Donald A. Novak

Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta

Concentrative absorption of glutamate by the developing placenta is critical for proper fetal development. The expression of GLAST1, GLT1, EAAC1, and EAAT4, known to be capable ofd-aspartate-inhibitable and Na+-coupled glutamate transport (system [Formula: see text]), was evaluated in day 14 vs. day 20 rat chorioallantoic placenta. Steady-state mRNA levels were greater at day 20 for all transporters. Immunohistochemistry determined that the expression of GLAST1, GLT1, and EAAC1 was greater throughout the day 20 placenta and was asymmetric with respect to cellular localization. EAAT4 protein was not detected. System[Formula: see text] activity was responsible for most of the Na+-dependent glutamate uptake and was greater in day 20 than in day 14apical and basal membrane subdomains of the labyrinth syncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 protein were identified on day 20, and quantities were greater in basal than in apical membranes. GLT1 expression, unchanged in apical membranes, was decreased in basal membranes. These data correlate transporter mRNA and protein content with transport activity and demonstrate an increasing capacity for glutamate absorption by the developing placenta.

Ontogeny of amino acid transport system A in rat placenta

Amino acid transport System A has previously been demonstrated in apical membranes derived from rat placenta, as well as in apical and basal membranes derived from human placenta. We have studied Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) transport in apical and basal predominant membrane fractions prepared from 14 and 20 day gestation rat placenta. Marker enzyme recoveries did not differ significantly between age groups. Markers for intracellular organelles were also found to be comparable. Na(+)-dependent MeAIB transport was not sensitive to freezing and could be found in all membrane components tested. Kinetic parameters were studied--Km = 852 +/- 215 microM, Vmax = 718 +/- 126 pmol/5 sec/mg protein--20 day apical; Km = 748 +/- 269 microM, Vmax = 610 +/- 176 pmol/5 sec/mg protein--20 day basal-predominant; Km 614 +/- 261 microM, Vmax = 123 +/- 45 pmol/5 sec/mg protein-14 day apical. Kinetic parameters could not be determined in the 14 day gestation basal-predominant fraction because of the small amount of uptake present. We conclude that System A like activity is found in both apical and basal predominant membrane fractions derived from rat placenta, and that this activity increases over the last one third of gestation.

Demonstration of system y+L activity on the basal plasma membrane surface of rat placenta and developmentally regulated expression of 4F2HC mRNA.

Na(+)-independent cationic amino acid transport in the rat placenta occurs by leucine-sensitive and leucine-insensitive pathways. The ontogeny of these transport mechanisms within the rat placenta has been described recently. To assign the leucine-inhibitable portion of uptake definitively the uptake of [3H]arginine was studied in the presence of both BCH (to inhibit system Bo,+) and varied concentrations of leucine. Uptake of arginine into basal-enriched membrane vesicles derived from rat placenta was, in the presence of sodium, inhibited by micromolar concentrations of leucine, consistent with assignment of this activity to system y+L. In contrast, the majority of arginine uptake into apical-enriched membrane vesicles was leucine insensitive. Messenger RNA derived from rat placenta at days 14, 16, 18 and 20 of gestation was hybridized with full-length rat cDNA probes against NBAT and 4F2HC (thought to encode proteins associated with system bo,+ and y+L activities, respectively). No NBAT mRNA was detected, whereas 4F2HC mRNA was present at all gestational stages, increasing 12-fold over the last third of gestation. It is concluded that system y+L is present in the basal plasma membrane of the rat placenta syncytium and is subject to developmental regulation by a mechanism that alters the steady content of 4F2HC mRNA.

Glutamine transport in human and rat placenta

Glutamine plays an important role in fetal nutrition. This study explored the transport of [3H]glutamine into apical and basal predominant membrane vesicles derived from rat and human placenta. Na+-dependent glutamine transport was present in both apical and basal predominant vesicles derived from 20- and, to a lesser degree, 14-day gestation rat placenta. Amino-acid transport systems A, ASC-like, B(o,+) (in apical membrane vesicles) and, perhaps, y+L were involved in Na+-dependent glutamine transport. Na+-dependent glutamine uptake into human placental microvillus and basolateral membrane vesicles also occurred via several distinct transport activities. Glutamine transport via system N was not detected in either rat or human placental preparations. Na+-dependent glutamine transport in the rat was more pronounced in basal as compared to apical membrane vesicles. Conversely, in the human preparations, activity was significantly higher in microvillus as compared to basolateral membrane vesicles. It is concluded that Na+-dependent glutamine transport occurs through a variety of transport agencies in both the rat and human placenta. Transport varies with ontogeny and between species.

Antibodies to Escherichia coli outer membrane porin C in the absence of anti-Saccharomyces cerevisiae antibodies and anti-neutrophil cytoplasmic antibodies are an unreliable marker of Crohn disease and ulcerative colitis.

Objectives: Antibodies to Escherichia coli outer membrane porin C (anti-OmpC), Saccharomyces cerevisiae, and neutrophil-specific nuclear antigens are associated with inflammatory bowel disease (IBD) in children and young adults. We hypothesized that anti-OmpC, in the absence of anti–S cerevisiae antibodies (ASCA) and antineutrophil cytoplasmic antibodies (ANCA), is an assay that overestimates the presence of Crohn disease (CD) and ulcerative colitis (UC). Patients and Methods: A retrospective review of patients evaluated at our institution between January 2002 and June 2006 revealed that 170 had serodiagnostic immunological assays performed as part of an evaluation for possible IBD. The assays were screened for a pattern in which anti-OmpC was present in the absence of ASCA and ANCA. Results: Seven patients between 3 and 20 years of age were discovered to be positive for anti-OmpC but negative for ASCA and ANCA. These patients were determined to have significant medical conditions without combined radiographic, endoscopic, or histological evidence of IBD. Despite the reported 85% positive predictive value of anti-OmpC for IBD, none of the 7 patients with isolated anti-OmpC had a diagnosis of CD or UC. Conclusions: Anti-OmpC, in the absence of ASCA and ANCA, is a serological pattern noted in a subset of medically complex cases in children and young adults without CD or UC.

Gestational programming of offspring obesity/hypertension

The intrauterine milieu impacts fetal growth directly during gestation. It is now clear, however, that postnatal phenotype is also influenced by prenatal conditions. A variety of disorders in the adult have been linked to fetal size at birth; these include glucose intolerance, cardiovascular disease, and the subjects of this review, obesity and hypertension. We will review recent data regarding these associations and the pathophysiologic mechanisms underlying them in humans as well as in animal models.

PERCEPTIONS OF PARENTING STRESS AND FAMILY RELATIONS BY FATHERS OF CHILDREN EVALUATED FOR ORGAN TRANSPLANTATION

18 fathers of children evaluated for solid organ or bone marrow transplantation completed measures of parenting stress and family functioning. Comparisons with normative data indicated that these fathers reported less parenting stress, less family conflict, more concern about family finances, and more limitations in family activities. These data highlight the need for family-based assessments in pediatric transplantation.

Effect of chronic cocaine administration on amino acid uptake in rat placental membrane vesicles

This study evaluated the effects of chronic exposure to cocaine during pregnancy on amino acid uptake in placental membrane vesicles. Pregnant rats received 62 mg/kg of cocaine hydrochloride by intraperitoneal (IP) injection as a divided daily dose on gestation days 8-19 inclusive. Fetal body weights were significantly decreased by 19% in the cocaine group, while placental weights were unchanged. Placental apical membrane vesicles were prepared from control and cocaine-treated animals, and marker enzyme enrichments for alkaline phosphatase and [3H]-dihydroalprenolol binding did not differ between cocaine and control groups. Rates of uptake (10 sec) of selected radiolabeled amino acids were measured utilizing a rapid filtration technique. Na(+)-dependent apical membrane [3H]-glutamine transport (50 microM) was reduced by 95% (p < 0.05) in cocaine-treated compared to control placentas. Uptake of 50 microM [3H]-methyl aminoisobutyric acid (MeAIB) into apical membranes was also decreased by 43% (p < 0.05) in cocaine membranes. Na(+)-independent [3H]-arginine transport (10 microM), however, did not differ between control or cocaine-treated groups. In summary, chronic cocaine administration selectively inhibited the transport of glutamine and MeAIB into apical membrane vesicles, but had minimal effect on arginine transport. We postulate that this diminution in uptake may contribute to the fetal growth retardation noted in our model.

Nutrition in early life: How important is it?

While few would argue the importance of nutrition during adult life, temporary excess or deficiency has typically been thought to be of little long-term consequence. Recent data, summarized above, suggests that this may not be the case during in utero life, when alterations in the quantity or quality of nutrients provided may have life-long consequences. Perhaps even more surprisingly, decisions made in the neonatal period, such as whether to breastfeed or bottle feed, may have impacts on later health that, while small individually, have huge public health implications. Clarification of the links between adult health and fetal/neonatal nutrition are clearly required. Prospective studies, though difficult because of the time involved, will play a key role in this process, as will more basic research on the mechanisms underlying both normal and pathologic fetal development.

Relationship of the polymerase chain reaction for cytomegalovirus to the development of hepatitis in liver transplant recipients

In a pilot study, the polymerase chain reaction was found to be more sensitive than standard viral culture methods for the detection of cytomegalovirus, particularly from blood and tissues. We therefore applied this technique to 71 serially collected liver biopsies from 16 orthotopic liver transplant patients. All patients were CMV-seropositive (n=15) or seroconverted (n=1). Seven patients (9 biopsies) had histologically proved CMV hepatitis, and all these biopsies were CMV PCR—positive. Six of these 7 patients had a prior liver biopsy that was CMV PCR-positive, but culture and histology-negative, an average of 13.2±6.9 days before the histologically positive biopsy. The 7th patient was not biopsied prior to the diagnostic biopsy. Three patients had 7 liver biopsies that were CMV PCR-positive, but histologically negative for CMV hepatitis. Two of these three had CMV infection confirmed by viral culture of blood or liver biopsy. The remaining 6 patients had a total of 26 liver biopsies that were negative for CMV by PCR, culture, and histology. Among liver transplant patients, CMV PCR performed on liver biopsy specimens correctly identified all histologically proven cases of CMV hepatitis. CMV PCR positivity in liver tissue did not correlate with latent infection and preceded the development of CMV hepatitis or other meaningful CMV infection in 8 of 10 patients.