Developmental changes in polyamines and autophagic marker levels in normal and growth-restricted fetal pigs.

Polyamines are essential for embryonic and fetal survival, growth, and development. Additionally, polyamines may induce autophagy in mammalian cells. However, little is known about the availability of polyamines or autophagy in the porcine conceptus with intrauterine growth restriction (IUGR). The present study was performed to evaluate the developmental changes of polyamine concentrations in IUGR and normal porcine fetuses as well as autophagic marker levels in the fetal intestinal mucosa during the second half of gestation when most fetal growth occurs. Allantoic fluid (ALF), amniotic fluid (AMF), umbilical vein, and the small-intestinal mucosa were obtained from both IUGR and normal fetal pigs at d 60, 90, and 110 of gestation. Concentrations of polyamines in fetal fluids as well as protein abundances of microtubule-associated protein light chain 3B (LC3B), an autophagic marker, in the fetal small-intestinal mucosa were determined. Concentrations of polyamines varied greatly in different fetal compartments and changed substantially with advancing gestation. Concentrations of polyamines in IUGR fetal fluids and the small-intestinal mucosa were markedly different from those in their normal counterparts at d 60 and 90 of gestation, whereas most of the differences were not detected by late (d 110) gestation. Specifically, polyamine levels were lower in the umbilical vein plasma but higher in ALF and AMF from IUGR fetuses. Furthermore, enhanced levels of an autophagic marker were observed in the small-intestinal mucosa of IUGR fetuses throughout mid and late gestation in association with abnormal spermidine levels in fetal plasma. These findings support the notion that enhanced autophagy may be an important survival mechanism in IUGR fetuses. Collectively, our findings provide a new framework for future studies to define the roles for polyamines in the prevention and treatment of IUGR in both human medicine and animal production.

Baggs ewes adapt to maternal undernutrition and maintain conceptus growth by maintaining fetal plasma concentrations of amino acids.

Adequate delivery of AA is essential for normal fetal growth and development. Recently, we reported that when ewes from the University of Wyoming flock (farm flock with adequate nutrition) were fed 50% (nutrient-restricted) or 100% (control-fed) of the NRC-recommended nutrient requirements between d 28 and 78 of gestation, fetal weights as well as concentrations of most AA in maternal and fetal blood were substantially reduced in nutrient-restricted vs. control-fed pregnancies. The current study utilized Baggs ewes, which were selected under a markedly different production system (range flock with limited nutrition), to test the hypothesis that adaptation of ewes to nutritional and environmental changes may alter placental efficiency and conceptus nutrient availability in the face of maternal nutrient restriction. Baggs ewes received 50 or 100% of the NRC nutrient requirements between d 28 and 78 of pregnancy. On d 78, maternal uterine arterial and fetal umbilical venous blood samples were obtained, and the ewes were euthanized. Amino acids and their metabolites (ammonia, urea, and polyamines) in plasma were analyzed using enzymatic and HPLC methods. The results showed that maternal plasma concentrations of 9 AA (Asp, Ile, Leu, Lys, Orn, Phe, Thr, Trp, and Val) as well as maternal and fetal plasma concentrations of ammonia and urea were reduced (P < 0.05) in nutrient-restricted compared with control-fed Baggs ewes. However, fetal plasma concentrations of all AA and polyamines did not differ (P = 0.842) between the 2 groups of ewes. Collectively, these findings suggest that Baggs ewes, by adapting to the harsh conditions and limited nutrition under which they were selected, were able to maintain fetal concentrations of AA in the face of a maternal nutrient restriction through augmenting placental efficiency.

Herpes simplex virus-mediated activation of human immunodeficiency virus is inhibited by oligonucleoside methylphosphonates that target immediate-early mRNAs 1 and 3.

IE1 and IE3 mRNAs and their protein products (IE110 and IE175, respectively) were detected in HSV-1-infected U937 cells at 4-15 hours postinfection. In transient expression assays with infectious HIV or an HIV-LTR-directed chloramphenicol acetyltransferase construction (HIV-LTRcat), HSV-1 caused HIV activation (86.7% +/- 6.4% conversion). Electrophoretic mobility shift assays with DNA sequences that encompass the LBP-1 binding site revealed increased levels of DNA-protein complex formation with nuclear extracts from HSV-1 infected as compared with uninfected U937 cells. Novel bands were not seen. HSV-1 mutants respectively deleted in IE110 (dl1403) or IE175 (d120) activated HIV as well as wild-type virus. However, HSV-1-mediated activation was inhibited (26% conversion) by simultaneous treatment with oligonucleoside methylphosphonates (ONMP) that specifically inhibit expression of IE110 (IE1TI) or IE175 (IE3TI). ONMP did not inhibit activation when used individually (83.8% and 67.8% conversion with IETI1 and IE3TI, respectively). Combinations of mutant ONMP that do not inhibit IE110 or IE175 expression did not reduce the levels of HSV-1-mediated activation. These findings suggest that HSV genes IE1 and IE3 can independently activate HIV in monocytic cells and ONMP that target HSV IE genes can be used to inhibit HIV activation.

The GGTCA palindrome and cognate cellular factors in trans-regulation of human immunodeficiency virus long terminal repeat by herpes simplex virus.

Molecular interactions between herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV) were investigated in the promonocytic cell line U937. HSV-1-mediated activation was observed in transient expression assays with hybrid constructions containing the HIV long terminal repeat (LTR)-directed chloramphenicol acetyltransferase gene. Comparison of constructions that differ in the GGTCA palindrome located within the negative regulatory region of the LTR revealed four- to eightfold lower activation levels for the wild-type as compared to the mutant sequence. Three protein species, 37K, 59K/64K and 75K, that bind to the wild-type GGTCA palindrome were resolved in nuclear extracts of uninfected U937 cells by gel retardation and u.v.-crosslinking experiments. The 37K protein did not bind to the mutant palindrome sequence. However, a distinct 120K protein was detected. The 37K and 59K/64K binding proteins were not resolved in similar experiments performed with nuclear extracts from HSV-1-infected U937 cells but there was a novel p50 species that binds only to the wild-type palindrome sequence. These findings raise the possibility that interaction of these proteins at the GGTCA palindrome is involved in HSV-1-mediated regulation of the HIV LTR in U937 cells.

NF-kappa B-binding proteins induced by HSV-1 infection of U937 cells are not involved in activation of human immunodeficiency virus.

Molecular interactions between the herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV) were investigated in U937 cells. Nonpermissive HSV-1 infection of U937 cells activated HIV as determined in transient expression assays with hybrid constructions containing the HIV-LTR-directed chloramphenicol acetyltransferase gene. Activation was independent of kappa B-enhancer elements whereas these elements were required for HSV-1-mediated activation in another cell line (C6). kappa B-binding proteins were induced in U937 cells by HSV-1 infection. Four species (45, 55, 75, and 75/80 kDa) were identified by DNA-protein cross-linking. Methylation interference analysis defined close contact only with the third residue of the previously established critical contact triplet GGG. Transient expression assays using mutants in HIV-LTR revealed the presence a cis-response element (GGTCA palindrome) in the negative regulatory region.

Intrinsic immunogenicity of an internal VP1 T-B epitope pair of type 1 poliovirus.

We describe the intrinsic immunogenicity of a poliovirus T-B epitope pair that is located in the N-terminus of the capsid protein VP1. This peptide is unusual in that it is located on the interior of the native virion at the VP1-VP3 interface in a region that becomes exposed after cell binding, proteolysis, or heating of the virus. Immunization of mice with either the virion or free peptide leads to anti-peptide antibody production. Anti-peptide immunity is under genetic control and 1-Ak restricted T cell proliferative responses have been identified. SJL/J (H-2s) mice that are low responders to this T-B epitope pair are also low responders to PSV-1 itself, suggesting that this site may be important in the production of neutralizing anti-PSV-1 antibodies. Interestingly, seropositive humans also have significant anti-peptide titers suggesting that immunization with poliovirus in a species permissive for infection also leads to anti-peptide antibody production. Collectively, these data suggest that a T-B epitope pair located on the interior of a protein or virion can be immunogenic. Several mechanisms whereby internal T-B epitope pairs might become immunogenic are discussed.

Immediate early and functional AP-1 cis-response elements are involved in the transcriptional regulation of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

Expression from the promoter of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (ICP10) is stimulated by co-transfection with DNA that encodes the virion protein Vmw65 previously shown to activate in trans the transcription of all IE genes (Wymer et al., 1989). Specific cis response elements involved in ICP10 transcriptional regulation were studied by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter/CAT structural gene constructions containing wild type or site-directed mutations of the promoter sequences. The data indicate that Vmw65 activation requires an intact TAAT-GARAT motif while complex formation requires an intact Oct-1 element, and the AP-1 consensus elements in the ICP10 promoter are functional in vitro. Thus, expression from the wild type and GA-rich mutant constructions was enhanced 10-20-fold by co-transfection with DNA encoding Vmw65. The GARAT and POU homeobox (PHB) binding motifs were required for Vmw65 mediated activation but the mutant in the POU specific box (PSB) binding motif was activated at higher concentrations of Vmw65 DNA (1.0-3.0 micrograms). The PHB and PSB binding motifs were necessary for complex formation as determined by gel retardation analysis with in vitro synthesized OTF-1 and Vmw65 proteins. The GARAT and GA-rich elements were not required. CAT expression from pICP10-cat was enhanced by co-transfection with jun and fos encoding DNA, and the ICP10 promoter complexed with in vitro synthesized jun protein.