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PubMed2005

Gut carbohydrases from the New Zealand marine herbivorous fishes Kyphosus sydneyanus (Kyphosidae), Aplodactylus arctidens (Aplodactylidae) and Odax pullus (Labridae).

Carbohydrase activities were examined in Odax pullus (Labridae), Kyphosus sydneyanus (Kyphosidae) and Aplodactylus arctidens (Aplodactylidae) collected from subtidal reefs in northeastern New Zealand. Enzyme extracts were prepared using two methods from gut wall, gut fluid and microbial pellet samples taken serially along the gut, and assayed against the substrates starch, laminarin, carrageenan, alginate and agarose. In all three fish species, starch degradation activity was substantially higher than for any other substrate tested. Activities of 500, 1294 and 3326 units g tissue(-1) were measured in anterior gut wall extracts of O. pullus, K. sydneyanus and A. arctidens, respectively. Starch degrading activity in gut fluid declined from 37, 313 and 284 units ml(-1) in anterior gut sections of O. pullus, K. sydneyanus and A. arctidens, respectively, to less than 50 units ml(-1) in terminal gut section of each species. Activity against structural polysaccharides was much lower than against starch and was detected mainly in posterior gut sections. The two methods of sample preparation differed little in enzyme activities; however, method of sample preparation did affect isoform patterns as displayed by zymogram analysis. Results suggest that these fish species fall on a continuum from maximizing throughput and digesting easily hydrolysed substrates in the foregut in A. arctidens to relying more heavily on microbial fermentation in the hindgut in K. sydneyanus.

Animals↗
PubMed2007

A double-blind placebo-controlled randomized study of Chinese herbal medicine as complementary therapy for reduction of chemotherapy-induced toxicity.

BACKGROUND: Chinese herbal medicine (CHM) is a common complementary therapy used by patients with cancer for reduction of chemotherapy-induced toxic effects. This study applied the highest standard of clinical trial methodology to examine the role of CHM in reducing chemotherapy-induced toxicity, while maintaining a tailored approach to therapy. PATIENTS AND METHODS: Patients with early-stage breast or colon cancer who required postoperative adjuvant chemotherapy were eligible for the study. Enrolled patients were randomly assigned to one of three Chinese herbalists who evaluated and prescribed a combination of single-item packaged herbal extract granules. Patients received either CHM or placebo packages with a corresponding serial number. The placebo package contained nontherapeutic herbs with an artificial smell and taste similar to a typical herbal tea. The primary end points were hematologic and non-hematologic toxicity according to the National Cancer Institute Common Toxicity Criteria Version 2. RESULTS: One hundred and twenty patients were accrued at the time of premature study termination. Patient characteristics of the two groups were similar. The incidence of grade 3/4 anemia, leukopenia, neutropenia, and thrombocytopenia for the CHM and placebo groups were 5.4%, 47.3%, 52.7%, and 1.8% and 1.8%, 32.2%, 44.7%, and 3.6%, respectively (P = 0.27, 0.37, 0.63, and 0.13, respectively). Incidence of grade 2 nausea was the only non-hematologic toxicity that was significantly reduced in the CHM group (14.6% versus 35.7%, P = 0.04). CONCLUSIONS: Traditional CHM does not reduce the hematologic toxicity associated with chemotherapy. CHM, however, does have a significant impact on control of nausea.

Adult↗
PubMed2004

Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR.

Currently used microbial markers cannot distinguish protozoal nitrogen (N) from bacterial N, thus limiting research on protozoal quantification in vivo by the lack of a repeatable, accurate marker for protozoal N. We report the development of a real-time PCR assay targeting the gene encoding 18S rDNA to quantify the amount of protozoal biomass in ruminal fluid and duodenal digesta. Protozoal cells were harvested from rumen fluid and concentrated for evaluation of recovery of rDNA in samples from the rumen and the duodenum. The DNA from concentrated cells was extracted with virtually 100% efficiency both before and after column purification. After serial spiking of protozoal cells into duodenal fluid over the entire range of quantification, the recovery was highly linear and constant at 81%. After serially spiking increasing quantities of protozoal rDNA into a constant volume of duodenal samples, nonlinear regression verified constant recovery of background rDNA in duodenal samples regardless of the ratio of target:nontarget rDNA. Recommendations for the procedure, including replication per sample, are described herein.

Animals↗
PubMed1989

Detection and identification of mycobacteria by amplification of mycobacterial DNA.

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.

Amino Acid Sequence↗
PubMed1996

Double-stranded RNAs in Korean isolates of Rhizoctonia solani AG4.

Eighty-one isolates of Rhizoctonia solani AG4 were obtained from soil samples with diverse geographic origins in Korea. Forty-five out of 81 isolates (56%) contained at least one dsRNA molecule with their sizes ranging from 2.3 to > 23.1 kb. Nine different sizes of dsRNA molecules were found and extensive variation in banding patterns was observed among isolates. By comparing the sizes and combinations of dsRNAs, 21 distinct banding patterns were observed. Cytoplasmic fractions from 3 isolates showed the same dsRNA band patterns as those from the total cell extracts. The dsRNAs were stable through 10 successive hyphal tip cultures and serial transfers onto the potato dextrose agar supplemented with cycloheximide or emetine. The presence or absence of dsRNAs was not apparently correlated with disease severity, phenol oxidase activity, and geographic origin.

Genetic Testing↗
PubMed1997

Computerized 3-D reconstruction of two "double teeth".

"Double teeth" is a root malformation in the dentition and the purpose of this study was to reconstruct three-dimensionally the external and internal morphology of two "double teeth". The first set of "double teeth" was formed by the conjunction of a mandibular molar and a premolar, and the second by a conjunction of a maxillary molar and a supernumerary tooth. The process of 3-D reconstruction included serial cross-sectioning, photographs of the sections, digitization of the photographs, extraction of the boundaries of interest for each section, surface representation using triangulation and, finally, surface rendering using photorealistic effects. The resulting three-dimensional representations of the two teeth helped us visualize their external and internal anatomy. The results showed: a) in the first case, fusion of the radical and coronal dentin, as well as fusion of the pulp chambers; and b) in the second case, fusion only of the radical dentin and the pulp chambers.

Anatomy, Cross-Sectional↗
PubMed1998

Herbal medicine for periodontal diseases.

Traditional Chinese Medicine (TCM) is a treasure-house which has shown miraculous clinical effects. It has aroused tremendous interests in elucidating its mechanisms both in China and abroad. This paper presents a review of published articles on the effects and mechanisms of herbal medicine on periodontal disease. Specifically, two modifications of an ancient compound prescription were serially studied, Guchiwan (Tooth-firming pills) and Guchigao (Tooth-firming extract). Some in vitro studies have shown suppressive effects of various herbal extracts on bone resorption by isolated osteoclasts. TCM should be intensively studied for their possible use in periodontics.

Animals↗
PubMed1970

Marihuana and temporal disintegration.

High oral doses of marihuana extract, calibrated for content of 1 (-)-Delta(1)-tetrahydrocannabinol, significantly impaired the serial coordination of cognitive operations during a task that required sequential adjustments in reaching a goal. This disintegration of sequential thought is related to impaired immediate memory.

Adult↗
PubMed2004

Oct-1 and nuclear factor Y bind to the SURG-1 element to direct basal and gonadotropin-releasing hormone (GnRH)-stimulated mouse GnRH receptor gene transcription.

The cis-regulatory element localized to position -292/-285 of the mouse GnRH receptor (mGnRHR) gene promoter, designated Sequence Underlying Responsiveness to GnRH 1 (SURG-1), has been shown previously to contribute to stimulation of mGnRHR gene expression by GnRH. We have identified three specific protein-DNA complexes on the SURG-1 element by EMSA using nuclear extracts from the gonadotrope-derived alphaT3-1 and LbetaT2 cell lines. Serial mutagenesis and supershift assays identified nuclear factor Y (NF-Y) binding to -288/-284 and Oct-1 binding to a TAAT sequence at -290/-287. Binding of these two transcription factors was confirmed in vivo by chromatin immunoprecipitation assay and increased in response to GnRH stimulation. To define the functional significance of these sequences in the regulation of mGnRHR gene transcription, transient transfection assays were performed in alphaT3-1 cells using a 1.2-kb mGnRHR (-1164/+62) gene promoter-luciferase reporter construct with selective mutations of the Oct-1, NF-Y, and/or the previously characterized activating protein 1 (AP-1) binding site (-274/-268). Individual mutations in the Oct-1, NF-Y, and AP-1 sites decreased both basal expression and stimulation by GnRH agonist, and the combined mutation of the Oct-1 and AP-1 binding sites further reduced basal transcriptional activity and abolished GnRH stimulation. Overexpression of NF-YA increased GnRHR promoter activity, whereas expression of a dominant negative NF-YA mutant decreased activity, further supporting a role of NF-Y in regulation of mGnRHR gene transcription. In addition, knockdown of Oct-1 by small interfering RNA confirmed that Oct-1 is important for mGnRHR gene expression. In conclusion, NF-Y and Oct-1 bind to the SURG-1 element to direct basal and GnRH-stimulated expression of the mGnRHR gene.

Animals↗
PubMed2001

Comparison of HER2/neu status assessed by quantitative polymerase chain reaction and immunohistochemistry.

We prospectively evaluated a series of 254 breast cancers by quantitative polymerase chain reaction (PCR) and immunohistochemistry using 3 antibodies: HercepTest, CB11, and TAB250. DNA was extracted from a 10-micron tumor section for PCR, and 4-micron serial sections were taken from the same block for immunohistochemistry. The immunohistochemical results were scored using a semiquantitative immunohistochemical system. A positive tumor by immunohistochemistry had a score of 5 or more. The manufacturer's recommended scoring system was used for the HercepTest. Tumors were positive for gene amplification if the ratio of the HER2/neu gene to control gene after normalization was 2 or more. Of 254 cases, 61 showed gene amplification. For immunohistochemistry, 23% of tumors were positive with CB11, 27% with TAB250, and 37% with the HercepTest. Results for each antibody were compared with PCR results. The overall concordance for the HercepTest was 82%, which was significantly lower than that for CB11 (88%) or TAB250 (87%). The specificity for the HercepTest was 80% compared with 90% for TAB250 and 93% for CB11, while the positive predictive value for the HercepTest was 57% compared with 71% and 76% for TAB250 and CB11, respectively.

Antibodies, Monoclonal↗
PubMed2004

Small amplified RNA-SAGE.

Serial analysis of gene expression (SAGE) is a powerful genome-wide analytic tool to determine expression profiles. Since its description in 1995 by Victor Velculescu et al., SAGE has been widely used. Recently, the efficiency of the method has been emphasized as a means to identify novel transcripts or genes that are difficult to identify by conventional methods. SAGE is based on the principle that a 10-base pair (bp) cDNA fragment contains sufficient information to unambiguously identify a transcript, provided it is isolated from a defined position within this transcript. Concatenation of these sequence tags allows serial analysis of transcripts by sequencing multiple tags within a single clone. Extraction of sequence data by computer programs provides a list of sequence tags that reflect both qualitatively and quantitatively the gene expression profile. Several modifications to the initial protocol allowed to start from 1 microg total RNA (or 10(5) cells). In order to reduce the amount of input RNA, protocols including extra polymerase chain reaction (PCR) steps were designed. Linear amplification of the mRNA targets might have advantage over PCR by minimizing biases introduced by the amplification step; therefore we devised a SAGE protocol in which a loop of linear amplification of RNA has been included. Our approach, named "small amplified RNA-SAGE" (SAR-SAGE) included a T7 RNA polymerase promoter within an adapter derived from the standard SAGE linker. This allowed transcription of cDNA segments, extending from the last NlaIII site of transcripts to the polyA tail; these small amplified RNAs then serve as template in a classical (micro)SAGE procedure. As the cDNAs are immobilized on oligo(dT) magnetic beads, several rounds of transcription can be performed in succession with the same cDNA preparation, with the potential to increase further the yield in a linear way. Except for the transcription step itself, the present procedure does not introduce any extra enzymatic reaction in the classical SAGE protocol, it is expected to keep the representation biases associated with amplification as low as possible.

Animals↗
PubMed1983

Inhalation studies of diesel exhaust and coal dust in rats.

The preliminary 12-month data of an ongoing two-year chronic inhalation study into the effects of diesel exhaust and coat dust on rats are reported. The study design consists of four exposure regimens: coal dust, diesel exhaust, coal dust plus diesel exhaust, and filtered air (control). Total respirable particulate is being maintained at 2 mg/m3 in all exposure groups. Animals are being serially sacrificed and subjected to immunologic, biochemical, physiological, microbiological, and pathological evaluations. Extracts of diesel emissions showed marked mutagenic activities in the Ames Salmonella his reversion assay. Mutagenic compounds were not however found in the urine of the exposed animals, nor were sister chromatid exchanges detected in peripheral lymphocytes. No changes were observed in cytochrome P450 enzyme activity in lung microsomes of exposed rats. Physiological tests showed no evidence of pulmonary function impairment in any group. Light microscopic examination of the lungs showed dust deposition with the formation of macules in all exposed groups. The preliminary results revealed few pathological effects of significance and no evidence of synergism between diesel exhaust and coal dust.

Animals↗
PubMed1975

Extraction of a factor from Ehrlich ascites tumor cells that increases the activity of the fetal isozyme of pyruvate kinase in mouse liver.

Isoelectrofocusing studies of mouse tissue extracts show mice to have a pyruvate kinase isozyme pattern very similar to that of the rat. Moreover, electrofocusing or kinetic assays conducted on liver extracts from normal mice and from mice bearing Ehrlich ascites tumors show that the latter have a higher proportion of the fetal K-isozyme of pyruvate kinase. Serial injection of the supernatant remaining after centrifugation of homogenized tumor cells at 100,000 x g, or of the phenolic extracts from the latter, produced a similar shift in the liver isozyme pattern involves both a decrease in L-isozyme activity and an increase in K-isozyme activity. However, only the increase in activity of the K-isozyme appears to be a specific response to injection of the extracts. The presence of a specific factor in these extracts was confirmed by the observation that similar extracts prepared from normal adult tissues did not increase activity of the K-isozyme. On the other hand, phenolic extracts from fetal mice did increase K-isozyme activity as did injections of serum from tumor-bearing mice or of the cell-free ascites fluid. Evidence is presented supporting the concept that the factor is proteinaceous in a nature, and that it acts by deprepressing synthesis of the K-isozyme.

Animals↗
PubMed1992

Standardized extracts.

Until recently, physicians practicing allergy have not had the benefit of using allergenic extracts with potencies expressed in uniform, meaningful units. Technologic advances have provided accurate and reproducible methods for qualitative and quantitative analyses of these products, and assignment of meaningful units can be accomplished by using a threefold serial dilution titration skin test and appropriate in vitro methods. Although the perfect method for standardization of allergenic extracts has not yet been developed and some confusion has been created in the process, significant improvement in the quality of allergenic extracts has resulted. Collaborative efforts of regulatory agencies, manufacturers, specialty organizations, and clinicians should continue to produce standardized extracts that offer significant advantages to physicians practicing allergy.

Allergens↗
PubMed1999

Three-dimensional magnetic resonance imaging of the interosseous membrane of forearm: a new method using fuzzy reasoning.

We now report newly developed three-dimensional magnetic resonance imaging (3D-MRI) system which is based on semiautomatic tissue extraction from the axial MR images utilizing the fuzzy reasoning calculation method and 3D-image reconstruction with surface rendering. We also studied normal in vivo dynamic changes of the interosseous membrane (IOM) of forearm during rotation using this 3D-MRI. Serial axial MRI of right forearms of five healthy volunteers was obtained in five rotational positions, and extraction and 3D-reconstruction of the radius, ulna, and IOM was made using the system. Extraction results were well with the fuzzy reasoning method. 3D-MRI of the radius and ulna, IOM were reconstructed from these images respectively, and their 3D-shapes were almost identical to the anatomic shape. 3D-MRI showed there were wavy deformities on the IOM in pronation position in the all five subjects and dorsiflexion on the most dorsal portion of the IOM at maximum supination in three forearms. In neutral position, the IOM of all five volunteers was almost flat. From anatomic orientation, these dynamic changes of the IOM mainly occurred at the membranous portion, which is soft, thin, and elastic. Otherwise, the tendinous portion which is a thick and strong complex of 5 to 10 bundles run from proximal one third of the radius to distal one fourth of the ulna, demonstrated minimal dynamic changes on the 3D-MRI. Therefore, the tendinous portion is considered to be taut during rotation to provide stability between the radius and the ulna, while the membranous portion is easy to deform and allowing smooth rotation. Furthermore, because of wide-use, our 3D-MRI system is useful for in vivo analysis of soft tissue kinesiology in normal and abnormal musculoskeletal systems.

Adult↗
PubMed1997

Comparative analysis of plasminogen activator inhibitor-1 expression in different types of atherosclerotic lesions in coronary arteries from human heart explants.

OBJECTIVES: Clinical manifestations of coronary heart disease result primarily from the progressive development of atherosclerotic plaques and subsequent thrombus formation: processes which may be accelerated by an enhanced expression of plasminogen activator inhibitor (PAI-1) in the vessel wall. In the present study, content and expression of PAI-1 were comparatively analyzed in human coronary arteries in relation to the presence and severity of atherosclerotic lesions. METHODS: Segments of coronary arteries obtained from heart explants (n = 15) were classified by the presence and types of atherosclerotic lesions. Antigen and activity levels of PAI-1 were determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. RESULTS: Total PAI-1 antigen consistently increased from macroscopically normal areas (MNAs) to early lesions (ELs) and to maximal levels in fibrous (FPs) and calcified (CPs) plaques. No PAI activity was detected, although PAI-1 in its free form was present in all vascular specimens. Both free PAI-1 and PAI-1 complexed with plasminogen activators were significantly increased in extracts of advanced lesions. However, there was a 2-3 fold molar excess of free versus complexed PAI-1 in FPs and CPs. These findings suggest the presence of relevant amounts of PAI-1 in its substrate rather than in its inhibitor conformation in areas of advanced lesions. Compared with MNAs, PAI-1 mRNA was strongly expressed within the thickened intima of ELs. The highest PAI-1 expression was observed in FPs and CPs, being mainly localized in areas surrounding the necrotic cores in co-localization with infiltrating macrophages. CONCLUSIONS: PAI-1 content is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. The concomitant elevation of PAI-1 mRNA suggests that the PAI-1 increase in regulated by local synthesis in the areas of atherosclerotic lesions.

Antigens↗
PubMed1980

Deposition of ragweed pollen and extract on nasal mucosa of patients with allergic rhinitis: effect on nasal airflow resistance and nasal mucus velocity.

This study was undertaken to ascertain whether nasal mucus velocity (NMV) could be altered by short-term exposure to antigen. Asymptomatic patients with a history of allergic rhinitis who had a positive cutaneous reaction to ragweed extract were investigated. The plan was to achieve approximately a fourfold elevation of nasal airflow resistance (NAR) with antigen challenge and then obtain serial measurements of NAR and NMV. NMV was not significantly altered when the antigen was introduced by nasal inhalation of (1) ragweed pollen grains, (2) nebulized ragweed extract for 10 breaths, and (3) nebulized ragweed extract for 30 min on each of 3 successive days. When ragweed extract was introduced by direct instillation of the solution into the nose, NMV fell below baseline values at either 0.5 or 1.5 hr, or at both times after administration. Persistence of impairment of mucociliary transport at a time when nasal airway constrictor response had dissipated suggested that a chemical mediator might have been responsible for the alteration of clearance. The failure to demonstrate depression of mucus transport with the inhalation studies might have been due to insensitivity of the radiopaque Teflon disk method or to a qualitatively different allergic reaction to direct instillation of antigen solution.

Adult↗
PubMed1994

"Lupinosis"-like lesions in mice caused by ustiloxin, produced by Ustilaginoieda virens: a morphological study.

A crude toxin, obtained by methanol-extraction of a water extract of false smut balls (INA-KOUJI in Japanese) caused by Ustilaginoidea virens on rice panicles, was injected into mice intraperitoneally. Single injection of 100 or 200 mg/kg body weight of the extract was not lethal but caused acute, occasional necrosis of hepatocytes and renal tubular cells, followed by increased number of mitotic figures with occasional multinuclear giant cells. Erosions and ulceration of the forestomach and atrophy of the thymus were observed a week later. Repeated intraperitoneal injection of the crude toxin at the levels of 12 and 25 mg/kg body weight induced more severe necrosis of the liver and kidneys, with delayed occurrence of mitosis. Forestomach erosion also occurred. Serial injection for 10-12 days of either 3 or 6 mg/kg of the crude extract or 400 micrograms/kg of ustiloxin A, using the purified crystals, caused relatively mild but definite liver and kidney lesions similar to those described above. The lesions in the liver and kidney were quite similar to those observed in lupinosis caused by phomopsin A, a mycotoxin produced by Phomopsis leptostromiformis. Isolation of the toxic substance indicates that the contaminated rice panicles may cause toxicosis of cattle, although no field outbreaks have occurred yet.

Animals↗