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1.  Components Required for the Formation of CH4 from Methylcobalamin by Extracts of Methanobacillus omelianskii 
Journal of Bacteriology  1966;92(3):696-700.
Wood, J. M. (University of Illinois, Urbana), and R. S. Wolfe. Components required for the formation of CH4 from methylcobalamin by extracts of Methanobacillus omelianskii. J. Bacteriol. 92:696–700. 1966.—Optimal conditions for the formation of CH4 from methylcobalamin (methyl-cobalt-5,6-dimethylbenzimidazolylcobamide) by partially purified extracts of Methanobacillus omelianskii (Methanobacterium omelianskii) were achieved by the addition of two protein fractions, adenosine triphosphate, Mg++, and a reduced flavin adenine dinucleotide-generating system. Adenosine diphosphate and adenosine monophosphate play an important role in the regulation of C1 transfer in this reaction. The possible position of adenosine triphosphate in B12-dependent methyl transfer is discussed.
PMCID: PMC276310  PMID: 4288494
2.  Insertion proQ220::Tn5 alters regulation of proline porter II, a transporter of proline and glycine betaine in Escherichia coli. 
Journal of Bacteriology  1989;171(2):947-951.
Mutation pro-220::Tn5, which increases the resistance of Escherichia coli to 3,4-dehydroproline (M. E. Stalmach, S. Grothe, and J. M. Wood, J. Bacteriol. 156:481-486, 1983), is not linked to putP, proP, or proU. It was located at 40.4 min on the E. coli chromosomal linkage map, by conjugational and transductional mapping, and is now denoted proQ220::Tn5. Proline porter II was not detectable when proQ220::Tn5 proP+ bacteria were cultivated under optimal conditions or with nutritional stress (amino acid limitation). Toxic proline analog sensitivity and proline porter II activity were partially restored to proQ220::Tn5 proP+ bacteria, but not to a proQ220::Tn5 proP219 strain, by a hyperosmotic shift and by growth under osmotic stress. Elevated expression of a proP::lacZ gene fusion, for bacteria grown under osmotic stress, was not influenced by the proQ220::Tn5 insertion. We propose that the proQ locus encodes a positive regulatory element which elevates proline porter II activity.
PMCID: PMC209686  PMID: 2536686
3.  Impact of Brain Tumor Location on Morbidity and Mortality: A Retrospective Functional MR Imaging Study 
fMRI is increasingly used in neurosurgery to preoperatively identify areas of eloquent cortex. Our study evaluated the efficacy of clinical fMRI by analyzing the relationship between the distance from the tumor border to the area of functional activation (LAD) and patient pre- and postoperative morbidity and mortality.
The study included patients with diagnosis of primary or metastatic brain tumor who underwent preoperative fMRI-based motor mapping (n = 74) and/or language mapping (n = 77). The impact of LAD and other variables collected from patient records was analyzed with respect to functional deficits in terms of morbidity (paresis and aphasia) and mortality.
Significant relationships were found between motor and language LAD and the existence of either pre- or postoperative motor (P < .001) and language deficits (P = .009). Increasing age was associated with motor and language deficits (P = .02 and P = .04 respectively). Right-handedness was related to language deficits (P = .05). Survival analysis revealed that pre- and postoperative deficits, grade, tumor location, and LAD predicted mortality. Motor deficits increased linearly as the distance from the tumor to the primary sensorimotor cortex decreased. Language deficits increased exponentially as the distance from the tumor to the language areas decreased below 1 cm. Postoperative mortality analysis showed an interaction effect between motor or language LAD and mortality predictors (grade and tumor location, respectively).
These findings indicate that tumors may affect language and motor function differently depending on tumor LAD. Overall, the data support the use of fMRI as a tool to evaluate patient prognosis and are directly applicable to neurosurgical planning.
PMCID: PMC3305992  PMID: 21885713
4.  The oral HDAC inhibitor pracinostat (SB939) is efficacious and synergistic with the JAK2 inhibitor pacritinib (SB1518) in preclinical models of AML 
Blood Cancer Journal  2012;2(5):e69-.
Acute myeloid leukemia (AML) is currently treated with aggressive chemotherapy that is not well tolerated in many elderly patients, hence the unmet medical need for effective therapies with less toxicity and better tolerability. Inhibitors of FMS-like tyrosine kinase 3 (FLT3), JAK2 and histone deacetylase inhibitors (HDACi) have been tested in clinical studies, but showed only moderate single-agent activity. High efficacy of the HDACi pracinostat treating AML and synergy with the JAK2/FLT3 inhibitor pacritinib is demonstrated. Both compounds inhibit JAK-signal transducer and activator of transcription (STAT) signaling in AML cells with JAK2V617F mutations, but also diminish FLT3 signaling, particularly in FLT3-ITD (internal tandem duplication) cell lines. In vitro, this combination led to decreased cell proliferation and increased apoptosis. The synergy translated in vivo in two different AML models, the SET-2 megakaryoblastic AML mouse model carrying a JAK2V617F mutation, and the MOLM-13 model of FLT3-ITD-driven AML. Pracinostat and pacritinib in combination showed synergy on tumor growth, reduction of metastases and synergistically decreased JAK2 or FLT signaling, depending on the cellular context. In addition, several plasma cytokines/growth factors/chemokines triggered by the tumor growth were normalized, providing a rationale for combination therapy with an HDACi and a JAK2/FLT3 inhibitor for the treatment of AML patients, particularly those with FLT3 or JAK2 mutations.
PMCID: PMC3366067  PMID: 22829971
HDAC inhibitor; JAK2 inhibitor; FLT3 inhibitor; in vivo combination; AML
5.  Pacritinib (SB1518), a JAK2/FLT3 inhibitor for the treatment of acute myeloid leukemia 
Blood Cancer Journal  2011;1(11):e44-.
FMS-like tyrosine kinase 3 (FLT3) is the most commonly mutated gene found in acute myeloid leukemia (AML) patients and its activating mutations have been proven to be a negative prognostic marker for clinical outcome. Pacritinib (SB1518) is a tyrosine kinase inhibitor (TKI) with equipotent activity against FLT3 (IC50=22 n) and Janus kinase 2 (JAK2, IC50=23 n). Pacritinib inhibits FLT3 phosphorylation and downstream STAT, MAPK and PI3 K signaling in FLT3-internal-tandem duplication (ITD), FLT3-wt cells and primary AML blast cells. Oral administration of pacritinib in murine models of FLT3-ITD-driven AML led to significant inhibition of primary tumor growth and lung metastasis. Upregulation of JAK2 in FLT3-TKI-resistant AML cells was identified as a potential mechanism of resistance to selective FLT3 inhibition. This resistance could be overcome by the combined FLT3 and JAK2 activities of pacritinib in this cellular model. Our findings provide a rationale for the clinical evaluation of pacritinib in AML including patients resistant to FLT3-TKI therapy.
PMCID: PMC3256753  PMID: 22829080
Pacritinib; SB1518; FLT3; JAK2; AML
6.  Bilateral cataract surgery and driving performance 
The British Journal of Ophthalmology  2006;90(10):1277-1280.
Cataract surgery is one of the most common medical procedures undertaken worldwide.
To investigate whether cataract surgery can improve driving performance and whether this can be predicted by changes in visual function.
29 older patients with bilateral cataracts and 18 controls with normal vision were tested. All were licensed drivers. Driving and vision performance were measured before cataract surgery and after second eye surgery for the patients with cataract and on two separate occasions for the controls. Driving performance was assessed on a closed‐road circuit. Visual acuity, contrast sensitivity, glare sensitivity and kinetic visual fields were measured at each test session.
Patients with cataract had significantly poorer (p<0.05) driving performance at the first visit than the controls for a range of measures of driving performance, which significantly improved to the level of the controls after extraction of both cataracts. The change in contrast sensitivity after surgery was the best predictor of the improvements in driving performance in patients with cataract.
Cataract surgery results in marked improvements in driving performance, which are related to concurrent improvements in contrast sensitivity.
PMCID: PMC1857457  PMID: 16825273
7.  Pupil dilatation does affect some aspects of daytime driving performance 
The British Journal of Ophthalmology  2003;87(11):1387-1390.
Aims: To examine the effects of pupil dilatation on driving performance and determine whether this was related to changes in standard measures of visual function.
Methods: The driving and vision performance of 16 young, visually normal participants was measured with both normal and dilated pupils. Pupils were dilated with 1% tropicamide. Driving performance was measured under daytime conditions on a closed road circuit that was free of other vehicles and has been used in previous studies of driving performance. Measures included road sign detection and recognition, hazard detection and avoidance, gap perception and negotiation, driving reaction times and time to complete the circuit. Visual performance measures included high contrast visual acuity, Pelli-Robson letter contrast sensitivity, and glare sensitivity.
Results: Pupil dilatation significantly (p<0.05) decreased the ability of participants to recognise low contrast hazards and avoid them, decreased their visual acuity and contrast sensitivity and increased glare sensitivity. The decreases in vision performance were not, however, significantly related to the decrement in driving performance.
Conclusion: Pupil dilatation can impair selected aspects of driving and vision performance and patients should be cautioned about these possible effects.
PMCID: PMC1771881  PMID: 14609840
dilated pupils; driving assessment; visual function
8.  Studies with inactivated equine influenza vaccine. 1. Serological responses of ponies to graded doses of vaccine. 
The Journal of Hygiene  1983;90(3):371-384.
Serological responses to three bivalent aqueous equine influenza vaccines of different potency and an adjuvanted bivalent vaccine containing inactivated A/equine/Prague/56 (H7N7) and A/equine/Miami/63 (H3N8) viruses, were examined in seronegative ponies. Potencies of the vaccines, measured by single-radial-diffusion tests, ranged from 4 to 56 micrograms of haemagglutinin (HA) antigen activity/virus strain per dose. Serological responses to vaccination were examined by haemagglutination-inhibition (HI) and single-radial-haemolysis (SRH) tests. Four weeks after a primary dose, HI responses to both vaccine viruses were barely detectable; after a second dose the HI responses to A/Miami/63 virus were low or undetectable but HI responses to A/Prague/56 virus were higher (17/20 ponies with titres greater than or equal to 1:16). In contrast SRH tests revealed dose-related antibody responses to both virus strains after one and two vaccine doses; levels after the second dose were 2- to 5-fold higher than after the primary dose. Highest post-vaccination antibody titres were obtained with the adjuvanted vaccine which contained 2- to 4-fold less antigen (13-23 micrograms HA) than the most potent aqueous vaccine. Post-vaccination antibody reacted well in SRH tests with recent antigenic variants of equine influenza virus. A remarkable finding was the high rate of decline in antibody, detected by HI or SRH tests, following one or two doses of vaccine. Even in animals with the highest post-vaccine antibody levels 2-4 weeks after a booster dose, antibody levels had declined to low or indetectable levels 14 weeks later. The low antibody titres detected at 14-32 weeks after vaccination were nevertheless vaccine dose-related.
PMCID: PMC2134282  PMID: 6345659
9.  Developing vaccines against pandemic influenza. 
Pandemic influenza presents special problems for vaccine development. There must be a balance between rapid availability of vaccine and the safeguards to ensure safety, quality and efficacy of vaccine. Vaccine was developed for the pandemics of 1957, 1968, 1977 and for the pandemic alert of 1976. This experience is compared with that gained in developing vaccines for a possible H5N1 pandemic in 1997-1998. Our ability to mass produce influenza vaccines against a pandemic threat was well illustrated by the production of over 150 million doses of 'swine flu' vaccine in the USA within a 3 month period in 1976. However, there is cause for concern that the lead time to begin vaccine production is likely to be about 7-8 months. Attempts to reduce this time should receive urgent attention. Immunogenicity of vaccines in pandemic situations is compared over the period 1968-1998. A consistent feature of the vaccine trials is the demonstration that one conventional 15 microg haemagglutinin dose of vaccine is not sufficiently immunogenic in naive individuals. Much larger doses or two lower doses are needed to induce satisfactory immunity. There is some evidence that whole-virus vaccines are more immunogenic than split or subunit vaccines, but this needs substantiating by further studies. H5 vaccines appeared to be particularly poor immunogens and there is evidence that an adjuvant may be needed. Prospects for improving the development of pandemic vaccines are discussed.
PMCID: PMC1088574  PMID: 11779397
10.  Effects of acidification on the mobility of metals and metalloids: an overview. 
The exchange rates for metals and metalloids between sediments, soils, water and aquatic biota are discussed in terms of normal and acidified ecosystems. Where it is possible, the pathways for a number of toxic elements are presented with special emphasis on the impact of acidification on changing chemical speciation, and on the potential toxicity of such acid-generated chemical species. Concerns for the impact of acidification on the stability of ecosystems, the safety of drinking water, bioaccumulation in fish and in plants are addressed.
PMCID: PMC1568487  PMID: 4076077
11.  Macrophage electrophoretic mobility (MEM) with myelin basic protein. 
British Journal of Cancer  1976;34(6):613-618.
Lymphocytes from a total of 161 subjects, including normal controls and patients with malignant and non-malignant conditions, have been investigated for their response to myelin basic protein, using the macrophage electrophoretic mobility (MEM) test. It has been confirmed that there was a high level of association between clinically evident cancer and a positive response. Lymphocytes from 24/25 patients with non-malignant inflammatory and ischaemic diseases also gave positive responses. In 46 patients with breast lumps studied before mastectomy or biopsy, the test was positive in 15/19 cases which proved to be malignant and in 5/27 which proved benign on histological examination. In its present form the test is not sufficiently reliable for the diagnosis of early cancer. Our results suggest that tissue necrosis in malignant and non-malignant conditions may be one of the factors resulting in sensitization to antigenic determinants present in preparations of myelin basic protein. Despite its technical difficulties, the test may provide a means of examing some aspects of immune recall not readily revealed by other test systems.
PMCID: PMC2025217  PMID: 1008988
13.  The next influenza pandemic: lessons from Hong Kong, 1997. 
Emerging Infectious Diseases  1999;5(2):195-203.
The 1997 Hong Kong outbreak of an avian influenzalike virus, with 18 proven human cases, many severe or fatal, highlighted the challenges of novel influenza viruses. Lessons from this episode can improve international and national planning for influenza pandemics in seven areas: expanded international commitment to first responses to pandemic threats; surveillance for influenza in key densely populated areas with large live-animal markets; new, economical diagnostic tests not based on eggs; contingency procedures for diagnostic work with highly pathogenic viruses where biocontainment laboratories do not exist; ability of health facilities in developing nations to communicate electronically, nationally and internationally; licenses for new vaccine production methods; and improved equity in supply of pharmaceutical products, as well as availability of basic health services, during a global influenza crisis. The Hong Kong epidemic also underscores the need for national committees and country-specific pandemic plans.
PMCID: PMC2640700  PMID: 10221870
14.  Sequential infection or immunization of ferrets with a series of influenza A (H3N2) strains (report to the Medical Research Council's Sub-Committee on influenza Vaccines (CDVIP/IV)). 
Epidemiology and Infection  1987;99(2):501-515.
Previous studies of boys at Christ's Hospital school have indicated that annual immunization with influenza virus vaccines did not significantly reduce the total incidence of influenza infection compared to unimmunized subjects. In view of the implications of this result, a similar study was conducted in ferrets to clarify these findings. Groups of ferrets were immunized or infected with a series of influenza A (H3N2) viruses over an 18-month period, and the immunity to subsequent live virus challenge was measured after each virus or vaccine exposure. The results indicated that live virus infection gave a more solid immunity than immunization with inactivated vaccine; and the serum haemagglutination-inhibiting antibody response was greater following immunization than following infection. In addition, differences in immunity could not be explained by measurements of cross-reacting and specific antibody, since the incidence of these antibodies was similar in both infected and immunized animals. The results do not suggest an explanation for the different levels of immunity induced following infection or immunization or the results obtained from the Christ's Hospital study. However, the relative contribution of various immune responses to virus or virus antigen is discussed, and it is suggested that the difference in immunity may lie in the ability of live virus infection to stimulate local antibody.
PMCID: PMC2249279  PMID: 3315713
15.  Protection against experimental infection with influenza virus A/equine/Miami/63 (H3N8) provided by inactivated whole virus vaccines containing homologous virus. 
Epidemiology and Infection  1988;100(3):501-510.
Thirty-one ponies immunized with inactivated virus vaccine containing A/equine/Miami/63 (H3N8) virus and six seronegative ponies were experimentally challenged with the homologous virus strain. All 6 unvaccinated ponies and 11 out of 31 vaccinated ponies became infected. A clear relationship between pre-challenge antibody, measured by single radial haemolysis (SRH), and protection was demonstrated as judged by virus excretion, febrile responses and antibody responses. Those ponies with SRH antibody levels greater than 74 mm2 were completely protected against challenge infection by the intranasal route.
PMCID: PMC2249356  PMID: 2837409
16.  Characterization of virulent and avirulent A/chicken/Pennsylvania/83 influenza A viruses: potential role of defective interfering RNAs in nature. 
Journal of Virology  1985;54(1):151-160.
In April 1983, an influenza virus of low virulence appeared in chickens in Pennsylvania. Subsequently, in October 1983, the virus became virulent and caused high mortality in poultry. The causative agent has been identified as an influenza virus of the H5N2 serotype. The hemagglutinin is antigenically closely related to tern/South Africa/61 (H5N3) and the neuraminidase is similar to that from human H2N2 strains (e.g., A/Japan/305/57) and from some avian influenza virus strains (e.g., A/turkey/Mass/66 [H6N2]). Comparison of the genome RNAs of chicken/Penn with other influenza virus isolates by RNA-RNA hybridization indicated that all of the genes of this virus were closely related to those of various other influenza virus isolates from wild birds. Chickens infected with the virulent strain shed high concentrations of virus in their feces (10(7) 50% egg infective dose per g), and the virus was isolated from the albumin and yolk of eggs layed just before death. Virus was also isolated from house flies in chicken houses. Serological and virological studies showed that humans are not susceptible to infection with the virus, but can serve as short-term mechanical carriers. Analysis of the RNA of the viruses isolated in April and October by gel migration and RNA-RNA hybridization suggested that these strains were very closely related. Oligonucleotide mapping of the individual genes of virulent and avirulent strains showed a limited number of changes in the genome RNAs, but no consistent differences between the virulent and avirulent strains that could be correlated with pathogenicity were found. Polyacrylamide gel analysis of the early (avirulent) isolates demonstrated the presence of low-molecular-weight RNA bands which is indicative of defective-interfering particles. These RNAs were not present in the virulent isolates. Experimental infection of chickens with mixtures of the avirulent and virulent strains demonstrated that the avirulent virus interferes with the pathogenicity of the virulent virus. The results suggest that the original avirulent virus was probably derived from influenza viruses from wild birds and that the virulent strain was derived from the avirulent strain by selective adaptation rather than by recombination or the introduction of a new virus into the population. This adaptation may have involved the loss of defective RNAs, as well as mutations, and thus provides a possible model for a role of defective-interfering particles in nature.
PMCID: PMC254772  PMID: 3973976
17.  A comparison of live and inactivated influenza A (H1N1) virus vaccines. 2. Long-term immunity. 
The Journal of Hygiene  1983;90(3):361-370.
Groups of volunteers were immunized with one of three influenza virus vaccines, and the resistance to challenge infection with attenuated influenza A (H1N1) virus was measured 8 months later. The vaccines were aqueous subunit influenza A/USSR/77 (H1N1) vaccine, aqueous subunit influenza B/Hong Kong/73 vaccine, or attenuated influenza virus A (H1N1) vaccine. The B virus vaccine was included as a control to assess the incidence of natural A virus infection during the study period. A proportion of the B virus vaccinees had pre-existing A (H1N1) virus antibody and were used to study the immunity conferred by natural infection to the live virus challenge. The serum antibody responses were measured at 1 and 8 months after immunization. The results showed that all the vaccines induced serum HI antibody in a proportion of the volunteers; however, after 1 month, higher titres of serum antibody were found in volunteers given inactivated A vaccine than in those given live attenuated A virus vaccine. Eight months post-immunization the titres of serum antibody in volunteers given inactivated vaccine had declined significantly, but there were no changes in the antibody titres of those given live virus vaccine. The incidence of infection by the challenge virus at 8 months post-immunization was directly related to the serum antibody titres 1 month post-immunization; no evidence was obtained to suggest that those given live virus vaccine had a more solid immunity than those given inactivated vaccine.
PMCID: PMC2134281  PMID: 6863910
18.  A comparison of live and inactivated influenza A (H1N1) virus vaccines. 1. Short-term immunity. 
The Journal of Hygiene  1983;90(3):351-359.
Groups of volunteers were immunized subcutaneously with one of three inactivated influenza virus A/USSR/77 (H1N1) vaccine preparations; a whole virus vaccine, a surface-antigen subunit adsorbed vaccine, or an aqueous surface-antigen subunit vaccine. The reactions to immunization were recorded, and the antibody response was measured 1 month later. A fourth group of volunteers were inoculated intranasally with live attentuated A/USSR/77 (H1N1) influenza virus; the reactions and antibody response of these volunteers were also measured. One month after immunization, the incidence of infection by challenge with homologous live attentuated virus was determined for all groups of volunteers. The results showed that all four vaccines used were relatively non-reactogenic, and that inactivated vaccines induced higher titres of serum antibody than the live attenuated vaccine. All the vaccines induced significant protection against challenge virus infection which was directly related to the level of serum HI antibody response.
PMCID: PMC2134273  PMID: 6863909
19.  Studies with inactivated equine influenza vaccine. 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8). 
The Journal of Hygiene  1983;90(3):385-395.
Forty ponies immunized with inactivated virus vaccine containing A/equine/Miami/63 (H3N8) virus and six unvaccinated, seronegative ponies were experimentally challenged with a representative of recent equine H3N8 virus isolates, A/equine/Newmarket/79. All unvaccinated ponies became infected as judged by virus excretion, febrile responses and antibody responses, but only two of the vaccinated ponies were fully protected. Pre-challenge antibody levels to A/Newmarket/79 virus detected by single radial haemolysis (SRH) correlated well with the degree of clinical protection but the levels required for complete protection (SRH zones greater than 65 mm2) were high. The importance of these results in relation to conventional vaccination procedures against equine influenza is discussed.
PMCID: PMC2134271  PMID: 6306098
20.  Recent studies on biomethylation and demethylation of toxic elements. 
Methylcobalamin (methyl-B12) has been implicated in the biomethylation of the heavy metals (mercury, tin, platinum, gold, and thallium) as well as the metalloids (arsenic, selenium, tellurium and sulfur). In addition, methylcobalamin has been shown to react with lead, but the lead-alkyl product is unstable in water. Details of the kinetics and mechanisms for biomethylation of arsenic are presented, with special emphasis on synergistic reactions between metal and metalloids in different oxidation states. This study explains why synergistic, or antagonistic, processes can occur when one toxic element reacts in the presence of another. The relative importance of biomethylation reactions involving methylcobalamin will be compared to those reactions where S-adenosylmethionine is involved.
PMCID: PMC1637412  PMID: 908310
21.  Influence of Side-Chain Substituents on the Position of Cleavage of the Benzene Ring by Pseudomonas fluorescens1 
Journal of Bacteriology  1969;97(3):1192-1197.
Pseudomonas fluorescens was grown on mineral salts media with phenol, p-hydroxybenzoic acid, p-hydroxy-phenylacetic acid, or p-hydroxy-trans-cinnamic acid as sole carbon and energy source. Each compound was first hydroxylated, ortho to the hydroxyl group on the benzene ring, to give catechol, protocatechuic acid (3,4-dihydroxy-benzoic acid), homoprotocatechuic acid (3,4-dihydroxy-phenylacetic acid), and caffeic acid (3,4-dihydroxy-trans-cinnamic acid), respectively, as the ultimate aromatic products before cleavage of the benzene nucleus. Protocatechuic acid and caffeic acid were shown to be cleaved by ortho fission, via a 3,4-oxygenase mechanism, to give β-substituted cis, cis-muconic acids as the initial aliphatic products. However, catechol and homoprotocatechuic acid were cleaved by meta fission, by 2,3-and 4,5-oxygenases, respectively, to give α-hydroxy-muconic semialdehyde and α-hydroxy-γ-carboxymethyl muconic semialdehyde as initial aliphatic intermediates. Caffeic acid: 3,4-oxygenase, a new oxygenase, consumes 1 mole of O2 per mole of substrate and has an optimal pH of 7.0. The mechanism of cleavage of enzymes derepressed for substituted catechols by P. fluorescens apparently changes from ortho to meta with the increasing nephelauxetic (electron donor) effect of the side-chain substituent.
PMCID: PMC249834  PMID: 5776526
22.  Gene products that promote mRNA turnover in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1992;12(5):2165-2177.
We showed previously that the increased rate of mRNA turnover associated with premature translational termination in the yeast Saccharomyces cerevisiae requires a functional UPF1 gene product. In this study, we show that the UPF1 gene codes for a 109-kDa primary translation product whose function is not essential for growth. The protein contains a potential zinc-dependent nucleic acid-binding domain and a nucleoside triphosphate-binding domain. A 300-amino-acid segment of the UPF1 protein is 36% identical to a segment of the yeast SEN1 protein, which is required for endonucleolytic processing of intron-containing pre-tRNAs. The same region is 32% identical to a segment of Mov-10, a mouse protein of unknown function. Dominant-negative upf1 mutations were isolated following in vitro mutagenesis of a plasmid containing the UPF1 gene. They mapped exclusively at conserved positions within the sequence element common to all three proteins, whereas the recessive upf1-2 mutation maps outside this region. The clustering of dominant-negative mutations suggests the presence of a functional domain in UPF1 that may be shared by all three proteins. We also identified upf mutations in three other genes designated UPF2, UPF3, and UPF4. When alleles of each gene were screened for effects on mRNA accumulation, we found that the recessive mutation upf3-1 causes increased accumulation of mRNA containing a premature stop codon. When mRNA half-lives were measured, we found that excess mRNA accumulation was due to mRNA stabilization. On the basis of these results, we suggest that the products of at least two genes, UPF1 and UPF3, are responsible for the accelerated rate of mRNA decay associated with premature translational termination.
PMCID: PMC364388  PMID: 1569946
23.  Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate. 
Applied and Environmental Microbiology  1987;53(10):2445-2451.
We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.
PMCID: PMC204127  PMID: 3322192
24.  Induction of contact dermatitis in guinea pigs by quaternary ammonium compounds: the mechanism of antigen formation. 
Eight quaternary ammonium compounds were tested for their ability to induce contact dermatitis in guinea pigs by using a modified Freund's complete adjuvant test together with the guinea pig maximization test. Only two quaternary ammonium salts of the eight tested could be designated as strong allergens. These two active substances were shown to be capable of stable association with membrane lipids in forming immunogenic complexes. This surface complexation phenomenon was confirmed by using a spin-labeled quaternary ammonium salt which competed for binding sites at the surface of epidermal cells in vivo. Electron spin resonance was used to demonstrate that stable "ion-pairs" are formed between binding sites and the two allergenic preservatives. Furthermore, information was obtained on the kinetics of immunogenic complex formation as well as on the position and orientation of the quaternary ammonium ion at the cell surface.
PMCID: PMC1474270  PMID: 3830108
25.  Proline transport and osmotic stress response in Escherichia coli K-12. 
Journal of Bacteriology  1986;166(1):253-259.
Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.
PMCID: PMC214584  PMID: 3514577

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