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Biomedical subjects

Jennifer L Martin

Publications and source records attributed to Jennifer L Martin.

At least 19 recordsLinked to original sources
PubMed2006

The sigmoidally transformed cosine curve: a mathematical model for circadian rhythms with symmetric non-sinusoidal shapes.

We introduce a family of non-linear transformations of the traditional cosine curve used in the modelling of biological rhythms. The non-linear transformation is the sigmoidal family, represented here by three family members: the Hill function, the anti-logistic function, and the arctangent function. These transforms add two additional parameters that must be estimated, in addition to the acrophase, MESOR, and amplitude (and period in some applications), but the estimated curves have shapes requiring many more than two additional harmonics to achieve the same fit when modelled by harmonic regression. Particular values of the additional parameters can yield rectangular waves, narrow pulses, wide pulses, and for rectangular waves (representing alternating 'on' and 'off' states) the times of onset and offset (hence duration, as when modelling the duration of the large night-time melatonin secretory epoch). We illustrate the sigmoidally transformed cosine curves, and compare them to harmonic regression modelling, in a sample of eight activity recordings made on patients in a nursing home.

Aged↗
PubMed2006

Surfactant protein D increases fusion of Mycobacterium tuberculosis-containing phagosomes with lysosomes in human macrophages.

Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst.

Animals↗
PubMed2006

Human cytosolic sulfotransferase SULT1A1.

Sulfonation is an important conjugation reaction required for a range of biological processes including phase II metabolism, whereby sulfo-conjugation renders a compound more hydrophilic to aid its excretion. The major enzyme responsible for xenobiotic sulfonation is the widely expressed cytosolic sulfotransferase SULT1A1. The SULT1A1 crystal structure has provided insights into this enzyme's substrate specificity and catalytic function, including its role in the sulfonation of endogenous substrates such as oestrogens. Contrary to its metabolic role, SULT1A1 can also bioactivate compounds; it is known to sulfonate pro-carcinogens such as hydroxymethyl polycyclic aromatic hydrocarbons leading to highly reactive intermediates capable of forming DNA adducts, potentially resulting in mutagenesis. Given the role of SULT1A1 in these diverse functions and the discovery of allelic variants with differing catalytic activities, this enzyme has been the focus of numerous polymorphic studies investigating the link between inter-individual SULT1A1 variance and the etiology of a variety of cancers.

Arylsulfotransferase↗
PubMed2006

Focusing in on structural genomics: the University of Queensland structural biology pipeline.

The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis.

Animals↗
PubMed2006

Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase.

3-Fluoromethyl-7-(N-substituted aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines (14, 16, and 18-22) are highly potent and selective inhibitors of phenylethanolamine N-methyltransferase (PNMT). Molecular modeling studies with 3-fluoromethyl-7-(N-alkyl aminosulfonyl)-1,2,3,4-tetrahydroisoquinolines, such as 16, suggested that the sulfonamide -NH- could form a hydrogen bond with the side chain of Lys57. However, SAR studies and analysis of the crystal structure of human PNMT (hPNMT) in complex with 7 indicated that the sulfonamide oxygens, and not the sulfonamide -NH-, formed favorable interactions with the enzyme. Thus, we hypothesized that replacement of the sulfonamide -NH- with a methylene group could result in compounds that would retain potency at PNMT and that would have increased lipophilicity, thus increasing the likelihood they will cross the blood brain barrier. A series of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines (23-30) were synthesized and evaluated for their PNMT inhibitory potency and affinity for the alpha2-adrenoceptor. A comparison of these compounds with their isosteric sulfonamides (14, 16, and 18-22) showed that the sulfones were more lipophilic but less potent than their corresponding sulfonamides. Sulfone 24 (hPNMT Ki = 1.3 microM) is the most potent compound in this series and is quite selective for PNMT versus the alpha2-adrenoceptor, but 24 is less potent than the corresponding sulfonamide, 16 (hPNMT Ki = 0.13 microM). We also report the crystal structure of hPNMT in complex with sulfonamide 15, from which a potential hydrogen bond acceptor within the hPNMT active site has been identified, the main chain carbonyl oxygen of Asn39. The interaction of this residue with the sulfonamide -NH- is likely responsible for much of the enhanced inhibitory potency of the sulfonamides versus the sulfones.

Animals↗
PubMed2006

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking.

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.

Amino Acid Sequence↗
PubMed2006

Disrupting life events and the sleep-wake cycle in depression.

BACKGROUND: Social rhythm disruption life events are significant predictors of mood relapse in bipolar patients. However, no research has examined the relationship between these events and their hypothesized mechanism of action: disrupted sleep-wake patterns. The goal of this study was to test whether participants with major depressive disorder have a greater disruption of daily sleep and motor activity following disrupting life events when compared to normal controls. METHOD: Over the course of 2 weeks, 39 normal controls and 39 individuals with major depressive disorder completed life events interviews and wore actigraphs to obtain estimates of sleep/wake activity. RESULTS: Statistically significant interactions indicated that the presence of at least one disrupting life event in the previous 4 months correlated with elevations in the amount of time spent awake after sleep onset [beta=0.45, deltaF(1,73)=4.80, p<0.05], and decreases in the percentage of time spent asleep [beta=-0.53, deltaF(1,73)=6.57, p<0.05], in depressed individuals but not in normal controls. CONCLUSIONS: The results indicated that depressed individuals may be more susceptible to the effects of life events on sleep than normal controls. This is the first study to date to correlate life events with objective measures of sleep. However, prospective longitudinal research is necessary to clarify the temporal relationship among these variables.

Adult↗
PubMed2006

Protein disulfide isomerase: the structure of oxidative folding.

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.

Animals↗
PubMed2006

Capturing user requirements in medical device development: the role of ergonomics.

Measuring and fulfilling user requirements during medical device development will result in successful products that improve patient safety, improve device effectiveness and reduce product recalls and modifications. Medical device users are an extremely heterogeneous group and for any one device the users may include patients and their carers as well as various healthcare professionals. There are a number of factors that make capturing user requirements for medical device development challenging including the ethical and research governance involved with studying users as well as the inevitable time and financial constraints. Most ergonomics research methods have been developed in response to such practical constraints and a number of these have potential for medical device development. Some are suitable for specific points in the device cycle such as contextual inquiry and ethnography. Others, such as usability tests and focus groups, may be used throughout development. When designing user research there are a number of factors that may affect the quality of data collected, including the sample of users studied, the use of proxies instead of real end-users and the context in which the research is performed. As different methods are effective in identifying different types of data, ideally more than one method should be used at each point in development. However, financial and time factors may often constrain this.

Equipment Design↗
PubMed2006

Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals.

Investigations into the roles of Ca(2+) and Zn(2+) in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca(2+) or Zn(2+) in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a probe for the targeted ion. Here, we investigated metal-ion responses by screening nominally Zn(2+)- or Ca(2+)-selective fluorophores in solutions containing various concentrations of Ca(2+), as a potential interferent for Zn(2+), or Zn(2+), as a potential interferent for Ca(2+). The results suggested that Zn(2+)-sensitive dyes were more specific for their targeted ion than dyes that targeted Ca(2+). Ca(2+)-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn(2+), even responding to Zn(2+) in the presence of high concentrations of Ca(2+). We demonstrate that these Ca(2+) indicators can effectively measure dynamic changes of cytosolic Zn(2+). Our results appeal for a new generation of Ca(2+) fluorophores that are more specific for Ca(2+) over Zn(2+). One implication of these results is that data obtained using Ca(2+)-sensitive dyes may need to be re-examined to determine if results previously attributed to Ca(2+) could, in part, be due to Zn(2+).

Animals↗
PubMed2006

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins.

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites.

Amino Acid Sequence↗
PubMed2006

A method for screening the temperature dependence of three-dimensional crystal formation.

Temperature is an important parameter controlling protein crystal growth. A new temperature-screening system (Thermo-screen) is described consisting of a gradient thermocycler fitted with a special crystallization-plate adapter onto which a 192-well sitting-drop crystallization plate can be mounted (temperature range 277-372 K; maximum temperature gradient 20 K; interval precision 0.3 K). The system allows 16 different conditions to be monitored simultaneously over a range of 12 temperatures and is well suited to conduct wide (approximately 20 K) and fine (approximately 3 K) temperature-optimization screens. It can potentially aid in the determination of temperature phase diagrams and run more complex temperature-cycling experiments for seeding and crystal growth.

Crystallization↗
PubMed2006

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase.

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 (unit-cell parameters a = b = 136.83, c = 99.82 A, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 A resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Animals↗
PubMed2006

An automatable screen for the rapid identification of proteins amenable to refolding.

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

Animals↗
PubMed2006

Daytime sleeping, sleep disturbance, and circadian rhythms in the nursing home.

OBJECTIVE: This study reports the frequency of abnormal daytime sleeping and identifies factors related to daytime sleeping, nighttime sleep disturbance, and circadian rhythm abnormalities among nursing home residents. METHODS: The authors conducted secondary analysis of data collected under usual care conditions within a nonpharmacologic sleep intervention trial. All residents from four Los Angeles nursing homes were screened for daytime sleeping (asleep>or=15% of observations, 9:00 am-5:00 pm). Consenting residents with daytime sleeping had two nights of wrist actigraphy to assess nighttime sleep disturbance (asleep<80%, 10:00 pm-6:00 am). Residents with nighttime sleep disturbance completed an additional 72-hour wrist actigraphy recording to assess circadian activity rhythms and light exposure. RESULTS: Sixty-nine percent of 492 observed residents had daytime sleeping, of whom 60% also had disturbed nighttime sleep. Sleep disturbance and daytime sleeping were rarely documented in medical records. Residents spent one-third of the day in their rooms, typically in bed, and were seldom outdoors or exposed to bright light. More time in bed and less social activity were significant predictors of daytime sleepiness. Ninety-seven percent of residents assessed had abnormal circadian rhythms. More daytime sleeping and less nighttime sleep were associated with weaker circadian activity rhythms. Later circadian rhythm acrophase (peak) was associated with more bright light exposure. CONCLUSION: Daytime sleepiness, nighttime sleep disturbance, and abnormal circadian rhythms were common in nursing home residents. Modifiable factors (e.g., time in bed) are associated with sleep/wake abnormalities. Mental health specialists should consider the complexity of factors causing sleep problems in nursing home residents.

Aged↗
PubMed2005

Mode of binding of methyl acceptor substrates to the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase: implications for catalysis.

Here we report three crystal structure complexes of human phenylethanolamine N-methyltransferase (PNMT), one bound with a substrate that incorporates a flexible ethanolamine side chain (p-octopamine), a second bound with a semirigid analogue substrate [cis-(1R,2S)-2-amino-1-tetralol, cis-(1R,2S)-AT], and a third with trans-(1S,2S)-2-amino-1-tetralol [trans-(1S,2S)-AT] that acts as an inhibitor of PNMT rather than a substrate. A water-mediated interaction between the critical beta-hydroxyl of the flexible ethanolamine group of p-octopamine and an acidic residue, Asp267, is likely to play a key role in positioning the side chain correctly for methylation to occur at the amine. A second interaction with Glu219 may play a lesser role. Catalysis likely occurs via deprotonation of the amine through the action of Glu185; mutation of this residue significantly reduced the kcat without affecting the Km. The mode of binding of cis-(1R,2S)-AT supports the notion that this substrate is a conformationally restrained analogue of flexible PNMT substrates, in that it forms interactions with the enzyme similar to those observed for p-octopamine. By contrast, trans-(1S,2S)-AT, an inhibitor rather than a substrate, binds in an orientation that is flipped by 180 degrees compared with cis-(1R,2S)-AT. A consequence of this flipped binding mode is that the interactions between the hydroxyl and Asp267 and Glu219 are lost. However, the amines of inhibitor trans-(1S,2S)-AT and substrate cis-(1R,2S)-AT are both within methyl transfer distance of the cofactor. These results suggest that PNMT catalyzes transfer of methyl to ligand amines only when "anchor" interactions, such as those identified for the beta-hydroxyls of p-octopamine and cis-AT, are present.

Amino Acid Substitution↗
PubMed2005

Human sulfotransferases and their role in chemical metabolism.

Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some recent advances on the molecular transcription mechanism controlling the individual SULT promoters. Interindividual variation in sulfonation capacity may be important in determining an individual's response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

Animals↗