Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Alkali-burned collagen produces a locomotory and metabolic stimulant to neutrophils.

Polymorphonuclear leukocytes (PMNs) invade the cornea following an alkali burn apparently undergoing a respiratory burst and degranulation, which is thought to lead to corneal ulceration. The supernatant obtained from burned Sigma collagen (Miller type 1) or from bovine cornea produced a significant locomotory stimulus to PMNs. Citrate inhibited this locomotory stimulus by 69.5% and 98%, respectively. PMNs were stimulated to undergo a respiratory burst without the concomitant release of beta-glucuronidase when exposed to the supernatant from alkali-burned commercial collagens, or from bovine or porcine corneas. This stimulation is reduced by 72% (Sigma collagen) or 89% (bovine cornea) when the supernatant is dialyzed against distilled water and reinstated when the osmolality is increased. The degree of the respiratory burst is partially dependent on the volume of the supernatant, the duration of alkali exposure, and/or the concentration of NaOH used. The respiratory burst of PMNs stimulated by alkali-burned Sigma collagen supernatant is inhibited by trifluoperazine but not by citrate or EDTA. Light and electron microscopy of these stimulated PMNs show many large blebs and hairlike projections. The authors hypothesize that collagen breakdown product(s) from alkali burning might be the initial, or one of the initial stimuli, for PMN invasion into the cornea and the subsequent activation of the respiratory burst.

Nutrition support team management of enterally fed patients in a community hospital is cost-beneficial.

OBJECTIVE: To determine whether nutrition support team (NST) management of enterally fed patients is cost-beneficial and to compare primary outcomes of care between team and nonteam management. DESIGN: A quasi-experimental study was conducted over a 7-month period. SETTING: A 400-bed community hospital. SUBJECTS: A convenience sample of 136 subjects who had received enteral nutrition support for at least 24 hours. Forty-two patients died; only their mortality data were used. Ninety-six patients completed the study. INTERVENTION: Outcomes, including cost, for enterally fed patients in two treatment groups--those managed by the nutrition support team and those managed by nonteam staff--were compared. MAIN OUTCOME MEASURES: Severity of illness level was determined for patients managed by the nutrition support team and those managed by nonteam staff. For each group, the following measures were adjusted to reflect a significant difference in average severity of illness and then compared: length of hospital stay, readmission rates, and mortality rates. Complication rates between the groups were also compared. The cost benefit was determined based on savings from the reduction in adjusted length of hospital stay. STATISTICAL ANALYSES PERFORMED: Parametric and nonparametric statistics were used to evaluate outcomes between the two groups. RESULTS: Differences were statistically significant for both severity of illness, which was at a higher level in the nutrition support team group (P < .001), and complication rate, which was greater in the nonteam group (P < .001). In the nutrition support team-managed group, there was a 23% reduction in adjusted mortality rate, an 11.6% reduction in the adjusted length of hospital stay, and a 43% reduction in adjusted readmission rate. Cost-benefit analysis revealed that for every $1 invested in nutrition support team management, a benefit of $4.20 was realized. APPLICATIONS: Financial and humanitarian benefits are associated with nutrition support team management of enterally fed hospitalized patients.

The effects of citrate on fMLP-induced polymorphonuclear leukocyte stimulation and locomotion.

Citrate reduces the incidence of corneal ulceration and perforation in alkali burned eyes and prevents polymorphonuclear leukocyte (PMN) accumulation in certain inflamed ocular tissues. Chelation of Ca2+ and Mg2+ by citrate appears to be the mechanism causing strong inhibition of in vitro PMN stimulation by opsonized zymosan. It is important to know if other activating agents of PMNs, with differing sensitivity to divalent cations, are inhibited by citrate. Citrate (12 mM) partially inhibits fMLP stimulation of the respiratory burst (50%) and degranulation (65%) of PMNs in a divalent cation free media, while having no effect or only a small effect in the presence of 1 mm Ca2+. Citrate also caused significant inhibition of fMLP (12 mM = 50%) induced locomotion of PMN when incubated in media containing 500 microM Ca2+ and 600 microM Mg2+ in a modified Boyden chamber. When used together, Ca2+ (6 mM) and Mg2+ (6 mM) reduced this inhibition to only 20%. Citrate apparently inhibits fMLP-induced stimulation in cation free media by chelating CA2+ effluxed from intracellular storage sites. In the chemotactic studies, citrate probably chelates extracellular Ca2+ and Mg2+. The divalent cation requirements of the activating agents and/or the PMN function may determine the degree of inhibition by citrate. It is therefore important to identify the mediators in alkali burned corneas as well as other inflammatory conditions.

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.