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PubMed2005

Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium.

Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.

Cell Culture Techniques↗
PubMedDate not supplied

Immunological and pathological evaluation of rhesus macaques infected with Leishmania major.

Cutaneous leishmaniasis, a parasitic infection causing ulcerating skin lesions, is an important disease worldwide and urgently requires a vaccine. Animal models that closely mimic human disease are essential for designing preventive vaccines against Leishmania major. We have evaluated both biologic and immunologic parameters of cutaneous L. major infection in nonhuman primates. Naïve rhesus macaques or monkeys previously exposed to L. major were infected with varying doses of L. major metacyclic promastigotes, and lesion size was assessed over a 10-week period. Monkeys previously infected with L. major had much smaller lesions that resolved faster compared with those of naïve monkeys in response to the two higher doses of infection. Moreover, eight of nine naïve monkeys had parasites detected in their lesions during the course of the infection. In addition, the cellular infiltrate within the lesions was qualitatively and quantitatively different in naïve versus previously infected monkeys. Finally, an ELIspot assay determined that the magnitude and kinetics of responses differed between previously infected and naïve monkeys.

Animals↗
PubMed2001

Are publicly-insured psychiatric outpatients in Japan satisfied?

Japan has a government financed outpatient program for people with mental disorders, called the 'publicly-insured' program. This study was performed to examine whether the target patient population used this publicly-insured program properly and to compare the degree of satisfaction of publicly-insured psychiatric outpatients with generally-insured psychiatric outpatients. The characteristics and satisfaction of 97 (43.9%) publicly-insured psychiatric outpatients and 124 (55.1%) generally-insured outpatients in Japan were studied. Psychiatrists rated sociodemographic and diagnostic information and patients were asked to complete the Japanese version of Client Satisfaction Questionnaire (CSQ-8J). The publicly-insured were longer-term and lower functioning patients and were significantly more dissatisfied with the services they received than the generally-insured patients. The publicly-insured program was successful in that patients with lower functioning (the primary target population of this program) were cared for and because they received treatment for longer periods of time. However, the program does not sufficiently satisfy the consumers of the services, despite its high costs. In this respect, this program needs to focus more on patients' points of view. More information on programs their enrollment procedures for patients may be helpful in educating consumers and citizens, clarifying expectations of services, and in influencing satisfaction.

Adult↗
PubMed2002

Optimization of DNA vaccination against cutaneous leishmaniasis.

The present studies were designed to examine the requirements of dose, route of inoculation and constituent antigens for the maintenance of complete and long lasting protection against cutaneous leishmaniasis due to Leishmania major conferred by a cocktail DNA vaccine encoding the Leishmania antigens LACK, LmST11 and TSA. Vaccination of C57Bl/6 mice with LACK DNA alone resulted in partial protection, whereas the combination of LmST11 and TSA provided stronger, though still incomplete protection compared to the combination of all three Ag DNAs. When intradermal (i.d), intramuscular (i.m.), and subcutaneous (s.c.) vaccination routes were compared, i.d. immunization reduced by five-fold the dose necessary to maintain complete protection. In vivo depletion of CD4+ or CD8+ T cells provided direct evidence that both populations are necessary to mediate complete protection. These results establish intradermal vaccination using DNA encoding multiple Leishmania antigens as a way to optimize priming of CD4+ and CD8+ T cells necessary for potent and durable protection against cutaneous leishmaniasis.

Animals↗
PubMedDate not supplied

Prevalence of aortic regurgitation by color flow Doppler in relation to aortic root size.

To determine whether there is a correlation between aortic root size and the prevalence of aortic regurgitation, we performed color flow Doppler echocardiographic studies on 1015 consecutive patients during a 3-month period. Patients were grouped according to their M-mode aortic root diameter as measured in the left parasternal position. The measured groups ranged from 2.0 to 4.5 cm, grouped at 0.1 cm intervals. As the aortic root size enlarged, the prevalence of aortic regurgitation increased linearly (p less than 0.001; correlation coefficient, r = 0.75). At an aortic root size in the "small normal" range of 2.0 to 2.4 cm, the prevalence of aortic regurgitation was 0% to 15%. In the "intermediate" and "top normal" ranges of 2.9 to 3.7 cm, the prevalence of aortic regurgitation increased linearly from 15% to 47%. With aortic root dilation, the prevalence of aortic root regurgitation was generally more than 50%. The severity of aortic regurgitation was semiquantified. Aortic root size was not a good indicator for the severity of aortic regurgitation. Patients with moderate and severe aortic regurgitation had variable aortic root sizes. Throughout the range of aortic root sizes, mild aortic regurgitation predominated. We conclude that aortic regurgitation is a common finding in patients with aortic roots that are dilated or are in the "top normal" size range, that the prevalence of aortic regurgitation increases linearly with aortic root size, and that aortic root size does not correlate with the severity of aortic regurgitation.

Aorta↗
PubMed2000

DNA vaccines: a key for inducing long-term cellular immunity.

Over the past few years, major advances in several areas of immunology have provided a foundation for the rational design of vaccines against diseases requiring cellular immunity. Among these advances are the cellular mechanisms by which DNA vaccines can sustain long-term humoral and cellular immunity.

Animals↗
PubMed2003

DNA vaccination: the answer to stable, protective T-cell memory?

Distinct populations of both CD4(+) and CD8(+) memory T cells have been identified on the basis of their location (lymphoid versus non-lymphoid tissues) and function. There have been several recent advances in our understanding of the factors that govern the generation and maintenance of T-cell memory in vivo. Utilising this knowledge could contribute to the refinement of DNA vaccine technology to induce more robust and longer-lasting protective T-cell based immunity.

Animals↗
PubMed1981

Differential thermal analysis of dipalmitoylphosphatidylcholine--fatty acid mixtures.

Mixtures of dipalmitoylphosphatidylcholine (DPPC) with palmitic, stearic, and myristic acids and the sodium salts of these acids were analyzed by differential thermal analysis (DTA) over a wide range of lipid compositions, all in excess water. All three fatty acids raise the liquid-crystal phase transition temperature and form sharp-melting complexes, with 1:2 DPPC--fatty acid stoichiometry observed for palmitic and stearic acids and suggested for myristic acid. Phase diagrams of the peritectic type, indicating nonideal mixing, was fitted to the DPPC--palmitic acid and DPPC--stearic acid data. In contrast, DPPC forms nearly ideal mixtures with the putative DPPC--myristic acid complex. At levels of only a few mole percent, both sodium stearate and myristate remove the pretransition and main transition and produce new peaks at approximately 30 and approximately 48 degrees C; the relative areas of the new peaks were unreproducible for the DPPC--myristate system. Sodium palmitate is the least disruptive of any of the sodium soaps or fatty acids; up to 80 mol % palmitate, the transition is lowered 3 degrees C and approximately doubled in width. The pretransition is detectable up to 36 mol %, and the main transition persists up to 88 mol % palmitate. The apparent pK of palmitic acid (12 mol %) in DPPC bilayers was determined to be 10.2 by direct pH measurement of ternary DPPC mixtures with known palmitic acid/sodium palmitate ratios; the intrinsic pK is estimated to be less than or approximately 8.5.

Fatty Acids, Nonesterified↗
PubMed1997

Assessing outcomes in clinical practice.

We are witnessing a remarkable explosion in interest and activity in quantifying outcomes and using these measures to enhance the value of clinical care. Outcomes assessment has become an imperative for clinical practice. This paper first will offer criteria for an ideal system of outcomes assessment. The paper will then review the principal domains of assessment for psychiatric practice and provide examples of instruments available in each domain. We will then describe the use of two instruments, one for clinical outcome and one for interpersonal aspects of patient satisfaction, developed and used at McLean Hospital. The relation between outcomes assessment and outcomes management will then be discussed. Finally, we will discuss the fundamental questions a clinical group or facility might consider in choosing outcomes measurement instruments.

Delivery of Health Care↗
PubMed2004

Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N.

The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue, pp. 95-102], this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.

Arabidopsis Proteins↗
PubMed2000

Vaccines against intracellular infections requiring cellular immunity.

Vaccines against a variety of infectious diseases represent one of the great triumphs of medicine. The immune correlates of protection induced by most current vaccines seem to be mediated by long-lived humoral immune responses. By contrast, there are no currently available vaccines that are uniformly effective for diseases such as HIV, malaria and tuberculosis, in which the cellular immune response might be crucial in mediating protection. Here we examine the mechanisms by which long-lived cellular immune responses are generated and maintained in vivo. We then discuss current approaches for vaccination against diseases in which cellular immune responses are important for protection.

Animals↗
PubMed1998

Vaccine requirements for sustained cellular immunity to an intracellular parasitic infection.

The humoral immunity induced by many viral and bacterial vaccines mediates protection that is maintained over a long period of time. In contrast, for other intracellular infections (such as with Leishmania major or Mycobacterium tuberculosis) for which cell-mediated immunity is required for protection, the mechanisms for developing durable responses after vaccination have not been well defined. Here we demonstrate that vaccination with plasmid DNA encoding a specific leishmanial antigen is more effective than leishmanial protein plus recombinant IL-12 in eliciting long-term immunity capable of controlling L. major infection. We also show that leishmanial protein plus IL-12 DNA produces an immunity that lasts much longer than does immunity elicited by leishmanial protein plus IL-12 protein, indicating that the persistence of IL-12 may be the essential determinant in maintaining durable cell-mediated immune responses for an intracellular parasitic infection.

Adjuvants, Immunologic↗
PubMed2002

Distinct lineages of T(H)1 cells have differential capacities for memory cell generation in vivo.

We studied here the long-term maintenance of distinct populations of T helper type 1 (T(H)1)-lineage cells in vivo and found that effector T(H)1 cells, defined by their secretion of interferon-gamma (IFN-gamma), are short-lived and do not efficiently develop into long-term memory T(H)1 cells. In contrast, a population of activated T(H)1-lineage cells that did not secrete IFN-gamma after primary antigenic stimulation persisted for several months in vivo and developed the capacity to secrete IFN-gamma upon subsequent stimulation. These data suggest that a linear differentiation pathway, as defined by the transition from IFN-gamma-producing to resting memory cells, is relatively limited in vivo and support a revised model for T(H)1 memory differentiation.

Animals↗