Vincent DeMarco

Glutamine: clinical applications and mechanisms of action

Supplementation of the conditionally essential amino acid glutamine may be beneficial for individuals who are highly stressed and have minimal energy and protein reserves. This includes elderly individuals, postoperative patients, individuals with cancer and very low birthweight infants. Individuals who are undergoing treatment with catabolic glucocorticoids may also benefit. Unfortunately, confusion exists as to situations in which glutamine may be beneficial because a clearly defined glutamine deficiency syndrome has not been described as for some other nutrients. In this review, we will discuss how glutamine affects protein metabolism under certain stressful conditions, how it affects intestinal mucosal integrity and how this might relate to sepsis and systemic inflammation. We will also discuss nutrients that are closely related to glutamine such as glutamate, nucleotides, arginine, glucosamines, and ornithine alpha-ketoglutarate and how and why they might be used as substitutes for glutamine.

Oviductal morphology and eggshell formation in the lizard, Sceloporus woodi

Despite a great deal of work in recent years on the structure of reptilian eggshells, few studies have examined the structure and regulation of the female reproductive tract in the formation of eggshell components, and none have examined the entire process from ovulation to oviposition. In this study, we examined oviductal structure in the oviparous lizard, Sceloporus woodi, followed changes in oviductal structure during gravidity, and determined uterine function in the formation of eggshell components. The endometrial glands of the uterus produce the proteinaceous fibers of the eggshell membrane mainly during the first 24 hours following ovulation, and the fibers are secreted intact and subsequently wrapped around the in utero eggs. Eggshell fibers of different thicknesses are layered around each egg, ranging from an inner layer of thick fibers that gradually become thinner medially and finally forms an outer layer of densely packed particulate matter. These changes in the fibrous layer are reflected by the thickness and length of fibers released from the endometrial glands. Calcium deposition occurs from 3 days following ovulation through day 14 (oviposition) and is accompanied by cellular changes in the luminal epithelium suggestive of secretory activity. Deposition of the eggshell components within the uterus occurs on all eggs simultaneously, rather than sequentially.

Glutamine Synthetase: A Key Enzyme for Intestinal Epithelial Differentiation?

BACKGROUNDnWe have previously shown that glutamine synthetase protein and mRNA are concentrated in the crypt region of the rat small intestine and that the activity of this enzyme is highest around the time of weaning. This anatomical location and time of peak activity are sites and periods of active enterocyte differentiation. This led to our current hypothesis that glutamine synthetase is important in the differentiation of enterocytes.nnnMETHODSnTo test our hypothesis, we treated Caco-2 cells with physiologic (0.6 mM) glutamine concentrations in cell culture medium. The experimental group was treated with methionine sulfoximine, an irreversible glutamine synthetase inhibitor, and the control group with phosphate buffered saline. Three standard and well-defined markers of intestinal differentiation-sucrase-isomaltase activity, microvillus formation, and electrical impedance in transwell plates-were compared between the two groups.nnnRESULTSnThe methionine-sulfoximine-inhibited group was found to have lower sucrase-isomaltase activity, a lower density of microvilli, and lower electrical impedance values over time compared with the control group.nnnCONCLUSIONnThe experimental group was found to be less differentiated by all three markers of differentiation. Therefore, glutamine synthetase is important for Caco-2 cell differentiation.

Glutamine supports recovery from loss of transepithelial resistance and increase of permeability induced by media change in Caco-2 cells1

Recent evidence suggests that the conditionally essential amino acid glutamine is important for intestinal barrier function. However, the mechanism remains undefined. To determine the effects of glutamine on permeability of intestinal epithelial cell monolayers, Caco-2 cells were grown on membrane filters and exposed to 4 mmol/L sodium butyrate in order to rapidly achieve high levels of alkaline phosphatase and high transepithelial resistance as seen in functionally mature enterocytes. A standard method of medium exchange consisting of removal and replacement resulted in a catastrophic loss of transepithelial resistance and increase of mannitol and dextran fluxes that required 2-4 hrs and protein synthesis to recover. The effect was attributed to exposure of the upper monolayer surface to atmosphere and could be avoided by refeeding by incremental perfusion. Spontaneously-differentiated Caco-2 monolayers were resistant to this stress. This novel stress test was employed as a sensitive assay for the requirement of glutamine for monolayer transepithelial resistance and mannitol permeability. Pre-stress glutamine availability was more important than Gln-availability during the recovery phase. Thus the transepithelial resistance and permeability of butyrate-induced monolayers is dynamically-regulated in response to atmospheric exposure, by a mechanism that depends on threshold levels of glutamine availability.

Exogenous progesterone or indomethacin delays parturition in the viviparous lizard Sceloporus jarrovi.

Female Sceloporous jarrovi in late pregnancy given an ip injection of progesterone (50 ng/g body wt) or indomethacin (4 micrograms/g body wt) exhibited no change in the length of pregnancy when compared to saline-treated controls. In contrast, pregnant females given subcutaneous implants (constant release pellets) containing progesterone or indomethacin exhibited significantly delayed parturition when compared with females given control implants. Indomethacin treatment also disrupted the normal birth process when it occurred, as all females receiving this compound exhibited parturition of only a portion (24-32%) of the total clutch. A similar phenomenon occurred in one experiment following implantation of progesterone pellets. Histological examination of the corpora lutea and oviduct indicated no obvious difference in structure among any of the treatment groups.

Annual variation in the seasonal shift in egg size and clutch size in Sceloporus woodi

SummaryEarly and late season clutch parameters were examined over a three year period in the Florida scrub lizard, Sceloporus woodi. Precipitation levels were monitored throughout the study. In the early and late season of 1984 and the early season of 1986 precipitation levels approximated long-term mean levels of precipitation. In 1985 a severe winter drought occurred. Clutch size was positively related to body size in all samples in every year. In 1984 and 1986, egg size was not related to clutch size, whereas, in 1985 egg size was negatively related to clutch size. In 1985, females produced large clutches of small eggs early in the season and small clutches of large eggs late in the season. In 1984, no seasonal changes in egg or clutch size occurred. In the late season of 1986, females produced the largest clutches and the smallest eggs of all the samples, but egg and clutch size were not statistically different from the early season egg and clutch size of 1986. Total clutch dry weight, an estimate of total clutch energy, was not different in any of the six sampling periods. These data do not support current adaptationist models that attempt to explain the control of clutch and egg size in lizards. It is argued in this paper that egg and clutch size may vary in response to past environments that affect a females physical condition, as well as, current resources that may be important for maintenance and reproduction. Egg and clutch size appear to be plastic traits selected to respond to proximal environmental variation, whereas, the investment of total dry matter/clutch has been optimized.

Estimating Egg Retention Times in Sceloporine Lizards

The extent to which squamates retain eggs in utero is poorly known. The purpose of this study was to develop a broadly applicable laboratory based method for estimating egg retention time relative to the total period of embryonic development. The first step in determining the range of egg retention times for a species is quantifying embryo stage at normal oviposition for a large sample of females. The next step involves measuring the total time taken to develop, from ovulation to hatching, at constant temperature. Finally, by calculating the fraction of total development time taken to achieve each embryonic stage from ovulation, relative egg retention time can be determined. The modal embryo stages at oviposition in Sceloporus woodi, S. virgatus, and S. scalaris are stage 27, 31, and 38, respectively. It was previously reported that total embryonic development times at 30 C for these three species are 61.6, 55.3, and 45.6 days, respectively. Additionally, embryos of each species achieve similar stages at similar pro- portions of total development time. Embryo stages at oviposition (ESO), reported in the literature, indicate that the modal ESO for lizards (N = 38 species) is stage 30, which corresponds to approximately 26% of total embryonic development time. Although there are a few species of squamates that exhibit extreme periods of egg retention, most squamates appear to retain eggs for an absolute period that may be slightly longer than the egg retention times of crocodilians and turtles. Unlike turtles and crocodilians, squamate embryos continue to develop in utero and achieve a more advanced degree of embryogenesis at oviposition

Effects of arachidonic acid, prostaglandin F2α, prostaglandin E2, and arginine vasotocin on induction of birth in vivo and in vitro in a viviparous lizard (Sceloporus jarrovi)

The ability of arachidonic acid (AA) and prostaglandins of the two series to induce parturition in vivo and oviducal contraction in vitro was studied in the viviparous lizard Sceloporus jarrovi. Injection of PGF2 alpha, PGE2, or AA during late pregnancy stimulated parturition within 2 hr in a threshold-dependent fashion. In contrast, during mid pregnancy, females did not respond. Surgically removed and cultured oviducts from females in late pregnancy gave birth in response to AA, PGF2 alpha, and PGE2. Oviducts from S. jarrovi in mid pregnancy responded to PGF2 alpha but not to AA. Addition of arginine vasotocin, a potent stimulator of PG synthesis, either alone or with AA to oviduct cultures stimulated rapid and complete birth in vitro from oviducts obtained from late, but not mid pregnant, females. Stage of pregnancy mediates the response of the lizard oviduct to prostaglandin stimulation.

Characterization of glutamine synthetase transcript, protein, and enzyme activity in the human placenta

This study characterizes the molecular mechanisms necessary for glutamine synthesis in the human placenta. RNA hybridization and protein immunoblotting were used to verify the presence of glutamine synthetase (GS) transcripts and protein, respectively. Additionally, the presence of GS was determined by immunohistochemistry. RNA hybridization demonstrated the presence of 1.8- and 2.8-kB transcripts and protein immunoblotting yielded a single 49-kDa band, characteristics of GS transcripts and protein, respectively. The mean (+/- s.d.) specific activity of placental GS, expressed as mumol gamma-glutamyl hydroxamic acid/mg protein/h was 1.80 +/- 0.59, which is comparable to other organs which are net glutamine producers. Immunohistochemical analysis indicated the presence of GS within the cytotrophoblast and mesenchyme layers of placental villi, but not in the syncytiotrophoblast. Although these results suggest that the human placenta is capable of synthesizing glutamine, the fate of glutamine produced by this organ remains speculative.

Effects of prostaglandin F2α prostaglandin E2 and arachidonic acid on the induction of oviposition in vivo and in vitro in oviparous lizards

Gravid females of four different species of oviparous lizard were treated in vivo with varying doses of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 or arachidonic acid (AA). In contrast to previous studies examining birds and viviparous lizards, no dosage induced oviposition in any of the treated females. All females, however, did exhibit behaviors associated with oviposition. Intact oviducts removed from gravid females and placed in organ culture did oviposit when treated with 30 or 100 ng PGF2 alpha/ml of culture media. Arachidonic acid at similar concentrations also was effective in stimulating birth. These data suggest that prostaglandins can stimulate oviposition in oviparous lizards but further suggest that their action may be inhibited by oviducal innervation until just prior to natural birth.