Secret life of cytochrome bc1.
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Animals↗
Biomedical subjects
J L Smith
Publications and source records attributed to J L Smith.
Adaptation of an enzyme to regulatory function: structure of Bacillus subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phosphoribosyltransferase.
BACKGROUND: The expression of pyrimidine nucleotide biosynthetic (pyr) genes in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR attenuation protein binds to specific sites in pyr mRNA, allowing the formation of downstream terminator structures. UMP and 5-phosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co-regulators with PyrR. The smallest RNA shown to bind tightly to PyrR is a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransferase (UPRTase), although the relationship between enzymatic activity and RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis. Elucidating the role of PyrR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuation. RESULTS: The 1.6 A crystal structure of B. subtilis PyrR has been determined by multiwavelength anomalous diffraction, using a Sm co-crystal. As expected, the structure of PyrR is homologous to those proteins of the large type I PRTase structural family; it is most similar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase). The PyrR dimer differs from other PRTase dimers, suggesting it may have evolved specifically for RNA binding. A large, basic, surface at the dimer interface is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the hood subdomain. These models of RNA and UMP binding are consistent with biological data. CONCLUSIONS: The B. subtilis protein PyrR has adapted the substrate- and product-binding capacities of a PRTase, probably an HGXPRTase, producing a new regulatory function in which the substrate and product are co-regulators of transcription termination. The structure is consistent with the idea that PyrR regulatory function is independent of catalytic activity, which is likely to be extremely low under physiological conditions.
Amino Acid Sequence↗
Enzymes utilizing glutamine as an amide donor.
Amide nitrogen from glutamine is a major source of nitrogen atoms incorporated biosynthetically into other amino acids, purine and pyrimidine bases, amino-sugars, and coenzymes. A family comprised of at least sixteen amidotransferases are known to catalyze amide nitrogen transfer from glutamine to their acceptor substrates. Recent fine structural advances, largely as a result of X-ray crystallography, now provide structure-based mechanisms that help to explain fundamental aspects of the catalytic and regulatory interactions of several of these aminotransferases. This chapter provides an overview of this recent progress made on the characterization of amidotransferase structure and mechanism.
Allosteric Regulation↗
Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli.
Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.
Adenosine Monophosphate↗
Getting organized: new insights into the organizer of higher vertebrates.
The Organizer of higher vertebrates (e.g., Hensen's node in birds and the node in mammals) functions much like the Organizer of lower vertebrates (e.g., embryonic shield in fish and dorsal lip of the blastopore in amphibians). In all classes of vertebrates, the Organizer displays a number of unique properties including the fate, migratory patterns, morphogenetic movements, and the level of commitment of its cells; its pattern of gene expression; its ability to induce neural differentiation; and its ability to organize and regionalize a secondary embryo when grafted ectopically. The importance of Organizer activity to formation of the neuraxis is highlighted by results from studies in which the Organizer is eliminated experimentally. Such studies demonstrate that an auxiliary system is present that can generate a reconstituted Organizer, which completely mimics the activity of the original Organizer. For almost 50 years after the discovery of Spemann's Organizer, the molecular nature of Organizer activity was virtually unknown. However, recent progress in identifying the morphoregulatory molecules underlying Organizer activity has been substantial, and a full understanding of the molecular basis of this activity is imminent. Thus, the intriguing question of how the Organizer organizes, raised by the seminal experiments of Spemann and Mangold, is finally being answered in this exciting renaissance of developmental biology driven by new molecular and genetic approaches.
Animals↗
Time domain analysis of embolic signals can be used in place of high-resolution Wigner analysis when classifying gaseous and particulate emboli.
The sample-volume length (SVL) of an embolic signal has previously been used to differentiate between gaseous and particulate emboli and has been calculated using high-resolution Wigner analysis. Although successful, this method of analysis is not widely available to other groups using transcranial Doppler ultrasound (TCD) to classify emboli. The SVL of embolic signals can also be calculated using time domain analysis, which is a far simpler method and potentially available to all TCD users. The aim of this study was to compare the SVL of embolic signals calculated using Wigner analysis and time domain analysis to assess whether or not time domain analysis can replace Wigner analysis to classify emboli. In total, 215 particulate and 100 gaseous emboli were recorded onto digital audiotape and analysed off-line. Two SVLs for each embolic signal were calculated by measuring embolic duration and velocity in the time domain and with Wigner analysis. Receiver operator characteristic (ROC) curves were plotted to assess the optimum SVL threshold for each method, and levels of sensitivity and specificity were defined. The optimum SVL threshold using Wigner analysis was 1.28 cm, yielding 93% sensitivity and 97% specificity. Using time domain analysis, the optimum threshold was 1.12 cm, yielding 90% sensitivity and 96% specificity. The methods were compared statistically (chi2) using their optimum thresholds, and were found not to be statistically different for classifying particles p=0.283) or gaseous emboli (p=0.700). This study has shown that the SVL of embolic signals, used to differentiate particulate from gaseous emboli, can be calculated more simply in the time domain, which yields as accurate results as calculating the SVL using Wigner analysis.
Blood Flow Velocity↗
Glutamine PRPP amidotransferase: snapshots of an enzyme in action.
Recent studies of glutamine PRPP amidotransferase have provided a new understanding of the function and mechanism of this rather complicated enzyme that may be a paradigm for other complex enzymes. New insights have been gained into the mechanisms of catalysis in the active sites of the two half-reactions, catalytic coupling, allosteric control by feedback inhibitors and the channeling of reaction and metabolic intermediates.
Allosteric Regulation↗
The effect of perioperative embolisation on visual function in patients undergoing carotid endarterectomy.
OBJECTIVES: To investigate the effect of carotid artery embolisation detected by transcranial Doppler (TCD) monitoring during carotid endarterectomy (CEA) on visual function. DESIGN: A prospective study. Visual function was assessed by an independent ophthalmalogist. MATERIALS: One hundred consecutive patients undergoing carotid endarterectomy. METHODS: All patients underwent pre- and postoperative retinal fundoscopy and automated visual field analysis. Intraoperative emboli were detected by continuous TCD monitoring of the middle cerebral artery. RESULTS: Preoperatively, six patients had fundoscopic evidence of silent retinal embolisation, five patients had visible emboli associated with amaurosis fugax. Visual field analysis found no significant difference between ipsi- and contralateral eye scores. Only patients with occlusions of major retinal vessels had clinically reduced visual function. Intraoperatively TCD identified embolisation in 83/91 of monitored operations. Postoperatively, ipsilateral visual field scores deteriorated while contralateral eye scores remained unchanged, however only one new retinal embolus was detected. The incidence of TCD detected embolisation was not associated with a corresponding deterioration in postoperative visual function. CONCLUSIONS: (1) A group of patients can be identified who experience "silent" retinal embolisation prior to CEA. (2) Clinically significant retinal embolisation resulting from CEA is uncommon. (3) CEA is associated with a deterioration in the visual field scores for the ipsilateral eye compared with the contralateral eye but the mechanism for this small but significant deterioration remains unclear.
Blindness↗
Experience with transcranial Doppler monitoring reduces the incidence of particulate embolization during carotid endarterectomy.
BACKGROUND: The aim of this study was to investigate whether the introduction of routine transcranial Doppler (TCD) ultrasonography during carotid endarterectomy reduces the incidence of microembolization by altering operative technique. METHODS: The number and nature of microemboli detected during the first 75 consecutive carotid endarterectomies performed with TCD monitoring during 1992-1993 (group 1) were compared with those in a similar series of 75 consecutive patients undergoing carotid endarterectomy in 1995 (group 2), after substantial experience (210 patients) with TCD monitoring. Emboli were classified as either particulate or gaseous. RESULTS: In patients with evidence of particulate emboli during the dissection phase of the operation, the total number of particulate emboli fell significantly in patients in group 2 (P = 0.019). Similarly, in patients in whom microembolization was detected on immediate opening of the shunt, the total number of microemboli also fell significantly in group 2 (P = 0.003). Overall, the median (95 per cent confidence interval) number of particulate emboli detected during the entire procedure fell significantly from 21 (16-29) in group 1 to 9 (7-14) in group 2 (P = 0.0008). CONCLUSIONS: TCD monitoring plays an important role in the training and quality control of carotid endarterectomy and helps significantly to reduce the amount of microembolization.
Aged↗
T cell receptor (TCR) Vbeta gene usage in bronchoalveolar lavage and peripheral blood T cells from asthmatic and normal subjects.
T cells are thought to play an important regulatory role in asthma, but little is known about the T cell repertoire of the human lung or whether asthma is associated with any specific repertoire changes. Flow cytometry and MoAbs to TCR VB (TCRBV) families were used to quantify bronchoalveolar lavage (BAL) and blood T cells from normal and atopic individuals. Clonality was then assessed by polymerase chain reaction (PCR) amplification of cDNA and gene scanning using consensus and family-specific TCRBV primers and confirmed by sequence analysis. In addition, blood and BAL T cell populations were studied pre- and post-allergen challenge in four patients with allergic asthma. The majority of TCRBV families detected in blood by MoAb staining were also represented in BAL. While differences between BAL and blood populations were evident in each individual studied, these differences were not consistent between individuals or between CD4+ and CD8+ T cell subpopulations. These results are in broad agreement with other published studies, but in contrast to previous work we found a consistent difference between TCRBV7 family usage in blood and BAL in all individuals studied, and a consistently increased proportion of CD4+ BAL T cells bearing BV5S2/3 in asthmatics only. After allergen challenge, the pattern of TCRBV gene usage was largely unchanged as judged by flow cytometry. Gene scanning of PCR products generated from consensus VB primers revealed polyclonal lymphocyte populations in blood and BAL from all seven atopic individuals: in one normal tested polyclonal populations were found in blood and oligoclonal populations in BAL. Selected families amplified with family-specific primers BV5S2/3, BV6 and BV7 (chosen because of their predominance in BAL compared with blood) were more variable and revealed predominant polyclonal populations in blood and polyclonal or oligoclonal populations in BAL. In one asthmatic patient a clonal BV5S2 family was found in BAL. Following allergen challenge there were no significant changes in polyclonality/oligoclonality/clonality in three cases, but in one case a clonal BV5S2 population was found after challenge, that had not been evident beforehand. The lung T cell repertoire is thus broadly representative of blood T cells, but shows population differences that may result from response to persistent exposure to airborne antigens common to normal and atopic individuals. Oligoclonal TCRBV family expansion appears to be primarily lung-specific but independent of atopic asthma, although our challenge data in one case support the concept that clonal populations may follow local allergen challenge. These data are consistent with selection and amplification of specific T cell families in the lung in response to local antigenic exposure.
Adult↗
Objective measurement of mood change induced by contemporary music.
A myriad of previous studies from a variety of disciplines has shown several effects of music on mind and body. This study investigated the relationship between different categories of contemporary music (n = 6) and the mood states of a group of students (n = 12), using the Profile of Mood States (POMS), to measure mood before and after exposure to these different pieces of music. When analysed together, all six pieces of music produced an overall change in mood (P = 0.008) as measured by 2-way repeated measures analysis of variance (ANOVA). When each category was examined individually, four categories of music produced highly significant changes in mood: the tense category (score -4.0 +/- 1.8 POMS Units; P < 0.001); depressed category (+0.5 +/- 0.2; P < 0.001); angry category (+0.9 +/- 1.6; P < 0.03); and the all moods category (1.6 +/- 0.3; P < 0.04). One piece of dance music produced changes in all mood categories, giving the largest positive mean mood change. By contrast, the popular/independent music, associated with the tense category, produced the largest negative mean mood change. The five POMS mood states were analysed separately for each piece of music. These findings are consistent with previous work. In addition, the finding of the effects of specific music categories on mood may have important implications for therapy in mental health and mental health nursing.
Adult↗
Estrogen profiles in postmenopausal African-American women in a wheat bran fiber intervention study.
High dietary fiber intake has been hypothesized to lower blood estrogen concentrations, an effect thought to be beneficial for decreasing breast cancer risk. This study investigated the association between dietary supplementation of wheat bran and circulating estrogen levels in postmenopausal African-American women participating in a community intervention trial. Seventeen postmenopausal women (aged 63 +/- 1.6 yr) participated in the study. Nutritional status was assessed and blood and 24-hour urine samples were collected before and after five to six weeks of daily supplementation of the diet with 35 g of wheat bran cereal (11.6 g insoluble dietary fiber) marked with 28 mg of riboflavin. Riboflavin confirmed that all postmenopausal participants adhered to the intervention protocol. Nine of the 17 postmenopausal women were taking some form of estrogen replacement therapy (PM-ERT). Baseline hormone levels in the PM-ERT group did not significantly change after the dietary intervention. Estradiol (96.8 +/- 20.3 vs. 113.8 +/- 23.3 pg/ml), androstenedione (0.47 +/- 0.06 vs. 0.45 +/- 0.06 ng/ml), and sex hormone-binding globulin (SHBG, 107 +/- 13.5 vs. 106.6 +/- 13.3 nmol/l) levels remained constant. In the eight postmenopausal women who were not receiving exogenous hormones (PM), wheat bran consumption was not associated with predicted decreased levels of estradiol (25.7 +/- 2.7 vs. 31.0 +/- 1.9 pg/ml), estrone (38.3 +/- 10.1 vs. 39.3 +/- 10.6 pg/ml), and androstenedione (0.78 +/- 0.08 vs. 0.68 +/- 0.11 ng/ml) or with increased concentrations of SHBG (35.2 +/- 6.4 vs. 34.8 +/- 6.5 nmol/l). Participants receiving ERT had baseline and postintervention levels of estradiol and SHBG significantly higher and androstenedione significantly lower than those not receiving ERT. No association between wheat bran supplementation and hormone levels was found in PM or PM-ERT African-American participants. These results in postmenopausal women are in contrast to findings of earlier studies in premenopausal women indicating that wheat bran fiber decreases serum sex hormones. Estrogen levels in postmenopausal women are only 5-10% of those in premenopausal women; therefore, a high wheat bran fiber diet alone may not be sufficient to depress these low levels even further.
Aged↗
Transcranial Doppler detected cerebral microembolism following carotid endarterectomy.
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Carotid Arteries↗
Forms of forward quadrupedal locomotion. II. A comparison of posture, hindlimb kinematics, and motor patterns for upslope and level walking.
To gain insight into the neural mechanisms controlling different forms of quadrupedal walking of normal cats, data on postural orientation, hindlimb kinematics, and motor patterns of selected hindlimb muscles were assessed for four grades of upslope walking, from 25 to 100% (45 degrees incline), and compared with similar data for level treadmill walking (0.6 m/s). Kinematic data for the hip, knee, ankle, and metatarsophalangeal joints were obtained from digitizing ciné film that was synchronized with electromyographic (EMG) records from 13 different hindlimb muscles. Cycle periods, the structure of the step cycle, and paw-contact sequences were similar at all grades and typical of lateral-sequence walking. Also, a few half-bound and transverse gallop steps were assessed from trials at the 100% grade; these steps had shorter cycle periods than the walking steps and less of the cycle (68 vs. 56%) was devoted to stance. Each cat assumed a crouched posture at the steeper grades of upslope walking and stride length decreased, whereas the overall position of the stride shifted caudally with respect to the hip joint. At the steeper grades, the range and duration of swing-related flexion increased at all joints, the stance-phase yield was absent at the knee and ankle joints, and the range of stance-phase extension at knee and ankle joints increased. Patterns of muscle activity for upslope and level walking were similar with some notable exceptions. At the steeper grades, the EMG activity of muscles with swing-related activity, such as the digit flexor muscle, the flexor digitorum longus (FDL), and the knee flexor muscle, the semitendinosus (ST), was prolonged and continued well into midswing. The EMG activity of stance-related muscles also increased in amplitude with grade, and three muscles not active during the stance phase of level walking had stance activity that increased in amplitude and duration at the steepest grades; these muscles were the ST, FDL, and extensor digitorum brevis. Overall the changes in posture, hindlimb kinematics, and the activity patterns of hindlimb muscles during upslope walking reflected the need to continually move the body mass forward and upward during stance and to ensure that the paw cleared the inclined slope during swing. The implications of these changes for the neural control of walking and expected changes in hindlimb kinetics for slope walking are discussed.
Animals↗
Forms of forward quadrupedal locomotion. III. A comparison of posture, hindlimb kinematics, and motor patterns for downslope and level walking.
To gain further insight into the neural mechanisms for different forms of quadrupedal walking, data on postural orientation, hindlimb kinematics, and motor patterns were assessed for four grades of downslope walking, from 25% (14 degrees slope) to 100% (45 degrees), and compared with data from level and downslope walking at five grades (5-25%) on the treadmill (0.6 m/s). Kinematic data were obtained by digitizing ciné film, and electromyograms (EMGs) synchronized with kinematic records were taken from 13 different hindlimb muscles. At grades from 25 to 75%, cycle periods were similar, but at the steepest grade the cycle was shorter because of a reduced stance phase. Paw-contact sequences at all grades were consistent with lateral-sequence walking, but pace walking often occurred at the steepest grades. The cats crouched at the steeper grades, and crouching was associated with changes in fore- and hindlimb orientation that were consistent with increasing braking forces and decreasing propulsive forces during stance. The average ranges of motion at the hindlimb joints, except at the hip, were often different at the two steepest slopes. During swing, the range of knee- and ankle-joint flexion decreased, and the range and duration of extension increased at the ankle joint to lower the paw downward for contact. During stance the range of flexion during yield increased at the ankle joint, and the range of extension decreased at the knee and metatarsophalangeal joints. Downslope walking was also associated with EMG changes for several muscles. The hip extensors were not active during stance; instead, hip flexors were active, presumably to slow the rate of hip extension. Although ankle extensors were active during stance, their burst durations were truncated and centered around paw contact. Ankle flexors were active after midstance at the steeper slopes before the need to initiate swing, whereas flexor and extensor digit muscles were coactive throughout stance. Overall the changes in posture, hindlimb kinematics, and activity patterns of hindlimb muscles during stance reflected a need to counteract external forces that would accelerate angular displacements at some joints. Implications of these changes are discussed by using current models for the neural control of walking.
Animals↗
A comparison of four methods for distinguishing Doppler signals from gaseous and particulate emboli.
BACKGROUND AND PURPOSE: Many reports in the medical literature have proposed methods of differentiating between gaseous and particulate emboli detected with the use of transcranial Doppler ultrasound. The purpose of this study was to compare the previously published methods with our own sample volume length (SVL) parameter to assess the accuracy of each method in classifying emboli. METHODS: A pure source of gaseous and particulate emboli was obtained from in vitro and in vivo studies, respectively, and recorded onto digital audiotape for off-line analysis. In total, 100 gaseous emboli and 215 particulate emboli were analyzed to measure four embolic parameters, namely, embolic duration, embolic velocity, relative signal intensity increase (measured embolic power [MEP]), and SVL of the embolic signal (= Duration x Velocity). Receiver operator characteristic analysis was used to assess the optimum threshold for each parameter to differentiate between particulate and gaseous emboli, and levels of sensitivity and specificity were calculated. RESULTS: Embolic duration and velocity produced the poorest levels of sensitivity and specificity compared with the MEP and SVL parameters. The optimum thresholds for embolic duration and velocity were 35 ms and 1 m/s, respectively, which produced a sensitivity (specificity) of 85.1% (87%) and 87% (67%), respectively. The optimum MEP and SVL thresholds were 30 dB and 12.8 mm, respectively, which produced a sensitivity (specificity) of 86.5% (95%) and 93% (97%), respectively. The SVL and MEP parameters were compared statistically (chi 2) at chosen specificity values of 90%, 95%, 97%, 99%, and 100%, which showed that the SVL sensitivities were statistically greater than MEP sensitivities (P < 0.01). CONCLUSIONS: SVL is the best parameter for differentiating between gaseous and particulate emboli but needs to be calculated with the use of a high-temporal-resolution spectral analyzer to measure embolic duration and velocity.
Diagnosis, Differential↗
Carcinoma of the gallbladder.
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Carcinoma↗
Foodborne illness in the elderly.
The elderly (> or = 65 years of age) are more susceptible to morbidity and mortality from foodborne-induced gastroenteritis than younger individuals. Several factors contribute to the increased susceptibility to foodborne infections as well as other infections in elderly populations. These include an age-associated decrease in humoral and cellular immunity, age-related changes in the gastrointestinal tract (decreased production of gastric acid and decreased intestinal motility), malnutrition, lack of exercise, entry into nursing homes, and excessive use of antibiotics. Data from foodborne outbreaks associated with nursing homes indicate that the elderly are more likely to die from foodborne Campylobacter, Clostridium perfringens, Escherichia coli O157:H7, Salmonella, and Staphylococcus aureus infections than the general population. Infections by Salmonella species are the most common cause of illness and death in nursing homes with Salmonella enteritidis as the major cause of both morbidity and mortality. While it is impossible to turn back the clock, practicing a healthy life-style with regular exercise, maintaining a balanced diet, receiving regular health care, paying attention to personal hygiene, and monitoring food preparation and handling should lead to a reduced incidence of foodborne and other infections in the elderly.
Aged↗