Conserved (CT)n.(GA)n repeats in the non-coding regions at the Gpdh locus are binding sites for the GAGA factor in drosophila melanogaster and its sibling species.

The (CT)n.(GA)n rich sequences in the upstream and 5' intron enhancer regions of the sn-glycerol-3-phosphate dehydrogenase (Gpdh) gene in Drosophila melanogaster, its sibling and distantly related species are conserved in their position and in the number of repeats. Using in vitro DNA-footprint analyses we show that the GAGA factor binds to these multiple closely spaced and overlapping conserved (CT)n.(GA)n repeats in D. melanogaster and D. erecta.

Noninvasive cardiac output by partial CO2 rebreathing after severe chest trauma.

BACKGROUND: In multiple trauma patients, early continuous cardiac output (CCO) monitoring is frequently desired but is difficult to routinely employ in most emergency departments because it requires invasive procedures. Recently, a noninvasive cardiac output (NICO) technique based on the Fick principle and partial CO2 rebreathing has shown promise under a variety of conditions. Since this method has not been tested after lung damage, we evaluated its utility in a clinically relevant model. METHODS: Anesthetized, ventilated swine (n = 11, 35-45 kg) received a unilateral blunt trauma via a captive bolt gun followed by a 25% hemorrhage. After 60 min of shock, crystalloid resuscitation was given as needed to maintain heart rate < 100 beats/min and mean arterial pressure > 70 mm Hg. Standard CCO by thermodilution (Baxter Vigilance, Irvine, CA) was compared with NICO (Novametrix Medical Systems Inc., Wallingford, CT) for 8 hr. RESULTS: The severity of the injury is reflected by seven deaths (average survival time = 4.25 hr). Trauma increased dead space ventilation (19%), airway resistance (30%), and lactate (3.2 mmol/L), and decreased dynamic compliance (48%) and Pao2/Fio2 (54%). In these extreme conditions, the time course and magnitude of change of CCO and NICO were superimposed. Bland-Altman analysis reveal a bias and precision of 0.01 +/- 0.69 liters/min. The linear relationship between individual CCO and NICO values was significant (p < 0.0001) and was described by the equation NICO = (0.74 +/- 0.1)CCO + (0.65 +/- 0.16 liters/min) but the correlation coefficient (r2 = 0.541) was relatively low. The cause for the low correlation could not be attributed to increased pulmonary shunt, venous desaturation, anemia, hypercapnia, increased dead space ventilation, or hyperlactacidemia. CONCLUSION: NICO correlated with thermodilution CCO, but underestimated this standard by 26% in extreme laboratory conditions of trauma-induced cardiopulmonary dysfunction; 95% of the NICO values fall within 1.38 liters/min of CCO; and with further improvements, NICO may be useful in multiple trauma patients requiring emergency intubation during initial assessment and workup.

Effects of a novel antioxidant during resuscitation from severe blunt chest trauma.

Previous work suggests that neutrophils (PMNs) and/or prostaglandins might mediate the progressive respiratory failure after severe pulmonary contusion. Since reactive oxygen metabolites are closely associated with both these factors, we examined the actions of a novel antioxidant after swine received a unilateral injury followed by 25% hemorrhage. An infusion (2mL/kg/h intravenously x 6 h) of either polynitroxylated 5% Dextran + Tempol (PND, n = 9), 5% Dextran (D, n = 6), or lactated Ringers (LR, n = 13) was begun 60 min post-injury to mimic 'pre-hospital resuscitation.' After 15 min, standard resuscitation was initiated (3x shed blood as LR in 30 min) plus further LR for 6 h to maintain hemodynamics. The total LR requirement was lower with PND (1,772+/-267 mL) versus D (3,040+/-689, P = 0.0563) or LR (4145+/-398, P = 0.0005). The ipsilateral bronchoalveolar lavage (BAL) PMN count with PND (8+/-2 x 10(5)/mL), was not different from its baseline (P = 0.131), but the counts with D (16+/-3) and LR (17+/-4) were both higher than their baselines (P = 0.0184 and 0.0431). Similarly, BAL protein with PND (1,560+/-350 mg %) was not elevated from its baseline (P = 0.0721), but the values with D (2,560+/-498) and LR (2,474+/-899) were both higher than their baselines (P = 0.0169 and 0.0325). In the contralateral (uninjured) lung, the effects were similar, but the increases were less for PMNs (8+/-2 versus 10+/-2 or 14+/-4 x 10(5)/mL) and for protein (609 +/-153 versus 1,955+/-671 or 1486+/-357 mg %). Despite these significant BAL changes, there was no obvious improvement in cardiopulmonary dysfunction. Thus oxidants probably have some role in the pathogenic mechanism of progressive secondary injury after thoracic trauma, but further work is needed to determine the therapeutic potential of antioxidants because no clinical improvement was detected.

Ocular motor signs in an infant with carbohydrate-deficient glycoprotein syndrome type Ia.

PURPOSE: To document the evolution of ocular motor abnormalities in an infant with carbohydrate-deficient glycoprotein syndrome. METHODS: Case report. An infant with carbohydrate-deficient glycoprotein syndrome type 1a underwent magnetic resonance imaging and infrared eye movement recording. RESULTS: A 10-month-old male with carbohydrate-deficient glycoprotein syndrome type Ia had rapid horizontal oscillations of the eyes when startled or awakened from sleep. Clinical examination confirmed this finding and disclosed congenital ocular motor apraxia with a reduced vestibulo-ocular reflex. Infrared eye movement recording showed ocular flutter and square wave jerks superimposed on a horizontal pendular nystagmus. Magnetic resonance imaging showed diffuse cerebellar hypoplasia. CONCLUSION: Carbohydrate-deficient glycoprotein syndrome type Ia can be associated with multiple cerebellar eye signs including ocular flutter, square-wave jerks, and congenital ocular motor apraxia.

Resuscitation of severe chest trauma with four different hemoglobin-based oxygen-carrying solutions.

BACKGROUND: The purpose of this study was to test whether polynitroxylation (PN) improved the therapeutic profile of hemoglobin-based oxygen-carrying compounds (HBOCs) that were unpolymerized (alphaalphaHb) or 70% polymerized (polyHb) in a clinically relevant model that combines pulmonary injury and reperfusion. To our knowledge, four different HBOC formulations have never been compared in the same trauma model. METHODS: Anesthetized, ventilated swine (n = 45) received a unilateral lung contusion + 25% hemorrhage. After 60 minutes, 250 mL of either PNalphaalphaHb (n = 5), alphaalphaHb (n = 10), PNpolyHb (n = 6), polyHb (n = 5), or normal saline (NaCl, n = 10) was administered for 20 minutes, followed by standard crystalloid resuscitation for 30 minutes, and supplemental crystalloid as required for 6 hours to maintain heart rate <100 beats/min and mean arterial pressure >70 mm Hg. RESULTS: Nine of 45 deaths occurred before resuscitation. Survival time was 395 minutes with NaCl versus 303 minutes with alphaalphaHb (p = 0.03) or 238 minutes with PNalphaalphaHb (p = 0.04). With both polymerized HBOCs, survival was 480 minutes (polyHb vs. alphaalphaHb, p = 0.005; PNpolyHb vs. PNalphaalphaHb, p = 0.006). All HBOCs were pressors (all p < 0.05) and all reduced the supplemental fluid required to maintain systemic hemodynamics during resuscitation (all p < 0.05). By 90 minutes postresuscitation, cardiac index was 112% of baseline with NaCl (p < 0.02), but was 78% with alphaalphaHb (p = not significant), 63% with PNalphaalphaHb (p < 0.01), 79% with PNpolyHb (p < 0.01), and 67% with polyHb p < 0.02). Relative to NaCI, no HBOC altered trauma-induced neutrophilia, thrombocytopenia, or the trauma-induced increases in bronchoalveolar lavage protein or bronchoalveolar lavage neutrophils. CONCLUSION: After resuscitation from chest trauma, we observed the following: (1) all HBOCs reduced fluid requirements and increased right and left ventricular afterload versus NaCl, which further compromised an already marginal cardiac performance; (2) mortality was less with polyHbs relative to alphaalphaHb, but the pressor action was unchanged; (3) the pressor action was less with polynitroxylated compounds relative to the unmodified HBOC, but this chemical modification had no effect on mortality; and (4) the pressor action of HBOCs must be attenuated by strategies other than polymerization or polynitroxylation for these compounds to be safe, effective resuscitants in humans.

Gastric outlet obstruction resulting from peptic ulcer disease requiring surgical intervention is infrequently associated with Helicobacter pylori infection.

BACKGROUND: Gastric outlet obstruction (GOO) secondary to peptic ulcer disease requiring therapeutic intervention remains a common problem. The incidence of Helicobacter pylori infection in this cohort has not been well defined. Pneumatic dilatation (PD) has been proposed as first-line therapy before surgical intervention. If H pylori infection in patients with GOO is infrequent, PD may not offer permanent control without the need for longterm antacid therapy. STUDY DESIGN: The purpose of this study was to examine the incidence of H pylori infection and surgical outcomes in patients undergoing resection for GOO. The records of all patients having resection (vagotomy and antrectomy) for benign disease from 1993 to 1998 for GOO at the University of Tennessee affiliated hospitals were reviewed retrospectively. Smoking history, NSAID use, weight loss, previous ulcer treatment, previous treatment for H pylori, and previous attempts at PD were among the factors examined. H pylori infection was documented by Steiner stain from either preoperative biopsy or, in most patients, final surgical specimens. Surgical complications and patient satisfaction were ascertained from inpatient records, postoperative clinical notes, and, where possible, followup telephone surveys. RESULTS: Twenty-four patients underwent surgical resection during the study period. There were 16 men and 8 women, with a mean age of 61 years (range 40 to 87 years). Weight loss was documented in 58% and averaged 27 lb. Five of 24 patients had previous attempts at PD, 3 of whom were H pylori negative. All five had further weight loss after these failed attempts. Of the 24 patients reviewed, only 8 (33%) were H pylori positive. There were no procedure-related deaths. Longterm clinical followup was possible in 16 of 24 patients, and all but one demonstrated dramatic clinical improvement by Visick score. CONCLUSIONS: We conclude the following: 1) In this cohort, H pylori infection was present in a minority; 2) previous attempts at PD were unsuccessful, which may be related to the H pylori-negative status of the patients; 3) mortality related to the operation was zero; and 4) patient satisfaction was positive by the Visick scale. Patients with H pylori-negative GOO resulting from peptic ulcer disease should be strongly considered for an early, definitive, acid-reducing surgical procedure.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

Structural changes in the promoter region mediate transvection at the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster.

Trans effects at the sn-glycerol-3-phosphate dehydrogenase locus (Gpdh) of Drosophila melanogaster give rise to an increase in GPDH activity and mRNA from the wild-type allele in heterozygotes with some low-activity alleles. Either the low-activity alleles that induce the effect have a defective P-element inserted between the promoter and a downstream intronic enhancer element or the promoter region is deleted. The trans effect is pairing dependent, characteristic of transvection at some other loci. The defective P-elements that mediate transvection are located between the promoter and at least up to 6 bp downstream of the transcription start site. Transvection at Gpdh appears similar to the "enhancer action in trans" mode at the yellow locus.

Expression of the GPDH-4 isozyme of sn-glycerol-3-phosphate dehydrogenase in three Drosophila species.

A fourth recently discovered isozyme of sn-glycerol-3-phosphate dehydrogenase (GPDH-4) in Drosophila melanogaster is shown to be a translational product of the Gpdh transcript which contains exons 1 through 7. This transcript was also found in two other Drosophila species, D. busckii and D. virilis. In contrast to D. melanogaster and D. busckii, the Gpdh transcript containing exons 1-6 is absent in D. virilis adults. The reason for this difference between D. virilis and the two other species is intriguing but remains elusive. We have ruled out the possibility that a replacement of an amino acid residue in exon 7 played any role in generating this interspecific variation.

sn-Glycerol-3-phosphate dehydrogenase in the honey bee Apis mellifera -an unusual phenotype associated with the loss of introns.

In comparison with the numerous Drosophila species and the mouse, the Gpdh gene in the honey bee Apis mellifera lacks most introns. This prevents the gene from producing different GPDH isoforms by alternative splicing, which occurs in Drosophila melanogaster. The sequences of the cDNA and genomic Gpdh of A. mellifera are described and show that at the amino acid level they share 84% similarity and 71% identity with D. melanogaster. The identity at the nucleotide level is 62% in the coding region, but no significant similarities were detected in the UTRs. Northern analyses revealed an accumulation of unspliced Gpdh pre-mRNA in the honey bee, probably reflecting splicing inefficiency, although it is also possible that splicing is a regulated step in Gpdh expression in A. mellifera. It is suggested that the intron loss occurred via reverse transcription of a mature Gpdh transcript.

Regulation of the expression of the sn-glycerol-3-phosphate dehydrogenase gene in Drosophila melanogaster.

P element-mediated transformation has been used to investigate the regulation of expression of the sn-glycerol-3-phosphate dehydrogenase gene of Drosophila melanogaster. A 13-kb construct containing the eight exons and associated introns, 5 kb of the 5' region, and 3 kb downstream from the structural gene produced normal levels of enzyme activity and rescued the poor viability of flies lacking the enzyme. All the regulatory elements essential for normal enzyme expression were located in a fragment that included the exons and introns and 1-kb upstream noncoding sequence. Deletions of the 1.6-kb second intron reduced activity to 25%. Transformants with fusion constructs between the sn-glycerol-3-phosphate dehydrogenase gene and the beta-galactosidase gene from E. coli revealed three elements that affected expression. A (CT)9 repeat element at the 5' end of the second intron increased expression in both larvae and adults, particularly at emergence. A second regulatory element, which includes a (CT)7 repeat, was located 5' to the TATA box and had similar effects on the gene's expression. A third, undefined, enhancer was located in the second intron, between 0.5 and 1.8 kb downstream of the translation initiation codon. This element increases enzyme activity to a similar extent in larvae and adults but has little effect when the enhancer at the 5' end of the intron is present.

A KP element inserted between the two promoters of the alcohol dehydrogenase gene of Drosophila melanogaster differentially affects expression in larvae and adults.

An allele of the Drosophila melanogaster alcohol dehydrogenase (Adh) gene has a 1.15-kb KP element inserted, in the same orientation as Adh transcription, five nucleotides upstream of the distal transcription start site. The target site--GTCCAAGT--in Adh between nucleotides -13 and -6 is duplicated at both ends of the insertion. Adult flies with either one or two copies of the allele have less than 12% of the alcohol dehydrogenase (ADH) activity of the controls. Activity levels are also reduced in larvae, but by a much smaller amount. Quantitative Northern analyses showed that the low activity level in adults was caused by reduced transcript levels from the distal promoter. 5' RACE experiments indicated that adult transcripts were mostly initiated downstream of the distal promoter but some skipped the first adult exon. In late third-instar larvae the transcripts present were from the proximal promoter. After the KP element was deleted by an appropriate breeding program, the distal transcript increased in adults to the control level, but ADH activity increased to only 50% of the control. No nucleotide changes were found in the gene that could explain this difference.

Nucleotide sequence and expression of the sn-glycerol-3-phosphate dehydrogenase gene in Locusta migratoria.

The sn-glycerol-3-phosphate dehydrogenase gene (Gpdh) in the locust (Locusta migratoria) encodes three mature transcripts and a number of isozymes. Gpdh expression is tissue- and developmentally regulated such that two transcripts are unique to flight muscle. Identical proteins are encoded by two transcripts which are generated by alternative splicing downstream of the stop codon in the penultimate exon.

Hepatic glutamine synthetase deficiency in fatal hyperammonemia after lung transplantation.

BACKGROUND: The cause of severe acquired hyperammonemia, an uncommon but often fatal complication of organ transplantation and chemotherapy for cancer, is obscure. OBJECTIVE: To test the hypothesis that liver glutamine synthetase deficiency may explain hyperammonemia in patients who have had organ transplantation or are receiving chemotherapy. DESIGN: Case report. PATIENTS: Two patients who had fatal hyperammonemia after orthotopic lung transplantation. MEASUREMENTS: Liver tissue was analyzed to determine the activities of two urea cycle enzymes and glutamine synthetase. Western blot assays for hepatic glutamine synthetase were performed to determine whether glutamine synthetase deficiency resulted from reduced enzyme levels. RESULTS: Activities of carbamoyl phosphate synthetase I and ornithine carbamoyltransferase in the liver were normal. The activity of hepatic glutamine synthetase was markedly reduced (in patient 1, 12% of the mean value in controls; in patient 2, 28% of the mean value in controls), and a concomitant reduction in the amount of glutamine synthetase protein was observed. CONCLUSION: Hyperammonemia after transplantation was associated with hepatic glutamine synthetase deficiency in two patients, but the causal relation between these two conditions must be further studied.

Quantitative assessment of whole body galactose metabolism in galactosemic patients.

We employed [1-13C] galactose in isotope kinetic studies to delineate whole body galactose metabolism in vivo in patients with galactose-1-phosphate uridyltransferase (GALT) deficiency. The data in three control and three adult galactosemic subjects, homozygous for the most common GALT gene defect, the Q188R mutation, and with absent RBC GALT activity, revealed an apparent endogenous galactose synthesis rate of 0.53-1.05 mg/kg per hour. Unlike normal children and adults who eliminated 3%-6% and 21%-47% of an intravenous bolus of [1-13C] galactose as 13CO, in expired air in 1 and 5 h respectively, classic galactosemic patients, either Q188R/Q188R or Q188R/unknown, released almost none in 1 h and 3%-6% in 5 h. In contrast, an African-American galactosemic variant patient with a S135L/S135L mutation and no residual RBC GALT activity oxidized [1-13C]galactose to 13CO2 at a rate comparable to control subjects. Individuals homozygous for the Duarte mutation, N314D/N314D and Q188R/ N314D. Q188R/+ and S135L/+ subjects also had normal breath test results. Not surprisingly, the Q188R/Q188R classic galactosemic patient cannot handle an acute galactose load, failing to match a control subject in the rapid conversion of [1-13C]galactose to [13C]glucose and 13CO2. However, classic patients synthesize substantial quantities of galactose de novo and on a lactose-free diet must oxidize comparable amounts of galactose to maintain steady-state levels of galactose and galactose metabolites such as galactose-1-phosphate, galactitol and galactonate. In vivo isotope kinetic analyses may allow us to understand better these aspects of galactose metabolism and, through the use of studies in variant galactosemics, perhaps allow us to begin to unravel the pathophysiology of galactosemia.

Sugar nucleotide concentrations in red blood cells of patients on protein- and lactose-limited diets: effect of galactose supplementation.

Uridine diphosphate (UDP) galactose, a pivotal compound in the metabolism of galactose, is the obligate donor of galactose in the formation of complex glycoconjugates. The cellular UDPgalactose concentration has been thought to be maintained by the interconversion of UDPglucose and UDPgalactose by UDPgalactose-4-epimerase. However, recent findings of lower average red blood cell (RBC) UDPgalactose concentrations in galactose-1-phosphate uridyltransferase-deficient patients suggest that other factors play a role in determining its concentration. To test the hypothesis that the amount of galactose traversing the Leloir pathway contributes to the cellular UDPgalactose pool, we determined RBC UDPgalactose in patients with maple syrup urine disease (MSUD), phenylketonuria (PKU), and other metabolic diseases who were treated with a low-protein, and consequently, low-lactose diet. Six patients with MSUD were also supplemented with 19 g galactose/d and their UDPhexose concentrations were measured at intervals. We show that young patients with MSUD or PKU have decreased average RBC UDPgalactose concentrations when compared with similarly aged healthy subjects. Galactose supplementation of MSUD patients significantly increased their UDPgalactose concentrations in both RBCs and white blood cells (WBCs) from 29.5 +/- 1.5 to 42.3 +/- 5.8 nmol/g hemoglobin and from 69.0 +/- 7.5 to 193.0 +/- 49.0 nmol/g protein, respectively. Discontinuation of supplementation was associated with a return to basal values in RBCs and a reattainment of the pretreatment ratio of UDPglucose to UDPgalactose in WBCs. These observations demonstrate that dietary galactose is a factor in establishing the steady state concentrations of the uridine sugar nucleotides and imply that galactose metabolism modulates the achievement of an epimerase-mediated equilibrium.

Molecular structure of rare but geographically widespread sn-glycerol-3-phosphate dehydrogenase 'ultra-fast' electrophoretic alleles in Drosophila melanogaster.

Six naturally occurring but rare alleles of sn-glycerol-3-phosphate dehydrogenase (Gpdh) in Drosophila melanogaster have been investigated in this study. They all belong to a class of GpdhUF (ultra-fast) alleles, because their electrophoretic mobilities are faster than that of the GpdhF (fast) allele. The GpdhUF variants are widespread, and have been reported from five continents. DNA sequence analysis has shown that the change in electrophoretic mobility was in each allele caused by a single amino acid residue substitution in the encoded protein. In the XiamenUF allele it is a substitution of lysine (AAA) to asparagine (AAT) in exon 1 (residue 3). An asparagine (AAT) to aspartate (GAT) change was found in exon 6 (residue 336) in the IowaUF and NetherlandsUF alleles. The mobility of the RaleighUF allele was altered by a valine (GTG) to glutamate (GAG) substitution in exon 3 (residue 76). Two mutations were detected in the BrazzavilleUF allele: a lysine (AAG) to methionine (ATG) substitution in exon 2 (residue 68) is responsible for the ultra-fast phenotype of this variant, while a tyrosine (TAT) to phenylalanine (TTT) substitution in exon 4 (residue 244) is not expected to alter the electrophoretic mobility of the encoded protein. These results indicate that the GpdhUF alleles originate from different mutational events, and only two of them--IowaUF and NetherlandsUF--might share a common ancestry. The GPDH activity of the IowaUF allele is intermediate between those of the GpdhS and GpdhF control stocks. The other GpdhUF variants have lower activities than the controls: XiamenUF--83%, RaleighUF--80% and BrazzavilleUF--73% of the GpdhF control.

Retrotransposon insertion induces an isozyme of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

The insertion of the blood retrotransposon into the untranslated region of exon 7 of the sn-glycerol-3-phosphate dehydrogenase-encoding gene (Gpdh) in Drosophila melanogaster induces a GPDH isozyme-GPDH-4-and alters the pattern of expression of the three normal isozymes-GPDH-1 to GPDH-3. The process of transcript terminus formation inside the retrotransposon insertion reduces the level of the Gpdh transcript that contains exon 8 and increases the level of the transcript that contains exons 1-7. The induced GPDH-4 isozyme is a translation product of the three transcripts that contain fragments of the blood retrotransposon. The mechanism of mutagenesis by the blood insertion is postulated to involve the pause or termination of transcription within the blood sequence, which in turn is caused by the interference of a DNA-binding protein with the RNA polymerase. Thus, we show the formation of a new functional GPDH protein by the insertion of a transposable element and discuss the evolutionary significance of this phenomenon.