Biomedical subjects
Publications and source records attributed to K Reilly.
This article focuses on the effect of daily living on pregnancy outcome. Although most patients can continue their everyday activities without any concern about effect on pregnancy outcome, all pregnant patients want and need their physician's advice about all of these areas.
Automation offers obvious advantages for the preparation of tablets prior to analysis by HPLC including unattended operation, minimization of human intervention and an electronic audit trail. However, significant effort has to be put in up front to develop and validate an automated method, particularly if it is required to closely follow an existing manual method. Here, method transfer for Roxifiban, a fibrinogen receptor antagonist, will be discussed. A Zymark tablet processing workstation II (TPWII) was used for all automated sample preparations. Manual methods for composite assay, content uniformity, weight variation and degradation products testing of a tablet formulation were transferred to the TPWII. The method involved weighing of the sample, disintegration of the dosage form by homogenization, extraction of the analyte in the homogenate solution, filtration of the homogenate, dilution of the filtrate and transfer to autosampler vials. Obstacles to a quick transfer included limitations in the volume capabilities of the TPWII, poor analyte solubility and achieving proper conditioning of the transfer lines and filter. After resolving these issues, a validated method was achieved. Spiked recoveries were from 99.4 to 101.1% (RSD's <0.5%). A cross-validation between automated and manual assay methods was compared by Westlake analysis giving a 0.7% calculated interval at the 95% confidence level. Carryover was 0.07% after 20 sample preparations at the highest tablet strength.
An alpha-amylase gene product was isolated from apple fruit by reverse-transcriptase PCR using redundant primers, followed by 5' and 3' RACE. The gene is a member of a small gene family. It encodes a putative 46.9 kDa protein that is most similar to an alpha-amylase gene from potato (GenBank accession M79328). In apple fruit this new gene was expressed at low levels, as detected by reverse-transcriptase PCR, in a number of plant tissues and during fruit development. Highest levels of mRNA for this transcript were observed 3 to 9 days after placing apple fruit at 0.5 degrees C. Phylogenetic analysis of amino acid sequence places the potato and apple proteins as a distinct and separate new subgroup within the plant alpha-amylases, which appears to have diverged prior to the split between monocotyledonous and dicotyledonous plants. These two divergent alpha-amylases lack the standard signal peptide structures found in all other plant alpha-amylases, and have sequence differences within the B-domain and C-domain. However, comparisons with structures of known starch hydrolases suggest that these differences are unlikely to affect the enzymatic alpha-1,4-amylase function of the protein. This is the first report of upregulation of a dicotyledonous alpha-amylase in response to low temperature, and confirms the presence of a new family of alpha-amylases in plants.
Neurofibromatosis type 1 (NF1) is a prevalent familial cancer syndrome resulting from germ line mutations in the NF1 tumor suppressor gene. Hallmark features of the disease are the development of benign peripheral nerve sheath tumors (neurofibromas), which can progress to malignancy. Unlike humans, mice that are heterozygous for a mutation in Nf1 do not develop neurofibromas. However, as described here, chimeric mice composed in part of Nf1-/- cells do, which demonstrates that loss of the wild-type Nf1 allele is rate-limiting in tumor formation. In addition, mice that carry linked germ line mutations in Nf1 and p53 develop malignant peripheral nerve sheath tumors (MPNSTs), which supports a cooperative and causal role for p53 mutations in MPNST development. These two mouse models provide the means to address fundamental aspects of disease development and to test therapeutic strategies.
A 35-nucleotide pseudoknot that binds vitamin B12 been isolated using systematic evolution of ligands by exponential enrichment (SELEX). Affinity chromatography was used to purify functional, properly folded molecules and the hanging-drop vapor-diffusion method was used to crystallize this aptamer. Two crystal forms have been obtained and the preliminary crystallographic characterization is reported here. Both crystal forms (space groups I222 or I212121 and C2221) diffract to 2.9 A and should prove sufficient for structure determination.
Two-dimensional (2-D) gel analysis was used to examine differences in the levels of 19 plasma proteins: before and after an acute inflammatory reaction (parenteral typhoid vaccination) in normal subjects, between rheumatoid arthritis (RA) patients and normals and in RA patients treated with tenidap (120 mg) and piroxicam (20 mg). Typhoid vaccination increased levels of SAA, haptoglobin alpha1, haptoglobin alpha2, haptoglobin beta and alpha1-anti-chymotrypsin but decreased transthyretin and apolipoprotein E. In RA patients, serum amyloid A (SAA), haptoglobin alpha2, haptoglobin beta, alpha1-antichymotrypsin and C3 proactivator levels were elevated while apolipoprotein A-I, apolipoprotein A-IV, transthyretin, Gc-globulin, alpha2-HS glycoprotein, alpha2-macroglobulin and alpha1-B glycoprotein levels were decreased, compared to normals. Compared to piroxicam, tenidap lowered levels of alpha1-antiprotease and SAA but raised the levels of transthyretin, Gc-globulin, alpha2-HS-glycoprotein and alpha2-macroglobulin in RA patients. C-reactive protein (CRP) could not be quantified on 2-D gels but, when measured by rate nephelometry, levels were reduced after treatment with tenidap compared to piroxicam. The general pattern of the acute phase protein response to an acute inflammatory response to typhoid vaccination is similar to that in the chronic inflammatory condition, RA. The impact of tenidap on both positive and negative acute-phase proteins in RA patients could clearly be distinguished from that of piroxicam.
A competitive PCR technique was used to enumerate the proteolytic bacterium Clostridium proteoclasticum from the rumen. A PCR primer, which circumscribes this organism and several closely related strains, was designed for a variable region within their 16S rRNA genes and was used in conjunction with a universal forward primer. This primer pair was tested for specificity against 85 ruminal bacterial strains. An internal control DNA was constructed for use in competitive PCRs and was shown to amplify under the same reaction conditions and with the same amplification efficiency as the target DNA. DNA from a known number of C. proteoclasticum cells was coamplified with the internal control to construct a standard curve. Rumen samples were collected from eight dairy cows fed four diets in rotation: high nitrogen, high nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen supplemented with carbohydrate. DNA extracted from these and spiked with internal control DNA was amplified with the C. proteoclasticum primer pair. The relative intensities of the PCR products were used to quantitate the numbers of C. proteoclasticum cell equivalents from the rumen samples. The numbers ranged from 2.01 x 10(6) ml-1 to 3.12 x 10(7) ml-1. There was no significant effect on the numbers of C. proteoclasticum detected in rumen samples among cows fed the four diets. The utility of the competitive PCR approach for quantifying ruminal bacterial populations in vivo and the occurrence of C. proteoclasticum in forage-fed dairy cows are discussed.
In order to characterize the thermodynamic constraints on the process of integral membrane protein folding and assembly, we have conducted a biophysical dissection of the structure of bacteriorhodopsin (BR), a prototypical alpha-helical integral membrane protein. Seven polypeptides were synthesized, corresponding to each of the seven transmembrane alpha-helices in BR, and the structure of each individual polypeptide was characterized in reconstituted phospholipid vesicles. Five of the seven polypeptides form stable transmembrane alpha-helices in isolation from the remainder of the tertiary structure of BR. However, using our reconstitution protocols, the polypeptide corresponding to the F helix in BR does not form any stable secondary structure in reconstituted vesicles, and the polypeptide corresponding to the G helix forms a hyperstable beta-sheet structure with its strands oriented perpendicular to the plane of the membrane. [The polypeptide corresponding to the C helix spontaneously equilibrates in a pH-dependent manner between a transmembrane alpha-helical conformation, a peripherally bound nonhelical conformation, and a fully water soluble conformation; the conformational properties of this polypeptide are the subject of the accompanying paper: Hunt et al. (1997) Biochemistry 36, 15177-15192.] Our observations suggest that the folding of alpha-helical integral membrane proteins may proceed spontaneously. However, the preference for a non-native conformation exhibited by two of the polypeptides suggests that the formation of some transmembrane substructures could require external constraints such as the links between the helices, interactions with the rest of the protein, or the involvement of cellular chaperones or translocases. Our results also suggest a strategy for improving the thermodynamic stability of alpha-helical integral membrane proteins, a goal that could facilitate attempts to overexpress and/or refold them.
This paper presents the results of a controlled trial of child stuttering treatment. The aim of the study was, first, to compare the effectiveness of three viable treatments, and, second, to compare these three treatments to a no-treatment control composed of children who stuttered of a similar age and sex ratio who were on treatment waiting lists. The three treatments investigated included intensive smooth speech, intensive electromyography feedback, and home-based smooth speech. The children/adolescents were assessed across three speaking contexts on measures of percentage syllables stuttered (% SS) and syllables spoken per minute (SPM) and outcomes were assessed 12 months later. Repeated measures analyses of variance demonstrated significant differences between the control group and all three treatment groups across time on conversations in the clinic, on the telephone, and at home (although home measures were not taken for the intensive smooth speech group). Although the controls' stuttering did not change across time, the treatment groups' stuttering was decreased to very low levels posttreatment (less than 1% syllables stuttered on average), with mean improvement in stuttering frequency of at least 85% to 90% across all assessment contexts. Stuttering did not increase significantly up to 3 months and one year posttreatment in the experimental groups, although levels did rise across time (less than 3% syllables stuttered on average). Speech naturalness results showed increasing naturalness across time as rated by the clinician and parent. This was not the case for the controls. The children were also less anxious across time following treatment. The results suggest that all three treatments for children aged 9-14 who stutter were very successful in the long term for over 70% of the group, though the EMG feedback and home-based treatments were superior when percentages falling below a cutoff point (2%SS) were used to discriminate between groups. Implications for child/adolescent treatment in the community are discussed. Long-term outcomes will be assessed up to 5 years after the treatment.
Hantavirus pulmonary syndrome is a newly recognized rodent-borne zoonosis. We report a case of hantavirus pulmonary syndrome in an employee of a California utility company who was probably occupationally exposed to Sin Nombre virus. Environmental assessment and genetic comparison of the patient's hantavirus isolates to hantavirus isolates from rodents trapped at possible sites of exposure suggested that the patient contracted his infection at the work site. The study revealed a close correspondence between the patient's viral genotype and that from a rodent trapped at the work site. This report alerts the public health and medical community to the fact that employees of utility companies and similar industries may be an important risk group in areas where hantavirus is endemic and emphasizes the need to incorporate strategies for preventing exposure to hantavirus and other emerging infections into occupational safety protocols.
A novel proteolytic bacterium was isolated from rumen contents of New Zealand cattle grazing fresh forage and was designated strain B316(T) (T = type strain). Strain B316(T) cells were straight to slightly curved rods (width, 0.4 to 0.6 microns; length, 1.3 to 3.0 microns) that were gram-positive and possessed a single subterminal flagellum. This isolate did not produce catalase, indole, ammonia, lipase, or lecithinase, or reduce nitrate, but it did produce a curd reaction with milk. Strain B316(T) was proteolytic, hydrolyzing casein and fraction I leaf protein. The crude proteinase was predominantly the serine type, but some cysteine proteinase and metallo-proteinase activities were also detected. The DNA base composition of strain B316(T) was 28 mol% G+C. A 16S ribosomal DNA sequence analysis of strain B316(T) indicated that it was most closely related to a member of clostridial cluster XIVa, viz., Clostridium aminophilum, an amino acid-fermenting organism isolated from the rumen; the similarity value was 92.2%. The results of the phenotypic characterization analysis, G+C content analysis, and phylogenetic analysis of the 16S ribosomal DNA sequence set strain B316(T) apart from all of the members of cluster XIVa. We propose that strain B316(T) should be designated a new species of the genus Clostridium, Clostridium proteoclasticum. Strain B316 is the type strain and has been deposited in the American Type Culture Collection as strain ATCC 51982.
The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.
The protease activities of 212 strains of rumen bacteria isolated from New Zealand cattle grazing pasture were measured. Thirty-seven per cent of strains had activity greater than or equal to the proteolytic rumen bacterium Prevotella ruminicola and 43 of these isolates were identified by morphology, carbon source utilization, Gram stain, biochemical tests and fermentation end-product analysis. Hierarchical Cluster Analysis showed that the strains formed four clusters: cluster A contained 26 strains and clustered with a reference strain of Streptococcus bovis; cluster C contained three strains and clustered with a reference strain of Butyrivibrio fibrisolvens, while clusters B (10 strains) and D (three strains) did not cluster with any of the remaining rumen bacterial type strains. Further tests identified strains of cluster B as Eubacterium budayi, while cluster D strains most closely resembled B. fibrisolvens and were described as B. fibrisolvens-like. An unclustered strain, C21a, was identified as P. ruminicola. The significance of these proteolytic bacterial populations is discussed in relation to protein breakdown in New Zealand ruminants.
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A patient presented with monarthric ankle pain, which took 2 years to diagnose by numerous physicians and imaging and laboratory tests. A review of the differential diagnosis for monarthritic joint pain with guidelines for the work-up of the patient are presented.
PURPOSE: To determine whether intravenously administered theophylline, when added to frequently nebulized albuterol and intravenously administered methylprednisolone, benefits children hospitalized with severe asthma. DESIGN: Prospective, randomized, placebo-controlled, parallel-group, double-blind study. SETTING: Inpatient pediatric service at a tertiary-care teaching hospital. PATIENTS: Twenty-one children 5 to 18 years of age. INTERVENTIONS: All patients received 2.5 to 5.0 mg of nebulized albuterol every 20 minutes to every 6 hours, intravenously administered methylprednisolone (1 mg/kg every 6 hours), and either intravenously administered theophylline (as aminophylline) or placebo for 36 hours. Serum theophylline concentrations were maintained between 55 and 110 mumol/L (between 10 and 20 micrograms/ml) by adjusting loading doses and continuous infusion rates. MEASUREMENTS AND MAIN RESULTS: Forced expired volume in 1 second (FEV1) and clinical score were measured at 0, 1, 3, 6, 12, 24, and 36 hours after the start of each individual study. The total number of nebulizations, total albuterol dosage, adverse effects, and duration of hospital stay were recorded. Twelve children received theophylline and nine received placebo. The two groups did not differ significantly in age, sex, or baseline FEV1. In both groups, clinical score significantly improved from baseline by 12 hours, and FEV1 by 24 hours (p < 0.05). There were no significant differences between the groups in FEV1 or clinical score at any of the measured time points. There were no significant differences in rate of improvement in FEV1, total number of nebulizations, total albuterol dosage, or duration of hospital stay. Adverse effects were mild and infrequent and did not differ significantly between the two groups. CONCLUSIONS: Theophylline, at therapeutic concentrations, did not additionally benefit children hospitalized with severe asthma who were being treated frequently with nebulized albuterol and with methylprednisolone intravenously.
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Common infectious disease problems that occur in pregnancy are outlined, including implications for pregnancy, appropriate diagnostic techniques, treatments, and methods for prevention of disease in mother and infant. Also included is general information about the use of immunizations and antibiotics in pregnancy.