Site-directed mutagenesis of phosphorylation sites of the branched chain alpha-ketoacid dehydrogenase complex

Regulation of the branched chain alpha-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1 alpha and E1 beta under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased Km for the alpha-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.     

Applications of amino acid derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Analysis of feed grains, intravenous solutions and glycoproteins

Primary and secondary amines are rapidly labelled by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate to form highly fluorescent asymmetric urea derivatives which are readily amenable to analysis by liquid chromatography. Derivatization consists of a simple, one-step procedure, and the resulting labelled amines can be analyzed without further cleanup. The adducts are extremely stable with no discernible loss in response after storage for one week at room temperature, making the reagent an ideal candidate for pre-column amino acid analysis. Chromatographic methods for protein hydrolysates have been developed for the analysis of samples containing many unusual amino acids including a number of cysteine derivatives, collagen hydrolysates containing hydroxyproline and hydroxylysine, performic acid oxidized samples and glycoprotein hydrolysates containing glucosamine and galactosamine. Samples with potentially interfering matrix components such as hydrolyzed feed grains and intravenous solutions are readily analyzed and are quantified with average per cent relative standard deviations in the 1-2% range. Comparative data on these samples are in good agreement with either ion-exchange amino acid analysis or label information.     

Maternal-fetal transfers: indications, appropriateness, and cost

The objective of this study was to assess the indications, appropriateness, and cost of maternal-fetal transfers to a tertiary care facility in an era of managed care. Our perinatal database was reviewed from January 1, 1996 through June 30, 1997 to determine maternal and fetal indications for transfer, referring institution characteristics, utilization of tertiary level services, and cost of transfer. There were 273 transfers from 53 referring hospitals ranging in distance from <20 miles (n = 102) to >100 miles (n = 41). Thirty-one patients were transferred by air (average cost $7656), 238 by ground (average cost $920), 4 by private car. The referring diagnosis was preterm premature rupture of membranes (PPROM) (n = 80), preterm labor (n = 76), preeclampsia (n = 42), medical complications (n = 25), or other (n = 50). Mean gestational age (GA) at transfer was 28.5+/-5.5 weeks. Patients were referred from hospitals with a self-designated nursery level I (n = 115), II (n = 111), III (n = 45), or none (n = 2). In 42 patients, (15%) no maternal or fetal indication for hospital transfer was identified after evaluation at the tertiary center. The most common referring misdiagnoses were preterm labor (n = 25), PPROM (n = 10) and preeclampsia (n = 3). One hundred and sixty-five patients delivered during transfer admission (mean GA = 29.6+/-4.8 weeks); 79 infants (48%) required admission to a level III, and 52 (31%) to a level II nursery. Most patients require the services of a tertiary facility after maternal fetal transfer. If delivered during transfer admission, the majority of neonates require care in an intermediate or intensive care nursery.     

Liver disease and compensatory growth: unexpected lessons from genetically altered mice

Over the last decade, several animal models have been established that permit exploration of liver biology and disease. Although these models have been developed using diverse strategies, including transgene targeting, homozygous gene disruption and administration of hepatotoxic chemicals, each approach creates an animal with hepatocyte damage, resulting in an hepatic microenvironment that supports proliferation of healthy hepatocytes. These models have been used to demonstrate: (1) the remarkable ability of adult hepatocytes to clonally proliferate in response to liver growth signals, (2) the effectiveness of transplanted donor hepatocytes in repopulating damaged liver parenchyma, and (3) the feasibility of reconstituting liver with xenogeneic hepatocytes. This paper reviews the development and use of these models, and outlines their potential future application to the study of hepatic stem cells, therapy of liver disease and hepatic toxicology.     

Common urological problems in women

BACKGROUND: Significant advances in the management of some common urinary problems have occurred in recent years. Problems that are likely to present to the general practitioner include urinary incontinence, voiding dysfunction, recurrent bacterial cystitis and interstitial cystitis. OBJECTIVE: The aim of this article is to highlight the advances that have occurred in the management of these common problems and their management strategy in the primary care setting. DISCUSSION: The management of incontinence, particularly stress urinary incontinence includes an accurate diagnosis and treatment tailored to the condition identified. Urinary incontinence due to the overactive and/or underactive bladder is more difficult to cure but significant improvement can be achieved in the majority of people. While severe incontinence requires an accurate diagnosis, non-operative strategies without a urodynamic diagnosis can be used in the treatment of milder forms. It is important to obtain an accurate diagnosis when the patient is not improving with conservative strategies. The problem of recurrent bacterial infections is common in women of all ages but cure should be possible in the majority of affected women. While the enigmatic entity of interstitial cystitis is increasingly recognised in out patients the exact basis for this condition remains obscure. Symptomatic improvement is achievable to a variable degree in most patients.     

TFF1 gene expression in human medullary thyroid carcinoma

This epidemiological study investigated the reasons why children in Northern Ireland who need orthodontic treatment do not receive treatment even when it is provided free by the state. A total of 1584 15- and 16-year-olds were examined in 23 high schools with the Index of Orthodontic Treatment Need. The characteristics of the adolescents who had received orthodontic treatment were compared with those who had a definite need for treatment and yet did not receive treatment or advice. One in 10 of the adolescents examined had an unmet need for orthodontic treatment. Logistic regression analysis was used to assess the influence of 11 variables including socioeconomic status, religion, and standard of dental health on the uptake of orthodontic care. This analysis revealed that the only significant predictors of whether an adolescent received orthodontic treatment was the dental attendance pattern of the adolescent, the adolescent's dental health, and the dental attendance pattern of the adolescent's mother. Those adolescents who had good dental health, who regularly attended a dentist, and whose mother regularly attended a dentist were more likely to receive orthodontic treatment.     

Validation of the in vivo intravascular ultrasound measurement of in-stent neointimal hyperplasia volumes

OBJECTIVES: This study was undertaken to validate the in vivo intravascular ultrasound (IVUS) measurement of in-stent neointimal hyperplasia (IH) volumes. BACKGROUND: Because stents reduce restenosis compared to balloon angioplasty, stent use has increased significantly. As a result, in-stent restenosis is now an important clinical problem. Serial IVUS studies have shown that in-stent restenosis is secondary to intimal hyperplasia. To evaluate strategies to reduce in-stent restenosis, accurate measurement of in-stent neointimal tissue is important. METHODS: Using a porcine coronary artery model of in-stent restenosis, single Palmaz-Schatz stents were implanted into 16 animals with a stent:artery ratio of 1.3:1. Intravascular ultrasound imaging was performed at 1 month, immediately prior to animal sacrifice. In vivo IVUS and ex vivo histomorphometric measurements included stent, lumen and IH areas; IH volumes were calculated with Simpson's rule. RESULTS: Intravascular ultrasound measurements of IH (30.4+/-11.0 mm3) volumes correlated strongly with histomorphometric measurements (26.7+/-8.5 mm3, r=0.965, p < 0.0001). The difference between the IVUS and the histomorphometric measurements of IVUS volume was 4.1+/-2.7 mm3 or 15.8+/-11% (standard error of the estimate=0.7). Both histomorphometry and IVUS showed that IH was concentric and uniformly distributed over the length of the stent. Intravascular ultrasound detected neointimal thickening of < or =0.2 mm in 5 of 16 stents. Sample size calculations based on the IVUS measurement of IH volumes showed that 12 stented lesions/arm would be required to show a 50% reduction in IVUS-measured IH volume and 44 stented lesions/arm would be required to show a 25% reduction in IH volume. CONCLUSION: In vivo IVUS measurement of IH volumes correlated strongly with ex vivo histomorphometry. Using volumetric IVUS end points, small sample sizes should be necessary to demonstrate effectiveness of strategies to reduce in-stent restenosis.     

Corticomotoneuronal contribution to the fractionation of muscle activity during precision grip in the monkey

1. During independent finger movements, the intrinsic muscles of the hand show a fractionated pattern of activity in which the timing and amplitude of electromyographic (EMG) activity varies considerably from one muscle to another. It has been suggested that, in the macaque monkey, corticomotoneuronal (CM) cells that produce postspike facilitation (PSF) of EMG in these muscles contribute to this fractionation. To test this hypothesis, we have investigated the relationship between the pattern of PSF exerted by a CM cell and the pattern of activity shown by the cell and by its target muscles. 2. The activity of 15 identified CM cells was recorded from two monkeys that performed a precision grip task. Spike-triggered averaging of rectified EMG during the hold period of this task showed that each cell produced PSF in at least two intrinsic hand muscles. 3. Segments of data were selected from the initial movement period of the task in which the EMG activity in one target muscle was substantially greater than that of the other, and the mean firing rate of each CM cell was determined for these periods. 4. CM cells showed bursts of activity in the movement period. Most of them (13/15) had a significantly (P < 0.001) higher firing rate when one of its target muscles was more active than the other. For nine of these cells (identified as set A), this muscle was the one receiving the larger PSF. In four cases (set B), the reverse was true. Two cells (set C), which produced PSF of equal size in their target muscles, showed no change in firing rate across the periods of fractionated EMG activity. 5. All set A and set B cells fired at significantly (P < 0.001) higher rates during the movement period, in association with fractionation of EMG activity, than in the hold period, in which a cocontracted pattern of muscle activity was observed. 6. There were pronounced differences in the strength of PSF exerted by the CM cells on their target muscles during the fractionation periods. One CM cell exerted PSF of a muscle during one period of fractionation, but postspike suppression of the same muscle during the other period. 7. It is suggested that changes in the firing rate of a CM cell and in the degree of facilitation it exerts could both contribute to the fractionation of activity in its target muscles. Cells of set A appear to be specifically recruited in a manner that directly reflects the pattern of facilitation they exert on the sampled target muscles. These results may explain why the CM system is so important for the performance of relatively independent finger movements.