Differential requirement of the cytoplasmic subregions of gamma c chain in T cell development and function.

The common cytokine receptor gamma chain (gammac), a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. The cytoplasmic domain of gammac consists of 85 aa, in which the carboxyl-terminal 48 aa are essential for its interaction with and activation of the Janus kinase, Jak3. Evidence has been provided that Jak3-independent signals might be transmitted via the residual membrane-proximal region; however, its role in vivo remains totally unknown. In the present study, we expressed mutant forms of gammac, which lack either most of the cytoplasmic domain or only the membrane-distal Jak3-binding region, on a gammac null background. We demonstrate that, unlike gammac or Jak3 null mice, expression of the latter, but not the former mutant, restores T lymphopoiesis in vivo, accompanied by strong expression of Bcl-2. On the other hand, the in vitro functions of the restored T cells still remained impaired. These results not only reveal the hitherto unknown role of the gammac membrane-proximal region, but also suggest the differential requirement of the cytoplasmic subregions of gammac in T cell development and function.

Uptake of glutathione by renal cortical mitochondria.

Transport of GSH into renal cortical mitochondria was studied. Mitochondria were highly enriched with little contamination from other subcellular organelles (as assessed by marker enzymes), they exhibited coupled respiration (respiratory control ratio greater than 3.0), and they had initial GSH concentrations of 5.71 +/- 0.65 nmol/mg protein (n = 47). Incubation of mitochondria with GSH in a triethanolamine, pH 7.4, buffer containing sucrose, potassium phosphate, MgCl2, and KCl, produced time- and concentration-dependent increases in intramitochondrial GSH content. Uptake was linear versus time for at least 2 min and exhibited kinetics consistent with one low-affinity, high-capacity process (Km = 1.3 mM, Vmax = 5.59 nmol/min per mg protein), although the results cannot exclude the presence of other, less quantitatively significant pathways. The initial rate of uptake of 5 mM GSH was not significantly altered by uncouplers (0.1 mM 2,4-dinitrophenol and 25 microM carbonyl cyanide m-chlorophenylhydrazone) or by 1 mM ADP. In contrast, incubation with 1 mM ATP, 1 mM KCN, 0.1 mM or 1 mM CaCl2 inhibited uptake by 41, 39, 43, or 55%, respectively. GSH uptake was markedly inhibited by gamma-glutamylglutamate and by a series of S-alkyl GSH derivatives. Strong interactions (i.e., both cis and trans effects) were observed with other dicarboxylates (i.e., succinate, malate, glutamate) but not with monocarboxylates (i.e., lactate, pyruvate). Preincubation of mitochondria with GSH protected against tert-butyl hydroperoxide- or methyl vinyl ketone-induced inhibition of state 3 respiration. These results demonstrate uptake of GSH into renal cortical mitochondria that appears to involve electroneutral countertransport (exchange) with other dicarboxylates. Functionally, GSH uptake into mitochondria can protect these organelles from various forms of injury, such as oxidative stress.

Enhanced levels of cathepsin B mRNA in murine tumors.

Relative amounts of mRNA for cathepsin B were measured in normal murine liver and three murine tumors, an invasive liver tumor (hepatoma, Hepa cl 9) and two melanoma variants (B16-F1 and B16 amelanotic melanoma, B16a). Using a human cDNA to the cathepsin B coding region as a hybridization probe, we detected two species of cathepsin B specific RNA transcripts (2.2 and 4.1 kb) in total RNA preparations of all four tissues. The concentrations of the 2.2 and 4.1 kb species were 3.6 and 2.7-fold greater in the highly metastatic B16a melanoma than in normal liver. The concentration of the 2.2 kb species in the invasive hepatoma was 1.7-fold greater than in normal liver. The increased levels of the 2.2 kb message were reflected in increases in activity of cathepsin B in both Hepa cl 9 and B16a.

Salivary phenytoin concentrations in geriatric patients.

The concentrations of phenytoin in saliva (Csal) and serum (C) in geriatric patients were compared. Salivary and venous blood samples were collected from 13 patients over 55 years old who were taking oral phenytoin for at least one week. The data of 10 patients were suitable for analysis. Phenytoin concentrations were determined with an immunoassay. Phenytoin Csal/C ratios were calculated. Based on previously reported Csal/C ratios of approximately 0.1 for general patient samples, an expected Csal for each patient was calculated as (C)(0.1). A Student's t test for paired data was used to test for differences between the expected and measured salivary concentrations. The mean Csal/C ratio was 0.098 (S.D. = 0.03, range 0.041-0.152). The correlation between the measured Csal and C was 0.96. There was no significant difference between the expected and measured salivary phenytoin concentrations (p greater than 0.6). This study suggests that age does not affect the ratio of salivary to serum phenytoin concentrations. The wide range of ratios observed, however, indicates that more research is needed before salivary phenytoin concentrations should be used clinically in geriatric patients.

Phenytoin concentrations in venous versus capillary blood of geriatric patients.

In 15 elderly patients who had seizure disorders, venous blood (standard venipuncture) was compared with capillary blood (finger lancet puncture) as the source of the specimen for determination of serum phenytoin concentration. The values obtained by the two procedures were similar (r = 0.99). The mean serum phenytoin levels in the venous samples was 17.3 micrograms/ml, and in the capillary samples 17.0 micrograms/ml; the paired t test showed no statistical difference. Either method provides an adequate blood sample for determination of phenytoin, and both may be used interchangeably. Capillary samples may be preferable for aged patients in whom venipuncture proves difficult or painful.