Leigh M. Sweet

Modulation of monocyte chemotactic protein-1 expression during lipopolysaccharide-induced preterm delivery in the pregnant mouse.

Preterm delivery is often associated with increased cytokine and chemokine production. These studies characterize the expression of the chemokine monocyte chemotactic protein-1 (MCP-1) in mice during lipopolysaccharide (LPS)—induced preterm delivery. Uterine and other tissues were harvested from CD-1 mice on gestational day 15 after intrauterine LPS injection. Quantitative real-time reverse-transcriptase polymerase chain reactions determined MCP-1 and toll-like receptor 4 (TLR4) mRNA expression during the 24 hours after LPS. MCP-1 protein expression was determined using a cytokine/chemokine protein array, enzyme-linked immunosorbant assay, and immunohistochemistry. Intrauterine LPS injection caused preterm delivery in CD-1 mice between 12 and 24 hours. Expression of MCP-1 mRNA significantly increased at 2 and 6 hours, while TLR4 expression did not significantly change over 24 hours. The MCP-1 protein levels peaked by 2 to 6 hours in maternal serum, liver, lung, kidney, and uterus. Immunohistochemistry confirmed MCP-1 in the myometrium and endometrium. These studies provide evidence suggesting that MCP-1 potentially plays an important role during the proinflammatory immune response, leading to preterm labor in the mouse.

The membrane-associated 40 KD fatty acid binding protein (Berk's protein), a putative fatty acid transporter is present in human skeletal muscle

Muscle tissue (1.1 +/- 0.1 grams) was obtained from seven healthy individuals (3 males, 4 females) using an open incision approach before and after ingestion of either 75 grams of dextrose (N=5) or water (N=2). Purified sarcolemmal membranes from the muscle were prepared using a sucrose step gradient. A polyclonal antibody raised against the purified (99%) rat hepatocyte 40 KD membrane fatty acid binding protein (mFABP-L) was used to probe for this putative transporter in the muscle membranes using Western blot. A single band at the 40 KD MW band was identified which reacted antigenically with the protein purified from rat livers. These response of Berks protein 60-75 minutes after dextrose ingestion (or water) was erratic and no specific trend could be identified. Our data demonstrate that the 40 KD mFABP-L originally isolated from rat liver is also present in human skeletal muscle membrane. This protein may be involved in transport of fatty acids across the membrane of skeletal muscle, however its physiological role in human fatty acid metabolism remains to be established.

Protease-activated receptor isoform expression in pregnant and nonpregnant rat myometrial tissue.

Objective: Three protease-activated receptor (PAR1, 3, and 4) isoforms have been shown to be responsible for the cellular effects of thrombin; another PAR isoform (PAR2) is responsible for the cellular effects of trypsin. The present studies sought to test the hypothesis that one (or more) of these PAR isoforms is expressed in myometrial tissue, thereby accounting for the uterotonic effects of these novel agonists. Methods: The rat PAR3 and 4 isoforms were cloned from a rat spleen cDNA library. PAR isoform mRNA expression was determined by using reverse-transcriptase polymerase chain reactions (PCR) in Sprague-Dawley rats. Confirmation of the identity of the amplified mRNA was done by sequence analysis. Relative quantification of the PAR1 and PAR2 isoforms was performed using a real-time quantitative reverse transcriptase PCR (RT-PCR) technique. PAR protein expression was confirmed by Western blots using polyclonal antibodies. Results: The rat PAR3 and 4 homologues showed significant sequence homology to the mouse and human amino acid and nucleotide sequences. The RT-PCR studies confirmed PAR1-4 expression in myometrium from rats in estrus. PAR3 was expressed in uterus, spleen, kidney, liver, lung, brain, and heart. PAR4 was expressed in uterus, spleen, and lung. Messenger RNA for the PAR1 and 2 isoforms was expressed during the second half of gestation in myometrium from timed-pregnant rats. In contrast, mRNA for the PAR3 and 4 isoforms was not detected in gestational myometrium. PAR protein expression appeared to match tissue mRNA expression patterns. Conclusion: These RT-PCR studies confirmed ubiquitous expression of the PAR1 and PAR2 isoforms in myometrium and other rat tissues; in contrast, the PAR3 and PAR4 isoforms are expressed in a tissue-specific and gestationally related pattern.

Expression of coagulation-related protein genes during LPS-induced preterm delivery in the pregnant mouse.

Preterm delivery (PTD) has been associated with inflammation along with activation of the coagulation pathway. These studies sought to characterize the expression of several coagulation pathway genes including plasminogen activator inhibitor 1 (PAI-1), tissue factor (TF), protease-activated receptor 1 (Par1), protease-activated receptor 2 (Par2), fibrinogen-like protein 2 (Fgl2), and thrombomodulin (TM) during lipopolysaccharide (LPS)-induced PTD in day 15 pregnant CD-1 mice. Western blot studies confirmed protein expression for PAI-1, Par1, Par2, Fgl2, and TM in the mouse uterus. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) confirmed increased PAI-1 messenger RNA (mRNA) in the uteri, lung, kidney, and liver tissues at 2 to 6 hours after LPS injection. In contrast, TF expression significantly decreased by 12 hours in uterine tissue; whereas, its expression was unchanged in the other maternal tissues. The uterine mRNA for Par1, Par2, Fgl2, and TM remained stable. In summary, these studies have confirmed expression of coagulation pathway genes within the pregnant uterus; some of which are modulated during LPS-induced PTD.

Estradiol and progesterone influence on influenza infection and immune response in a mouse model

Influenza infection severity may be mediated by estradiol and/or progesterone.

Role of Nonreceptor Protein Tyrosine Kinases During Phospholipase C-γ1-related Uterine Contractions in the Rat

Activated phospholipase C1, produced in response to tyrosine phosphorylation, appears to play an important role during uterine contractions. These studies sought to determine which non-receptor protein tyrosine kinases are involved in the activation of phospholipase C1 in rat uterine tissue. In vitro contraction studies were performed utilizing isoform specific protein tyrosine kinase inhibitors. Western blots were performed utilizing antibodies to phosphotyrosine-phospholipase C1, total phospholipase C1, c-Src kinase and Lck kinase. Spontaneous, stretch-stimulated, and bpV(phen) (tyrosine phosphatase inhibitor) enhanced uterine contractions were significantly suppressed in response to Damnacanthal (Lck kinase inhibitor) and PP1 (c-Src kinase inhibitor). Damnacanthal and PP1 also significantly suppressed bpV(phen)-enhanced tyrosine phosphorylation of phospholipase C1. Western blots confirmed expression of Lck kinase and c-Src kinase in uterine tissue. In conclusion, the Lck and c-Src kinases appear to play an important role in regulating tyrosine phosphorylation of phospholipase C1 and contractile activity in the rat uterus.

SHP Protein Tyrosine Phosphatase Expression in Rat Uterine Tissue

Objective: Enhanced tyrosine phosphorylation of phosopholipase C-γ1 (PLCγ1) is associated with increased spontaneous contractile activity. PLCγ1 phosphorylation is regulated by cellular protein tyrosine kinases and tyrosine phosphatases (PTPs). The studies in this report were undertaken to characterize the expression of two PTPs known to bind to PLCγ1: Src-homology phosphatase type-1 (SHP-1) and type-2 (SHP-2). Methods: Uterine and other tissues were obtained from non-pregnant (estrus) and pregnant (gestational day 12 through day 1 postpartum) Sprague-Dawley rats. PTP activity in myometrial homogenates was determined using an in vitro fluorometric PTP assay with and without bpV(phen) (a nonselective PTP inhibitor), or PTP-Inhibitor 1 (PTP-I1, a SHP selective inhibitor). Western blots were performed using polyclonal antibodies to SHP-1 and SHP-2. Immunoprecipitation studies were performed to demonstrate an association between PLCγ1 and the SHP proteins. Results: The in vitro PTP assays demonstrated comparable enzyme activity in myometrium from estrus and pregnant animals. BpV(phen) produced a 93% reduction in PTP activity (P <.05); similarly, PTP-11 produced an 86% reduction in enzyme activity (P <.05). Western blots confirmed robust expression of both SHP-1 and SHP-2 protein in rat uterus. SHP-1 expression decreased significantly at the end of gestation; in contrast, SHP-2 levels remained stable. Immunoprecipitation studies confirmed an association between the SHP proteins and PLCγ1. Conclusion: These studies have demonstrated that SHP-1 and SHP-2 are expressed in rat myometrium and appear to be responsible for the PTP activity in this tissue, thereby providing a molecular mechanism for the modulation of PLCγ1 phosphotyrosine levels in the rat uterus.