subunit of the annexin II tetramer plays a key role in the stimulation of t-PA-dependent plasminogen activation

Annexin II tetramer (AIIt) is an important endothelial cell surface protein receptor for plasminogen and t-PA. AIIt, a heterotetramer, is composed of two p36 subunits (called annexin II) and two p11 subunits. In this report, we have compared the ability of the isolated p36 and p11 subunits to stimulate t-PA-dependent [Glu]plasminogen activation. The fluid-phase recombinant p11 subunit stimulated the rate of t-PA-dependent activation of [Glu]plasminogen about 46-fold compared to an approximate stimulation of 2-fold by the recombinant p36 subunit and 77-fold by recombinant AIIt. The stimulation of t-PA-dependent activation of [Glu]plasminogen by the p11 subunit was Ca2+-independent and inhibited by epsilon-aminocaproic acid. [Glu]Plasminogen bound to a p11 subunit affinity column and could be eluted with epsilon-aminocaproic acid. Both AIIt and the p11 subunit protected t-PA and plasmin from inactivation by PAI-1 and alpha2-antiplasmin, respectively. A peptide to the C terminus of the p11 subunit (85-Y-F-V-V-H-M-K-Q-K-G-K-K-96) inhibited the p11-dependent stimulation of t-PA-dependent plasminogen activation. In addition, a deletion mutant of the p11 subunit, missing the last two C-terminal lysine residues, retained only about 15% of the activity of the wild-type p11 subunit. Similarly, a mutant AIIt composed of the wild-type p36 subunit and the p11 subunit deletion mutant possessed about 12% of the wild-type activity. These results, therefore, suggest that the C-terminal lysine residues of the p11 subunit bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen by AIIt.     

Sympathetic neuron survival and proliferation are prolonged by loss of p53 and neurofibromin

This review will discuss studies demonstrating that activation of opioid receptors within the central nervous system alters various immune system parameters. Specifically, natural killer cell cytolytic activity and lymphocyte proliferative responses to mitogen appear to be modulated predominantly, if not exclusively, through central opioid receptors. The potential mechanisms by which central opioid receptors appear to modulate these peripheral immune functions will be examined by evaluating the role of both the hypothalamic-pituitary-adrenal (HPA) axis and the autonomic nervous system. The studies discussed below indicate that acute administration of morphine or related compounds appears to primarily alter peripheral immune function through the sympathetic nervous system, while more prolonged exposure to opioids alter the immune system predominantly by activation of the HPA axis. Finally, the potential clinical relevance of these observations are discussed in relationship to both the therapeutic use, as well as the abuse of opioid compounds.     

A population-based twin study of self-esteem and gender

BACKGROUND: Self-esteem (SE), a widely used construct in the social sciences, is usually conceptualized as a reflection of socialization and interpersonal experiences that may differ considerably between the genders. METHODS: The Rosenberg self-esteem scale was assessed at personal interview in both members of 3793 unselected twin pairs (1517 male-male, 856 female-female and 1420 male-female) from the population-based Virginia Twin Registry. Gender effects on SE were assessed by both analysis of variance and biometrical twin modelling. RESULTS: The mean SE score was slightly but significantly lower in women v. men, and in women who grew up with a male v. a female co-twin. Twin modelling suggested that: (i) individual differences in self-esteem in both men and women were best explained by genetic and individual-specific environment factors; (ii) heritability estimates were similar in women (32%) and in men (29%); and (iii) the same genetic factors that influenced SE in women also influenced SE in men. Analyses supported the validity of the equal environment assumption for SE. The heritability of SE cannot be explained by the moderate correlation between SE and symptoms of depression. CONCLUSIONS: These results are inconsistent with prominent gender-related aetiological models for SE, which postulate that individual differences arise from socialization experiences both within and outside the home of origin which differ widely for the two genders. Instead, a significant proportion of the population variance in SE is due to genetically-influenced temperamental variables that are the same in men and women.     

Magnetic resonance imaging in breast cancer staging

For many solid carcinomas, high-resolution cross-sectional imaging has changed cancer staging, the evaluation of therapeutic response, the detection of recurrence, and even how therapy is selected and performed. Such imaging has not yet had similar effects on breast cancer. Evaluations of therapeutic response in breast carcinomas have been impeded by the current limited methods of evaluating breast tumor size and extent: clinical palpation, ultrasonography, and mammography. The use of magnetic resonance imaging (MRI) of the breast in the evaluation of breast tumors brings the advantages of high-resolution cross-sectional imaging to breast cancer staging and treatment evaluation and is likely to greatly enhance research efforts in this complex disease. MRI of the breast has evolved to be the most accurate noninvasive technique for local staging of breast cancer. MRI is most accurate in measuring tumor size and detecting multicentric disease. These staging characteristics affect the selection of therapy and initial determination of prognosis; therefore, MRI of the breast can change the assessment of fundamental parameters on which treatment is selected. Because clinical trials of new cancer treatments are predicated on proper and accurate characterization of the tumor, MRI also should affect how clinical trials are performed and evaluated.     

Translocation of inserted foreign epitopes by a channel-forming protein

Certain bacterial protein toxins are able to insert themselves into, and at least partially across, lipid bilayer membranes in the absence of any auxiliary proteins, by using unknown mechanisms to overcome the high energy barrier presented by the hydrophobic bilayer core. We have previously shown that one such toxin, colicin Ia, translocates a large, hydrophilic part of itself completely across a lipid bilayer in conjunction with the formation of an ion-conducting channel. To address the question of whether the colicin can translocate any arbitrary amino acid sequence, we have altered the translocated segment by inserting, singly, two different foreign epitopes. Colicins containing either epitope retain significant bactericidal activity and form channels of normal conductance in planar bilayers. Furthermore, antibodies added on the side of the bilayer opposite that to which the colicin was added interact specifically with the corresponding epitopes, producing an inhibition of channel closing. Thus, the inserted epitopes are translocated along with the rest of the segment, suggesting that a surprisingly small part of colicin Ia, located elsewhere in the molecule, acts as a nonspecific protein translocator.     

Effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver

We have previously shown that intravenous administration of keratinocyte growth factor (KGF) induces hepatocyte proliferation, allowing for efficient and noninvasive in vivo gene transfer with high-titer retroviral vectors in mice. The distinctive periportal distribution of transduced cells led us to investigate the ability of virus-sized particles to perfuse the liver adequately after growth factor treatment. We found that perfusion was adequate, and that transduction was limited to the periportal region because only those cells were stimulated to divide. Cells in this region also showed increased expression of Ram-1, the receptor for the murine Moloney leukemia virus (MoMLV) amphotropic envelope, after KGF treatment. In further studies we found that recombinant hepatocyte growth factor (HGF) induces a different population of hepatocytes to divide and upregulate Ram-1. The differential pattern of induction suggested that combining KGF and HGF would improve gene transfer efficiency further. Indeed, simultaneous delivery of both growth factors leads to an overall increase in the number of proliferating cells. Importantly, when coupled with MoMLV delivery, efficiency of gene transfer increased. These results confirm the utility of growth factors for noninvasive hepatic gene transfer in mice, and demonstrate how experiments to define the mechanism of transduction can be taken advantage of to develop improved gene transfer protocols.