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Respiratory and blood gas responses in exercising birds.

The effects of exercise in birds on changes in body temperature, ventilation, blood gases and air-sac gases are reviewed. Except in the case of isothermic exercise below the anaerobic threshold, birds hyperventilate during exercise. Exercise hyperventilation is greater at higher exercise intensities and at higher environmental temperatures. The domestic fowl appears to be a suitable model for the study of physiological responses to exercise in running birds. A prior period of training is necessary to accustom the birds to laboratory procedures. The possible neural and/or humoral mechanisms controlling exercise hyperpnea are listed. Intrapulmonary hypocapnia seems to exclude the possibility that lung chemoreceptors are responsible for the hyperpnea during exercise, but these receptors probably play a predominant role in the determination of ventilatory pattern.

Animals↗

Proliferation measurements with flow cytometry Tpot in cancer of the uterine cervix: correlation between two laboratories and preliminary clinical results.

PURPOSE: To assess the prognostic value of the pretreatment potential doubling time (Tpot) in carcinoma of the uterine cervix, relative to other established clinical factors. METHODS AND MATERIALS: Fifty-two patients with cervical cancer were studied prospectively from March 1991 to October 1993. Pretreatment evaluation included examination under anesthesia and tumor biopsy 6 h following the intravenous administration of bromodeoxyuridine (200 mg). Tpot was determined by deriving the labeling index (LI) and S-phase synthesis time (Ts) using flow cytometry. Six patients were not evaluable and excluded. The remaining 46 patients (average age 55 years) were treated uniformly with radical radiation therapy. There were 39 squamous carcinomas and 7 adenocarcinomas. Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stages were: Ib and IIa, 12 patients; IIb, 18 patients; III and IV, 16 patients. The median external beam dose was 50 Gy (range, 45-52.8 Gy) delivered in 25 fractions. The median intracavitary dose was 40 Gy (range. 25.5-40 Gy) delivered with a single line source to a point 2 cm lateral of the midline, with a mean dose rate of 0.71 Gy/h. The median overall treatment time was 45 days (range, 34-73 days). As of July 31, 1994, 12 patients had died of disease, and the average follow-up for alive patients was 1.4 years (range, 0.5-3.3 years). RESULTS: There were 27 tumors with diploid deoxyribonucleic acid (DNA) content and 19 tumors were aneuploid. The median and mean Tpot for the 46 patients were 5.5 and 6.6 days, respectively [range, 2.0-25.6 days; coefficient of variation (CV), 74%]. For 25 patients where Tpot measurements were performed at two separate laboratories, there was a fair correlation (r = 0.74), but systematic differences were detected suggesting that the lack of agreement was not simply due to intratumoral variation. To date, 30 patients remained disease free, while 8 patients had pelvic failure and 9 patients developed distant metastases as the first failure site (1 patient developed both at the same time). In univariate analysis, the only significant prognostic factor for disease-free survival was tumor size (p = 0.004). A short Tpot (or high LI) and long overall treatment time (OTT) were weakly associated with poorer disease-free survival, although not statistically significant (1/Tpot, p = 0.14; LI, p = 0.23; OTT, p = 0.04). Age, FIGO stage, hemoglobin level, S-phase fraction, DNA ploidy, and Ts were not associated with disease-free survival. Multiple regression analysis was not performed because of the relatively small number of patients and short follow-up. CONCLUSIONS: Tpot values determined with current techniques by different laboratories cannot be used interchangeably for the purpose of therapy decisions. Vigorous quality assurance and standardization of the laboratory procedures and analysis methods are important to reduce interlaboratory variation. In this uniformly treated group of patients with cancer of the uterine cervix, traditional clinical prognostic factors remain the most important. Preliminary data suggest that the flow cytometry-determined Tpot and labeling index predict for disease-free survival, although a larger number of patients with longer follow-up is required to assess the true prognostic significance of these assays and to determine if their effect is independent of other clinical factors.

Adenocarcinoma↗

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries.

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.

Blotting, Southern↗

The influence of aging on spontaneous shifts of attention from external stimuli to the contents of consciousness.

In a series of studies using laboratory procedures and retrospective reports it has been established that with increasing age adults less frequently have unbidden task-unrelated image and thought intrusions (TUITs). TUITs--also referred to as daydreams--have been linked to the "current concerns" and "unfinished business" of the individual, and old adults have been shown to express fewer current concerns than young adults. It has also been hypothesized that selective loss of neurons in old age might interfere with thought production, resulting in fewer unbidden thoughts and spontaneous shifts of attention to them. In this article we examine the extent to which intraindividual change in the frequency of TUITs over 6 to 8 years is consistent with the decrease expected from the prior cross-sectional studies. In particular, we examine the frequency of daydreams based upon retrospective self-reports using the Daydreaming Frequency scale of the Imaginal Processes Inventory. The longitudinal sample consisted of 93 women and 169 men. Significant and equivalent decreases in Daydreaming Frequency scale values occurred at all ages. Longitudinal decreases were consistent with cross-sectional age differences. Thus, spontaneous shifts of attention to the contents of consciousness were seen to decrease over a 6 to 8 year interval within individuals--a result consistent with a within-individual change in conditions leading to spontaneous shifts.

Adult↗

Manual approaches to rapid microbiology results.

Rapid generation of microbiology results does not require sophisticated automated instruments. Many common laboratory procedures, such as microscopic examination of clinical specimens, use of manual screening tests, macroscopic examination of isolated colonies, and performance of rapid biochemical tests, can provide the physician with clinically useful information. These tests are neither expensive nor time-consuming and should be used routinely to generate rapid test results.

Bacteria↗

Kaposi's sarcoma: its 'oncogenes' and growth factors.

Viral genes capable of inducing vascular tumors in the skin of transgenic mice are the tat gene of HIV-1 and polyoma virus' middle T antigen gene. Instead of vascular tumors, the tat gene of HTLV-I causes thymic atrophy and mesenchymal tumors in transgenic mice. No proof exists that any of these genes contribute to the induction of KS but HIV-1 tat is a strong suspect. The gene product K-FGF of the oncogene K-fgf/hst (int) uses bFGF receptors, is homologous with bFGF and acts as a mitogen for fibroblasts, endothelial cells and melanocytes. The overexpression of the K-fgf gene in KS is not proven unequivocally; some doubts exist suggesting the activation of this gene during the laboratory procedure of transfection with KS cell heavy DNA. Growth factor(s) not well identified (IL-6?) are released from HTLV-I- or II, or HIV-1- or 2-infected T4 lymphocytes and in particular from HIV-1-infected macrophages. This growth factor(s) promote(s) the continuous proliferation of endothelial cells and KS cells. AIDS-KS cells release other growth factors identical with or closely related to basic FGF, a major inducer of angioneogenesis. In addition, acidic FGF, IL-1 alpha and -beta, GM-CSF, PDGF-B and TGF-beta are released from AIDS-KS cells. The release of GM-CSF is induced by IL-1. GM-CSF promotes granulocytic, monocytic and endothelial cell proliferation. TGF-beta is known to suppress lymphocyte-mediated cytotoxicity and may act as a local immunosuppressive factor together with interferon inactivators. We theorize that when TGF-beta production ceases, TNF-beta (lymphotoxin) production switches on leading to programmed cell death (apoptosis) of KS cells resulting in regression of these lesions. The newly discovered angiogenesis factors VEGF/VPF may emerge as protooncogene-oncogene products analogous to PDGF and c-sis activation. AIDS-KS heavy DNA transfects NIH3T3 cells. NIH3T3 cells carrying this gene induced angiosarcomas when implanted in mice. An as yet unidentified large virus (mycoplasma?) was derived from these cells during passages in culture. No causative relationship between this agent and Kaposi sarcoma has as yet been established. Even though IFN-alpha exerts antiretroviral effects in AIDS, we propose that the therapeutic effect of IFN-alpha in AIDS-KS is based on antiangiogenesis activity by suppressing protooncogenes-oncogenes of the FGF family.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired Immunodeficiency Syndrome↗

Association of behavioral inhibition with hair pigmentation in a European sample.

Behavioral inhibition, a temperamental trait signalling a predisposition to childhood and adolescent anxiety disorders, is slightly more frequent in America among Caucasian children having blue irises. This paper examines a community sample of 101 German toddlers assessed for behavioral inhibition in a standardized laboratory procedure. Hair pigmentation was found to be significantly associated with behavioral inhibition in the sense that blond children exhibited higher fear scores. As in American samples, blue-eyed children had a higher fear score than did other children, but this difference was not statistically significant.

Anxiety Disorders↗

Monitoring drug-protein interaction.

A variety of therapeutic drugs can undergo biotransformation via Phase I and Phase II enzymes to reactive metabolites that have intrinsic chemical reactivity toward proteins and cause potential organ toxicity. A drug-protein adduct is a protein complex that forms when electrophilic drugs or their reactive metabolite(s) covalently bind to a protein molecule. Formation of such drug-protein adducts eliciting cellular damages and immune responses has been a major hypothesis for the mechanism of toxicity caused by numerous drugs. The monitoring of protein-drug adducts is important in the kinetic and mechanistic studies of drug-protein adducts and establishment of dose-toxicity relationships. The determination of drug-protein adducts can also provide supportive evidence for diagnosis of drug-induced diseases associated with protein-drug adduct formation in patients. The plasma is the most commonly used matrix for monitoring drug-protein adducts due to its convenience and safety. Measurement of circulating antibodies against drug-protein adducts may be used as a useful surrogate marker in the monitoring of drug-protein adducts. The determination of plasma protein adducts and/or relevant antibodies following administration of several drugs including acetaminophen, dapsone, diclofenac and halothane has been conducted in clinical settings for characterizing drug toxicity associated with drug-protein adduct formation. The monitoring of drug-protein adducts often involves multi-step laboratory procedure including sample collection and preliminary preparation, separation to isolate or extract the target compound from a mixture, identification and determination. However, the monitoring of drug-protein adducts is often difficult because of short half-lives of the protein adducts, sampling problem and lack of sensitive analytical techniques for the protein adducts. Currently, chromatographic (e.g. high performance liquid chromatography) and immunological methods (e.g. enzyme-linked immunosorbent assay) are two major techniques used to determine protein adducts of drugs in patients. The present review highlights the importance for clinical monitoring of drug-protein adducts, with an emphasis on methodology and with a further discussion of the application of these techniques to individual drugs and their target proteins.

Biotransformation↗

Patent foramen ovale closure: historical perspective.

The work of the pioneers in PFO closure finally may evolve into a front-line therapy for secondary prevention of stroke in selected patients who have PFO and embolic events. Current implants, although new to most of interventional cardiology and neurology, are the result of years of steady, progressive work. It seems that PFOclosure is here to stay and will become a key element in a collaborative approach between cardiology and neurology in the treatment of embolic stroke. The advances in device technology may make PFO closure sufficiently safe, effective,and durable that it may become one of the most frequent catheter laboratory procedures to be performed.

Heart Septal Defects, Atrial↗

Novel use of the Friedewald formula to tackle anomalous HDL-C results in two cases of paraproteinaemia.

OBJECTIVES: We report here two cases of paraproteinaemia with one falsely low and the other dubiously high HDL-cholesterol (HDL-C) results. The spurious results seemed to be related to the nature (IgG or IgM) as well as the concentration of the paraproteins. DESIGN AND METHODS: We have been using an alternative approach to estimate the HDL-C concentration by incorporating into it the LDL-cholesterol (LDL-C) value obtained by direct measurements and by back-calculation based on the time-honored Friedewald equation in these atypical specimens as an interim measure, pending optimization of the Roche direct HDL-C plus assay currently in use in our laboratory. RESULTS: This approach is convenient and does not require sophisticated instrumentation. What we are suggesting is to tackle this analytical problems on HDL-C assay due to paraprotein interference by back-calculating the HDL-C values from the measured LDL-C and triglyceride values using the Friedewald formula and is to be regarded as an alternative way to circumvent the interference issue without the need for more elaborative laboratory procedures. We do not intend to advocate screening every single HDL-C value obtained by the direct method for possible analytical errors using this approach. CONCLUSIONS: The back-calculation for HDL-C based on the Friedewald formula is conceived by the authors as an alternative and relatively simple way to estimate the HDL-C value in the presence of paraprotein interference, in particular when there is a minus HDL-C value or when the result is dubiously high. By the same token, when the measured HDL-C and the calculated HDL-C do not match further investigations would be warranted to safeguard the validity of the reported result. It is also, to the best of our knowledge, the first time extra bands due to the IgM and IgG paraproteins were demonstrated in the lipoprotein electrophoresis plate.

Aged↗

Albumin locust valley: a new case of analbuminemia.

OBJECTIVE: To discover and document cases of the rare disease analbuminemia. DESIGN AND METHODS: Blood specimens of a subject were analyzed by routine clinical laboratory procedures, by immunochemical tests for albumin, and by electrophoresis with immunofixation using anti-human serum albumin (HSA). Single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing of the 14 exons of the HSA gene were conducted on DNA from leukocytes. RESULTS: Albumin concentration was 0.003 g/L; serum globulins and cholesterol were elevated. Immunoelectrophoresis showed no trace of albumin in any of the serum components. The coding region plus all mRNA splice sites were normal and mutation-free, and SSCP and HA showed no abnormalities. CONCLUSIONS: Data define a bona fide case of analbuminemia. We suggest that the mutation causing the analbuminemic trait in this subject might involve a remote regulatory element.

Adult↗

Minimization of the inevitable residual monomer in denture base acrylic.

OBJECTIVES: Residual monomer ([MMA](R)) in denture base acrylic continues to be of concern. The response surface of concentration vs. time and temperature for the equilibration of methyl methacrylate (MMA) and its polymer (PMMA) allows a prediction of the time to the minimum at any temperature for a closed system. It was the purpose here to determine whether this prediction applies to normal denture base processing, and whether optimum conditions could be identified. METHODS: Denture bases were processed following normal laboratory procedures, including pre-cure for 3 h at 70 degrees C for all tests. Commercial powder and liquid were used at either 95 or 100 degrees C, or a plain PMMA powder and the same liquid at 95 degrees C, for times ranging from 5 to 192 h. Residual MMA was determined by gas chromatography. RESULTS: [MMA](R) decreased steadily from approximately 0.25% to as low as approximately 0.07% with increasing time at temperature, but did not approach equilibrium. The rate of diffusive loss of MMA appears to exceed the rate of depolymerization. SIGNIFICANCE: Residual monomer is inevitable for all PMMA-based products no matter what the curing conditions are. However, extended time at high temperature can allow low values to be attained, and the time allowed can compensate for processing temperatures somewhat lower than the ordinarily recommended 100 degrees C. It is suggested that overnight processing at 95 degrees C should be adopted to minimize [MMA](R) and save energy. This result is of importance for work at high altitude.

Acrylic Resins↗

Application of new in situ hybridization probes for Ku70 and Ku80 in tissue microarrays of paraffin-embedded malignant melanomas: correlation with immunohistochemical analysis.

Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis. Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01). We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.

Adult↗

Wrongful convictions, lessons learned: the Canadian experience.

Following the wrongful conviction of a man for the sexual assault and murder of a child, the Province of Ontario commissioned a public inquiry to prevent future miscarriages of justice. The implementation of several recommendations regarding forensic laboratory procedure and the presentation of expert evidence has proven to be beneficial to the Canadian criminal justice system.

Child↗

Comparison of liquid chromatography with fluorescence detection to liquid chromatography-mass spectrometry for the determination of fluoxetine and norfluoxetine in human plasma.

A comparison study on fluoxetine (FL) and norfluoxetine (NORFL) quantitation in human plasma was carried out between the recently developed liquid chromatographic method with fluorescence detection (LC-FLD) and an earlier established liquid chromatography-mass spectrometry (LC-MS) laboratory procedure. Comparative method evaluation was based on the analysis of plasma samples obtained from Parkinsonian patients receiving 20mg of FL per day. The LC-FLD method involves a two-step liquid extraction procedure without any derivatization, followed by direct chromatography on a Zorbax C8 reversed-phase column. The analytical results are discussed in terms of the method validation and the corresponding experimental protocol (r>/=0.998; CV<9%; LOQ 20 microg/l). There was good correlation between FL, as well as NORFL, plasma levels as determined by the LC-MS and LC-FLD techniques (r=0.9597, N=16 and r=0.9852, N=14 for FL and NORFL, respectively). The results confirm that direct FL/NORFL fluorimetric determination is acceptable for routine use in pharmacokinetic and clinical studies.

Calibration↗

PCR detection of human papillomavirus of the mucosa: comparison between MY09/11 and GP5+/6+ primer sets.

BACKGROUND AND OBJECTIVES: Human papillomaviruses (HPV) are the causal agent for the development of carcinomas in the cervix uteri and further pathological changes of the skin including mucosa, particularly warts, condylomas and dysplasias. Therefore, we investigated the efficacy of different consensus primers pairs for HPV detection by PCR using brushed samples from the oral cavity in comparison with samples from the cervix uteri. STUDY DESIGN: In the present study, we used two well-established sets of PCR primers in different combinations for the detection of HPV DNA in 106 non-invasive brush biopsy samples of the oral mucosa and 56 samples from the cervix uteri. Direct sequencing of PCR products in all cases determined HPV genotype and specificity. RESULTS: Overall, HPV was detected in 69 of 106 oral mucosa samples. HPV specific amplicons were obtained in 35.8% (N = 38) when using GP5+/6+ primers. The positivity rate was increased to 65.1% in a GP5+/6+ auto-nested PCR approach. In contrast, MY9/11 PCR and nested PCR with MY9/11 outer followed by GP5+/6+ inner primers yielded 2.2% and 16.1%, respectively. In gynaecological samples, PCR results were similar independent of the primer combination used. Thus, DNA quality and DNA content could be additional factors influencing the rate of positivity. CONCLUSION: For oral mucosa samples, auto-nested GP5+/6+ PCR is in our hands the most suitable approach for epidemiological studies because of its high sensitivity, high reliability and reproducibility as well as its relatively simple laboratory procedure.

Biopsy↗

Site-specific DNA-nicking mutants of the heterodimeric restriction endonuclease R.BbvCI.

The restriction enzyme R.BbvCI cleaves duplex DNA within a seven base-pair asymmetric recognition sequence, thus: CCTCAGC/GCTGAGG-->CC--TCAGC/GC--TGAGG. We show that R.BbvCI comprises two different subunits, R(1) and R(2); that each subunit contains a catalytic site for DNA strand hydrolysis; and that these sites act independently and strand-specifically. In turn, each catalytic site was inactivated by mutagenesis to form dimeric enzymes in which only one site remained functional. The altered enzymes hydrolyzed just one strand of the recognition sequence, nicking the DNA rather than cleaving it. Enzymes in which the catalytic site in the R(1) subunit remained functional nicked the bottom strand of the sequence, producing CCTCAGC/GC--TGAGG, while those in which the catalytic site in the R(2) subunit remained functional nicked the top strand, producing CC--TCAGC/GCTGAGG. These DNA-nicking enzymes could prove useful for investigation of DNA repair, recombination, and replication, and for laboratory procedures that initiate from nicks, such as DNA degradation, synthesis, and amplification.

Amino Acid Sequence↗

Multivariate voxel-based morphometry successfully differentiates schizophrenia patients from healthy controls.

Currently available laboratory procedures might provide additional information to psychiatric diagnostic systems for more valid classifications of mental disorders. To identify the correlative pattern of gray matter distribution that best discriminates schizophrenia patients from healthy subjects, we applied discriminant function analysis techniques using the multivariate linear model and the voxel-based morphometry. The first analysis was conducted to obtain a statistical model that classified 30 male healthy subjects and 30 male schizophrenia patients diagnosed according to current operational criteria. The second analysis was performed to prospectively validate the statistical model by successfully classifying a new cohort that consisted of 16 male healthy subjects and 16 male schizophrenia patients. Inferences about the structural relevance of the gray matter distribution could be made if the individual profile of pattern expression could be linked to the specific diagnosis of each subject. The result was that 90% of the subjects were correctly classified by the eigenimage, and the Jackknife approach revealed well above chance accuracy. The pattern of the eigenimage was characterized by positive loadings indicating gray matter decline in the patients in the lateral and medial prefrontal regions, insula, lateral temporal regions, medial temporal structures, and thalamus as well as the negative loadings reflecting gray matter increase in the patients in the putamen and cerebellum. When the eigenimage derived from the original cohort was applied to classify data from the second cohort, it correctly assigned more than 80% of the healthy subjects and schizophrenia patients. These findings suggest that the characteristic distribution of gray matter changes may be of diagnostic value for schizophrenia.

Adult↗