Diaphyseal non-union in the distal phalanx of a child.

Non-union after finger fracture is an infrequent complication. Although well described in adults, there are few reported cases in children. We describe a 6 year-old boy who presented with a non-union of the diaphysis of the distal phalanx three years after a finger tip crush injury.

Mechanisms governing the expression of the enzymes of glutamine metabolism--glutaminase and glutamine synthetase.

Whether on the scale of a single cell, organ or organism, glutamine homeostasis is to a large extent determined by the activities of glutaminase (GA, EC 3.5.1.2) and glutamine synthetase (GS, EC 6.3.1.2), the two enzymes that are the focus of this report. GA and GS each provide examples of regulation of gene expression at many different levels. In the case of GA, two different genes (hepatic- and kidney-type GA) encode isoforms of this enzyme. The expression of hepatic GA mRNA is increased during starvation, diabetes and high protein diet through a mechanism involving increased gene transcription. In contrast, the expression of kidney GA mRNA is increased post-transcriptionally by a mechanism that increases mRNA stability during acidosis. We found recently that several isoforms of rat and human kidney-type GA are formed by tissue-specific alternative RNA splicing. Although the implications of this post-transcriptional processing mechanism for GA activity are not yet clear, it allows for the expression of different GA isoforms in different tissues and may limit the expression of GA activity in muscle tissues by diverting primary RNA transcripts to a spliceform that produces a nonfunctional translation product. The expression of GS enzyme is also regulated by both transcriptional and post-transcriptional mechanisms. For example, the GS gene is transcriptionally activated by glucocorticoid hormones in a tissue-specific fashion. This hormonal response allows GS mRNA levels to increase in selected organs during catabolic states. However, the ultimate level of GS enzyme expression is further governed by a post-transcriptional mechanism regulating GS protein stability. In a unique form of product feedback, GS protein turnover is increased by glutamine. This mechanism appears to provide a means to index the production of glutamine to its intracellular concentration and, therefore, to its systemic demand. Herein, we also provide experimental evidence that GS protein turnover is dependent upon the activity of the 26S proteosome.

Glutamine.

Relatively little was known about glutamine metabolism until the 1930s, when Sir Hans Krebs first demonstrated glutamine hydrolysis and biosynthesis in the kidney. Subsequent studies by Rose in 1938 demonstrated that glutamine is a nonessential (dispensable) amino acid, as it can be readily synthesized de novo in virtually all tissues in the body. Because the body has the capacity to synthesize considerable quantities of glutamine, it has been assumed that glutamine is not required in the diet. However, this amino acid becomes quite depleted during the course of a catabolic insult such as injury or infection, indicating that the ability of glutamine production to meet demands during a variety of surgical illnesses is impaired. In states of health, the assumption that glutamine is not required in the diet is probably valid, although it is difficult to test the hypothesis, as glutamine is present in virtually all dietary proteins. Most naturally occurring food proteins contain 4% to 8% of their amino acid residues as glutamine; therefore less than 10 g of dietary glutamine is likely to be consumed daily by the average person. In contrast to this usual dietary availability, studies in stressed patients indicate that considerably larger amounts of glutamine (20-40 g/day) may be necessary to maintain glutamine homeostasis. Thus from a nutritional standpoint, glutamine may be thought of as a drug as well as a nutrient. This paper reviews the physiology and biochemistry of glutamine with an emphasis on its metabolism in surgical illnesses and its role as a conditionally essential amino acid.

Glutamine synthetase expression in muscle is regulated by transcriptional and posttranscriptional mechanisms.

Skeletal muscle exports glutamine (Gln) and increases the expression of the enzyme glutamine synthetase (GS) in response to physiological stress. Acute stress or direct glucocorticoid administration raises muscle GS mRNA levels dramatically without a parallel increase in GS protein levels. In the lung, this discrepancy is caused by feedback destabilization of the GS protein by its product Gln. It was hypothesized that muscle GS protein levels increase during stress only when the intracellular Gln pool has been depleted. Adult male rats were injected with the glucocorticoid hormone dexamethasone (DEX) to mimic the acute stress response and with the GS inhibitor methionine sulfoximine (MSO) to deplete muscle Gln stores. DEX increased GS mRNA levels by 2.8-fold but increased GS protein levels by an average of only 40%. MSO diminished muscle GLN levels by 68% and caused GS protein levels to rise in accordance with GS mRNA. Chronic stress was mimicked using 6 days of MSO treatment, which produced anorexia, 23% loss of body weight, and 64% decrease in muscle Gln levels, as well as pronounced increases in both muscle GS mRNA (26-fold) and protein levels (35-fold) without elevation of plasma glucocorticoid levels. Calorie-restricted pair-fed animals exhibited lesser increases in muscle GS mRNA (8-fold) and protein levels (5-fold) without a decline in muscle Gln content. Thus regulation of GS expression in both acute and chronic stress involved both transcriptional and posttranscriptional mechanisms, perhaps affected by muscle Gln content.

Glutamine synthetase expression in rat lung is regulated by protein stability.

During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500-700%, GS activity levels rise only 20-45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% (P < 0.01) and decreased lung tissue Gln levels by 70% (P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold (P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% (P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold (P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction (P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.

Sepsis increases lung glutamine synthetase expression in the tumor-bearing host.

Acute stresses such as trauma or endotoxemia augment GLN demand and are associated with increased release of this amino acid from skeletal muscle and lung as well as increased expression of glutamine synthetase (GS, the principal enzyme of GLN synthesis) in these tissues. Muscle GLN release is also increased during chronic catabolic states which are associated with depletion of lean body mass, such as starvation or malignancy. We hypothesized that the expression of GS in response to an acute stress would be altered in tumor-bearing rats (TBR) experiencing severe cachexia and therefore a previously heightened GLN demand. Male Fischer 344 rats were implanted with methylcholanthrene-induced fibrosarcoma tumors or underwent sham operations and pair-feeding (sham) with TBR partners. When tumor burden reached approximately 15% of carcass weight, animals received injections of either Escherichia coli lipopolysaccharide (LPS, 1 mg/kg body wt) or saline vehicle. Rats were sacrificed 8 h after injection and lung and muscle tissue were analyzed for GS mRNA and protein via Northern and Western blot techniques, respectively. LPS injection caused an equivalent 4- to 6-fold increase in lung and muscle GS mRNA in both TBR and sham rats (P < 0.01). LPS did not produce a significant increase in GS protein level in muscle tissue of either group or in lung tissue of sham rats. In contrast, endotoxin did lead to a 3.5-fold increase in GS protein levels in lung tissue of TBRs (P < 0.05). This increase in lung GS protein may signify the importance of the lung in maintaining GLN homeostasis during chronic catabolic states where muscle mass is diminished.

Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats.

During sepsis, the lung responds by exporting increased amounts of the amino acid glutamine. This response is accompanied by increased enzymatic activity of glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia. It is also known that GS expression in the rat lung can be induced by glucocorticoid hormones. To determine whether the septic response and the response to glucocorticoids are related, we have characterized the induction of GS expression during lipopolysaccharide (LPS)-induced endotoxemia in normal, neutropenic, and adrenalectomized rats. Normal rats exhibited a time- and dose-dependent induction of GS mRNA levels after a single intraperitoneal dose of LPS. Responses to LPS were maximal at doses of 0.1 mg/kg body wt and above. A single 10 mg/kg body wt dose of LPS led to a rapid, transient sevenfold increase in GS mRNA (P < or = 0.1) and a twofold increase in GS protein level 8 h postinjection. Induction of lung GS mRNA 4 h after LPS injection was approximately fivefold in neutropenic (P < or = 0.1) and fourfold in nonneutropenic control rats (P < or = 0.1), suggesting that infiltrating neutrophils or neutrophil-derived factors are not required for GS induction. In response to high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold (P < or = 0.02) compared with sixfold in sham-operated control rats (P < or = 0.02). However, in response to low-dose, long-term endotoxemia, adrenalectomized rat lung GS mRNA increased threefold (P < or = 0.02) compared with fourfold in sham-operated control rats (P < or = 0.02). Adrenalectomy did not affect the elevation of lung GS mRNA levels in response to dexamethasone. In addition, GS mRNA was induced four- and sixfold in rat microvascular pulmonary endothelial cells exposed to plasma from control and septic rats, respectively. The addition of a glucocorticoid antagonist, RU-38486, completely blocked GS gene induction in the presence of control plasma but only attenuated the response to plasma from septic animals by 30%. These results suggest that GS gene induction during sepsis is only partially mediated by adrenal-derived glucocorticoid hormones.

Catalysis of the hydrolysis of phosphorylated pyridines by alkaline phosphatase has little or no dependence on the pKa of the leaving group.

Bacterial alkaline phosphatase is an active catalyst for the hydrolysis of N-phosphorylated pyridines, with values of the second-order rate constant kcat/Km in the range 0.4-1.2 x 10(6) M-1 s-1 at pH 8.0, 25 degrees C. There is little or no dependence of the rate on the pKa of the leaving group; the value of beta 1g is 0 +/- 0.05, which may be compared with beta 1g = -1.0 for the nonenzymic reaction. Phosphorylated pyridines do not have a free electron pair available for protonation or coordination of the leaving group. Therefore, this result means that the similar, small dependence on leaving group structure for the enzyme-catalyzed hydrolysis of phosphate esters [Hall, A. D., & Williams, A. (1986) Biochemistry 25, 4784-4790) does not provide evidence for general acid catalysis or electrophilic assistance of leaving group expulsion. The results are consistent with the hypothesis that productive binding of the substrate, which may involve a conformational change, is largely rate limiting for turnover of the enzyme at low substrate concentrations.