Biomedical subjects
Publications and source records attributed to Jennifer L Martin.
Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABCDEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.
Sparse-matrix sampling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 diverse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.
STUDY OBJECTIVES: To examine the validity of self-reported survey estimates of sleep patterns in adolescents through a comparison of retrospective survey descriptions of usual school- and weekend-night sleep habits with diary-reported sleep patterns and actigraphically estimated sleep behaviors over a subsequent week. DESIGN AND SETTING: High school students completed a Sleep Habits Survey about the previous 2 weeks and then wore an actigraph (AMI, Ardsley, NY) for 8 days while keeping a daily sleep diary. Matched-pair t tests assessed average differences between survey and diary reports and between survey and actigraph estimates. Pearson correlations assessed the extent to which survey reports were in agreement with diary reports and actigraphy estimates. PARTICIPANTS: 302 high school students (196 girls, 106 boys) in grades 9-12 from five high schools. RESULTS: School-night survey total sleep times and wake times did not differ from sleep amounts reported in the diary or estimated by actigraphy; survey bedtimes were slightly earlier. On weekends, survey total sleep times and wake times were longer and later, respectively, than estimated with actigraphy and reported on diaries. Moreover, school- and weekend-night survey variables were significantly correlated both with diary and actigraphy variables. Strengths of the associations were consistently greater for school-night variables than the corresponding weekend-night variables. CONCLUSIONS: The findings support the validity of the Sleep Habits Survey estimates in comparison with diary and actigraphy. Strengths and limitations for survey measures of high school students' usual sleep/wake patterns are discussed.
Few reported inhibitors of secretory phospholipase A(2) enzymes truly inhibit the IIa human isoform (hnpsPLA(2)-IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D-tyrosine was derivatised to give a series of potent new inhibitors of hnpsPLA(2)-IIa. A 2.2-A crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca(2+) ion through carboxylate and amide oxygen atoms, H-bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T-shaped aromatic-group-His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.
Diffraction quality crystals are essential for crystallographic studies of protein structure, and the production of poorly diffracting crystals is often regarded as a dead end in the process. Here we show a dramatic improvement of poorly diffracting DsbG crystals allowing high-resolution diffraction data measurement. Before dehydration, the crystals are fragile and the diffraction pattern is streaky, extending to 10 A resolution. After dehydration, there is a spectacular improvement, with the diffraction pattern extending to 2 A resolution. This and other recent results show that dehydration is a simple, rapid, and inexpensive approach to convert poor quality crystals into diffraction quality crystals.
Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.
Sleep in the nursing home environment is extremely fragmented, possibly in part as a result of decreased light exposure. This study examined the effect of light on sleep and circadian activity rhythms in patients with probable or possible Alzheimer's disease. Results showed that both morning and evening bright light resulted in more consolidated sleep at night, as measured with wrist actigraphy. Evening light also increased the quality of the circadian activity rhythm, as measured by a 5-parameter extended cosine model (amplitude, acrophase, nadir, slope of the curve, and relative width of the peak and trough). Increasing light exposure throughout the day and evening is likely to have the most beneficial effect on sleep and on circadian rhythms in patients with dementia. It would behoove nursing homes to consider increasing ambient light in multipurpose rooms where patients often spend much of their days.
Sulfonation catalyzed by sulfotransferase enzymes plays an important role in chemical defense mechanisms against various xenobiotics but also bioactivates carcinogens. A major human sulfotransferase, SULT1A1, metabolizes and/or bioactivates many endogenous compounds and is implicated in a range of cancers because of its ability to modify diverse promutagen and procarcinogen xenobiotics. The crystal structure of human SULT1A1 reported here is the first sulfotransferase structure complexed with a xenobiotic substrate. An unexpected finding is that the enzyme accommodates not one but two molecules of the xenobiotic model substrate p-nitrophenol in the active site. This result is supported by kinetic data for SULT1A1 that show substrate inhibition for this small xenobiotic. The extended active site of SULT1A1 is consistent with binding of diiodothyronine but cannot easily accommodate beta-estradiol, although both are known substrates. This observation, together with evidence for a disorder-order transition in SULT1A1, suggests that the active site is flexible and can adapt its architecture to accept diverse hydrophobic substrates with varying sizes, shapes and flexibility. Thus the crystal structure of SULT1A1 provides the molecular basis for substrate inhibition and reveals the first clues as to how the enzyme sulfonates a wide variety of lipophilic compounds.
Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 A. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 A.
The S-adenosylmethionine-dependent methyltransferase enzymes share little sequence identity, but incorporate a highly conserved structural fold. Surprisingly, residues that bind the common cofactor are poorly conserved, although the binding site is localised to the same region of the fold. The substrate-binding region of the fold varies enormously. Over the past two years, there has been a significant increase in the number of structures that are known to incorporate this fold, including several uncharacterized proteins and two proteins that lack methyltransferase activity.
CcmG is unlike other periplasmic thioredoxin (TRX)-like proteins in that it has a specific reducing activity in an oxidizing environment and a high fidelity of interaction. These two unusual properties are required for its role in c-type cytochrome maturation. The crystal structure of CcmG reveals a modified TRX fold with an unusually acidic active site and a groove formed from two inserts in the fold. Deletion of one of the groove-forming inserts disrupts c-type cytochrome formation. Two unique structural features of CcmG-an acidic active site and an adjacent groove-appear to be necessary to convert an indiscriminately binding scaffold, the TRX fold, into a highly specific redox protein.
An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design.
OBJECTIVES: To determine whether fragmented sleep in nursing home patients would improve with increased exposure to bright light. DESIGN: Randomized controlled trial. SETTING: Two San Diego-area nursing homes. PARTICIPANTS: Seventy-seven (58 women, 19 men) nursing home residents participated. Mean age +/- standard deviation was 85.7 +/- 7.3 (range 60-100) and mean Mini-Mental State Examination was 12.8 +/- 8.8 (range 0-30). INTERVENTIONS: Participants were assigned to one of four treatments: evening bright light, morning bright light, daytime sleep restriction, or evening dim red light. MEASUREMENTS: Improvement in nighttime sleep quality, daytime alertness, and circadian activity rhythm parameters. RESULTS: There were no improvements in nighttime sleep or daytime alertness in any of the treatment groups. Morning bright light delayed the peak of the activity rhythm (acrophase) and increased the mean activity level (mesor). In addition, subjects in the morning bright light group had improved activity rhythmicity during the 10 days of treatment. CONCLUSION: Increasing exposure to morning bright light delayed the acrophase of the activity rhythm and made the circadian rhythm more robust. These changes have the potential to be clinically beneficial because it may be easier to provide nursing care to patients whose circadian activity patterns are more socially acceptable.
Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.
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A number of non-pharmacologic interventions have been developed and studied for the treatment of primary insomnia in adults. Fifty-four non-pharmacological intervention studies published over the last 20 years were reviewed to determine how primary insomnia was assessed and to characterize patients participating in the studies. The main objectives were to inform clinicians who look to the literature for information on the treatment of insomnia about selection and diagnosis of participants, and to examine differences between clinic-based and research-based studies. Mean age for all patients was 47 years; 65% were women. Studies typically used interview procedures to assess participants. The most common exclusion criteria overall was insomnia secondary to medical illness or psychiatric disorder. Methods for assessing these criteria varied widely across studies. Inclusion most commonly required a 6-month duration of insomnia, and sleep difficulty at least 3 nights per week. There were significant differences between clinic- and research-based studies in the screening assessments, exclusion criteria, and participant drop-out rate. The heterogeneity in assessment and diagnosis complicates comparison across studies. Some studies were more likely to include severe insomnia sufferers and/or individuals with secondary insomnia. Comprehensive assessment and complete reporting of research methods is essential to determining the relative efficacy and clinical utility of interventions. Clinicians should be aware of the differences across studies from research vs. clinical settings. To enhance the generalizability of research-based studies to clinical practice, careful screening and description of study participants is essential. When participants are well-described, research-based studies can be useful and informative to clinicians.
OBJECTIVE: Preliminary data suggest that morning bright light might improve symptoms of agitation, a serious problem in patients with dementia. The authors expand on an earlier pilot study by evaluating the effect of bright light therapy on agitated behavior in a large sample of patients with severe dementia. METHODS: Ninety-two patients were randomly assigned to morning bright light, morning dim red light, or evening bright light. Agitation was rated by research staff who observed the patients every 15 minutes throughout the treatment period and by caregivers at one time-point before and one time-point after treatment. RESULTS: Morning bright light delayed the acrophase of the agitation rhythm by over 1.5 hours. Bright light was associated with improved caregivers' ratings but had little effect on observational ratings of agitation. CONCLUSION: Although the result that light shifted the peak of the agitated behavior might be generalizable to patients with milder forms of AD, the fact that agitation was not ameliorated might not be. Because the suprachiasmatic nucleus (SCN) of patients with severe AD is likely to be more degenerated, and the circadian activity rhythms deteriorate as the disease progresses, it is still possible that patients with more intact SCNs, that is, patients with mild or moderate AD, might benefit from light treatment even more than those with severe AD.
OBJECTIVE: The authors examined the relationship between sleep-disordered breathing (SDB) and agitation in patients with Alzheimer disease (AD). METHODS: Thirty-eight AD patients (29 women, 9 men) in nursing homes in San Diego, CA participated. The mean age was 82.3 years, with a range of 61 to 95 years. Mean Mini-Mental State Exam score was 6.5 (range: 0-16). Sleep was recorded for one night, and agitation was measured with behavioral observations and ratings by nursing staff. RESULTS: SDB was very prevalent in this sample and was related to some types of agitation during the day but not in the evening or night. Aggressive Agitation on the Cohen-Mansfield Agitation Inventory and Manual Manipulation on the Agitated Behavior Rating Scale were greater with more severe SDB. Searching and wandering agitation decreased with more severe SDB. CONCLUSION: This study supports the hypothesis that SDB is related to agitation in AD, although the results are specific to certain types of agitation. Treatment of SDB may decrease agitation, easing the burden of caregiving and prolonging the time that patients are able to remain at home.