[Improvement of the dissolution behavior of problem drugs. 4. Differential thermal analysis of iomeglamic acid-succinic acid embedding].
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In studies on the onset and progression of cancer, molecular biology has proven to be very useful. Reasoning that tumorigenesis can be considered as a multistep process in which a normal cell gradually escapes from its delicately regulated growth pattern, one might describe this process in terms of gene expression. The group of genes that are good candidates for such studies are the proto-oncogenes. Recent progress in oncogene research, however, has made us believe that the group of oncogenes is much greater than the 50 so far characterized, since all growth factor-, growth factor receptor-, signal transducer- and transcription-regulating genes have a potency to become oncogenic. Alternatively, recessive genes, such as tumor suppressor genes, might be equally relevant to the onset and progression of cancer. Therefore, new approaches in molecular studies on the genesis and progression of malignancies are urgently needed. One of these methods is the so-called differential or subtraction hybridization analysis. Particularly, the rationale of differential hybridization and the first applications to prostate cancer research are discussed in this paper.
The purpose of this study was to apply a novel method of multiscale echo texture analysis for distinguishing benign (hemangiomas) from malignant (hepatocellular carcinomas (HCCs) and metastases) focal liver lesions in B-mode ultrasound images. In this method, regions of interest (ROIs) extracted from within the lesions were decomposed into subimages by wavelet packets. Multiscale texture features that quantify homogeneity of the echogenicity were calculated from these subimages and were combined by an artificial neural network (ANN). A subset of the multiscale features was selected that yielded the highest performance in the classification of lesions measured by the area under the receiver operating characteristic curve (Az). In an analysis of 193 ROIs consisting of 50 hemangiomas, 87 hepatocellular carcinomas and 56 metastases, the multiscale features yielded a high A: value of 0.92 in distinguishing benign from malignant lesions, 0.93 in distinguishing hemangiomas from HCCs and 0.94 in distinguishing hemangiomas from metastases. Our new multiscale texture analysis method can effectively differentiate malignant from benign lesions, and thus has the potential to increase the accuracy of diagnosis of focal liver lesions in ultrasound images.
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Long-term administration of antipsychotic drugs can induce differential expression of a variety of genes in the brain, which may underscore the molecular mechanism of the clinical efficacy and/or side effects of antipsychotic drugs. We used cDNA microarray analysis to screen differentially expressed genes in rat frontal cortex under 4 weeks' treatment of risperidone (1 mg/kg). Using real-time quantitative PCR, we were able to verify eight genes, whose expression were significantly upregulated in rat frontal cortex under chronic risperidone treatment when compared with control animals. These genes include receptor for activated protein kinase C, amida, cathepsin D, calpain 2, calcium-independent receptor for alpha-latrotoxin, monoamine oxidase B, polyubiquitin, and kinesin light chain. In view of the physiological function of these genes, the results of our study suggest that chronic risperidone treatment may affect the neurotransmission, synaptic plasticity, and proteolysis of brain cells. This study also demonstrates that cDNA microarray analysis is useful for uncovering genes that are regulated by chronic antipsychotic drugs treatment, which may help bring new insight into the molecular mechanism of antipsychotic drugs.
This article links clinical research to fundamental orthodontic concepts to give the clinician a workable differential diagnosis system. The clinical research, conducted by the Charles Tweed Foundation, attempted to establish a "profile" for the Class II malocclusion correction, which, because of certain characteristics, was destined to failure. The Cranial Facial Dental Analysis integrates this clinical research with the total space analysis to give the clinical orthodontist a useful tool for differential diagnosis.
OBJECTIVE: To investigate differential protein expression profiles of human colorectal carcinoma cell lines with different metastatic potentials and screen metastasis-associated proteins for analyzing the relationship between metastasis-associated proteins and the tumorigenesis, progression and metastasis of colorectal carcinomas. METHODS: With two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry, we analyzed the differentially expressed proteins in two human colorectal carcinoma cell lines SW620 and SW480 with high and low metastatic potentials, and screened for proteins associated with colorectal carcinoma metastasis. RESULTS: Image analysis software Malenie III demonstrated good match and reproducibility of the 2-DE maps obtained from 3 independent experiments, with 1316+/-62 spots detected for SW620 cells and 1332+/-74 spots for SW480 cells with the average matching rate of 82% and 80%, respectively. The spots distributed in the greatest density at the isoelectric points of 4-7 and relative molecular mass weight of 20,000-70,000. Twenty-five distinctly different protein spots (14 spots for SW620 and 11 for SW480) in-gel digested by TPCK trypsin and 23 peptide mass fingerprint maps were obtained by mass spectrometry. Three highly matched proteins and 14 preliminarily matched proteins or fragments were obtained by analysis with Mascot software in the NCBInr database. Some of these differentially expressed proteins were related to gene transcription, cell cycle, signal transduction, cell apoptosis, etc, with possible involvement of cell differentiation, proliferation, invasion, adhesion, and metastasis of colorectal carcinoma. CONCLUSION: The 2-DE protein expression profile of SW620 cells with high metastatic potential displays obvious difference from that of SW480 cells with low metastatic potential, and a variety of proteins can be involved in the metastasis of colorectal carcinoma.
We compared Doppler spectral parameters in acute inflammatory, reactive, lymphomatous, and metastatic lymph nodes, and evaluated pulsed Doppler sonography as a method for distinguishing between different causes of cervical lymphadenopathy. Spectral Doppler analysis with measurements of resistance index (RI), pulsatility index (PI), peak systolic velocity (PSV), and end-diastolic velocity (EDV) was performed in 197 patients with cervical lymphadenopathy. Results of Doppler analysis were compared with findings of cytology and histology or with clinical presentation and follow-up. Student's t-test was used to assess statistical significance of differences in Doppler parameters between groups of patients. Significant differences for RI and PI were shown between all groups of patients except between lymphomatous and reactive nodes. Specificity of 100% for metastatic nodal involvement was shown for cutoff values RI>0.80 and PI>1.80. A positive predictive value (PPV) of 100% for acute lymphadenitis was shown for cutoff values RI<0.50 and PI<0.60. An EDV>9 cm/s has 100% negative predictive value for nodal metastasis, and EDV<1 cm/s has 100% specificity and PPV for metastasis. Although there exist differences in RI, PI, PSV, and EDV between different nodal diseases, none of these parameters offer both good sensitivity and good specificity, and only extreme cutoff values may occasionally be helpful in differential diagnosis. Doppler spectral analysis is a valuable noninvasive adjunct which can help in differentiation between metastatic, lymphomatous, acute inflammatory, and reactive lymphadenopathy, but cannot obviate biopsy in the majority of cases.
PURPOSE: To identify differentially expressed genes in mouse bone marrow involved in radiation-induced injury. MATERIALS AND METHODS: Microarray analysis was used to identify the differentially expressed genes and other techniques, e.g., polymerase chain reaction (PCR), western-blotting and antisense were also used to validate the results. RESULTS: DNA microarray analysis demonstrated that the mRNA of 34 genes increased and 69 genes decreased in mouse bone marrow cells (BMC) from C57BL mice 6 h after a whole body dose of 6.5 Gy. These differentially expressed genes were involved in a number of processes including DNA replication/repair, proliferation/apoptosis, cell cycle control and RNA processing. In these experiments, a decline of the mammalian homolog Sir2a (an acronym for the silent mating type information regulation 2 homolog [SIRT1]) mRNA accompanied by an increase of P53 protein acetylation was observed in irradiated BMC. To determine whether the reduced SIRT1 is related to the higher acetylation status of P53 after irradiation, we designed and synthesized antisense oligonucleotides (AS) targeting human SIRT1 mRNA. Notably, AS transfection increased tumor protein 53 (P53) protein acetylation and bax-luciferase activity in human bone marrow stromal cell line (HS-5) after radiation. Furthermore, the AS transfer stimulated cell apoptosis in post-irradiation HS-5 cells. CONCLUSION: Ionizing radiation (IR) affects the expression of a series of genes including genes involved in G1/S transition and the P53 pathway. Among those, reduction of SIRT1 was seen to be involved in transactivation of P53.
Microarray analysis of RNA from diclofenac-administered mouse livers was performed to establish a global gene expression profile during injury and recovery stages at two different doses. A single dose of diclofenac at 9.5 mg/kg or 0.95 mg/kg body weight was given orally, and the liver samples were obtained after 6, 24, and 72 h. Histopathologic studies enabled the classification of the diclofenac effect into injury (6, 24 h) and recovery (72 h) stages. By using the Applied Biosystems Mouse Genome Survey Microarray, a total of 7370 out of 33,315 (22.1%) genes were found to be statistically reliable at p<0.05 by two-way ANOVA, and 602 (1.8%) probes at false discovery rate <5% by Significance Analysis of Microarray. Among the statistically reliable clones by both analytical methods, 49 genes were differentially expressed with more than a 1.625-fold difference (which equals 0.7 in log(2) scale) at one or more treatment conditions. Forty genes and two genes were identified as injury- and recovery-specific genes, respectively, showing that most of the transcriptomic changes were seen during the injury stage. Furthermore, multiple genes involved in oxidative stress, eicosanoid synthesis, apoptosis, and ATP synthesis showed variable transcript levels upon acute diclofenac administration.
Low-resolution pyrolysis gas-liquid chromatography (p.g.l.c.) can differentiate genera of aerobic food spoilage bacteria. Multivariate statistical techniques were applied but neither principal components nor furthest neighbour cluster analysis produced a consistent differentiation although both confirmed the reproducibility of p.g.l.c. When the distance between genera was redefined in terms of Mahalanobis D2- a generalized concept taking into account scatter around the mean-good differentiation was observed and could be displayed graphically by plotting the genus group means relative to the first two canonical variate axes. The coefficients of the canonical variates provide a strategy for discriminating between the genus groups. Some practical problems in the identification of unknowns using this technique are discussed.
We have used bidirectional transfer methods in concert with SMART total cDNA complex probes to sequentially screen differential display arrays. In this report we show the utility of this methodology in examining a manganese superoxide dismutase cDNA fragment which we detected while evaluating the effects of the proinflammatory cytokines IL1-beta, TNF-alpha, and IL6 on human umbilical vein endothelial cell (HUVEC) gene expression. By using parallel hybridization of the bidirectional blots with SMART total cDNA (32)P probes derived from untreated or cytokine-treated HUVECs, differential expression between cell treatments can be clearly evaluated. Subsequent screening using this bidirectional blot method results in detection of modulated cDNA clones. Northern and total cDNA blot hybridization with the cDNA clonal fragment confirmed both modulated expression and the efficacy of this screening method. These procedures allow one to use bidirectional blots to evaluate band modulation on agarose gels which are initially run to evaluate the reamplification of display fragments or to confirm cloned cDNA fragments. Thus, bidirectional blot analysis using SMART total cDNA probes allows direct evaluation of differential display bands from the initial reamplification through plasmid insert cloning, increasing the investigator's ability to eliminate false-positive bands during each step of analysis.
Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia.
This review deals with the application of differential display to investigate gene expression in plants. A substantial articles reports the isolation and profiling of various genes expressed in cells using this technique. Genes involved in physiological events, stress responses, signal transduction and secondary metabolism have been isolated and characterized. Some of the isolated genes encode transcription factors, membrane proteins and rare enzymes that were previously difficult to purify. These results suggest that differential display is a powerful tool used to investigate the rare genes involved in the plant life cycle without using information from proteins.
BACKGROUND: Microarray analysis is a popular tool to investigate the function of genes that are responsible for the phenotype of diseases. Keloid is an intricate lesion that is probably modulated by interplay of many genes. We ventured to study the differences of gene expressions between keloids and normal skin with the aid of a cDNA microarray to explore the molecular mechanism underlying keloid formation. MATERIALS AND METHODS: The polymerase chain reaction products of 8400 human genes were spotted on a chip in array. The DNAs were then fixed on the glass plate by a series of treatments. Total RNAs were isolated from freshly excised human keloids and normal skins and then were purified to mRNAs by Oligotex. Both the mRNAs from keloids and normal skins were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP for preparing the hybridization probes. The mixed probes were then hybridized to the cDNA microarray. After highly stringent washing, the cDNA microarray was scanned for the fluorescent signals to display the differences between two kinds of tissues. RESULTS: Among 8400 human genes, there were 402 genes (4.79%) with different expression levels between the keloids and normal skins in all cases, 250 genes, including TGF-beta1 and NGF, were upregulated (2.98%) and 152 downregulated (1.81%). Analyses of collagen, fibronectin, proteoglycan, growth factors, and apoptosis-related molecule gene expression confirmed that our molecular data obtained by cDNA microarray were consistent with the published biochemical and clinical observations of keloids. Higher expression of TGF-beta(1) and NGF in keloids versus normal skins was also testified with reverse transcription polymerase chain reaction method. CONCLUSIONS: DNA microarray technology is an effective technique in screening for differences in gene expression between keloid and normal skin. Many genes are involved in the formation of keloids. Further analysis of the obtained genes will help to understand the molecular mechanism of keloid formation.
BACKGROUND: Only sporadic cases of prostate carcinomas with squamous differentiation have been reported. DESIGN: The files of two institutions were reviewed for prostate cancers with squamous differentiation. RESULTS: A total of 33 cases were studied. The average age at diagnosis was 68 years (range 49-86 years). The most common presenting symptoms included bladder outlet obstruction and dysuria. Thirteen men had a positive digital rectal examination. Diagnosis was made by needle biopsy (n = 23); transurethral resection of the prostate (n = 5); needle and transurethral resection of the prostate (n = 1); transurethral resection of the bladder (n = 1); or biopsy of metastases (n = 3). In 21 of 33 cases, there was a prior diagnosis of adenocarcinoma of the prostate; 8 patients were treated with hormones, 4 were treated with radiation, and 1 received both radiation and hormone therapy. Of the 12 men without a prior diagnosis of adenocarcinoma, 2 patients had received hormonal therapy for benign prostatic hyperplasia. Eight of 33 cases were pure squamous carcinomas. The remaining cases were adenosquamous carcinoma (n = 16), adenosquamous and urothelial carcinoma (n = 3), and adenosquamous carcinoma and sarcoma (n = 6). The squamous carcinoma component of these mixed cases averaged 40% of the tumor volume (range 5%-95%) and had a range of cytologic atypia (mild [n = 6], moderate [n = 17], severe [n = 10]). In the 25 cases with adenocarcinoma, the glandular component tended to be high-grade (Gleason grade >6 in 19 cases). Immunohistochemistry for prostate specific acid phosphatase and prostate specific antigen was positive in a large percentage of the adenocarcinomas (85% and 75%, respectively) and only very focally positive in 12% of the squamous carcinomas. 34 beta E12 was diffusely positive in >95% of the squamous carcinomas and only focally positive in <10% of the adenocarcinomas. Cytokeratins 7 and 20 did not differentiate the squamous and adenocarcinoma components. Follow-up was available on 25 of 33 cases, with the average survival being 24 months (range 0-63 months). CONCLUSION: Squamous differentiation in prostate cancer is uncommon, often but not necessarily arising in the setting of prior hormone or radiation therapy, and is associated with a poor prognosis. In addition to pure squamous cell carcinoma and adenosquamous cancer, other patterns may be seen. Whereas the adenocarcinoma component is typically high grade, the squamous component has a wide range of differentiation.
Monoclonal antibodies were used to examine the immunoglobulin isotypes expressed by B lymphocyte precursors of IgM, IgG, IgA, and IgA2 plasma cells. Plasma-cell differentiation was induced by the addition of pokeweed mitogen to cultures of blood mononuclear cells. Anti-mu, -gamma, -alpha, and -alpha 1 antibodies were used in some experiments to inhibit differentiation of B lymphocytes bearing these heavy chain isotypes, and for selective removal of B lymphocyte precursors before culture with pokeweed mitogen in other experiments. Three major subpopulations of B lymphocyte precursors were identified: (a) a subpopulation of surface (s) IgM+ precursors of IgM plasma cells that did not express IgG or IgA isotypes, (b) a subpopulation of sIgG+ precursors of IgG plasma cells of which approximately one-half bore some IgM and none had detectable IgA receptors, and (c) a subpopulation of sIgA+ precursors of IgA plasma cells; one half of these precursors could be shown to express functional IgM receptors but none were found to express IgG receptors. The sIgA subpopulation could be further subdivided into sIgA1+ precursors of IgA1 plasma cells and IgA1-negative precursors of IgA2 plasma cells. These results suggest that normal human B cells can switch from mu directly to each of the other heavy chain isotypes, and that these represent the main switch pathways.
An open architecture mRNA profiling technology, LEAD (Ligation specificity-based Expression Analysis Display), was developed for studying differential gene expression. This method utilizes restriction enzymes with N(m) degeneracy in their recognition/cleavage sequences to fractionate cDNA population (N represents any one of the four possible bases; while m > or = 1, represents the number of degenerate bases). The fractionated cDNAs are subpooled by selective ligation with specific adapters, and then amplified and labeled by PCR. Fluorescent-labeled cDNA fingerprints are separated by electrophoresis as distinct bands with unique size and sequence, and quantified electronically by using the LEAD Finder program. The specificity of ligation and the uniform efficiency of PCR reaction allow precise quantification of differential gene expression among samples. Transcripts of low abundance (1/100,000 copies) can be detected, allowing the status of nearly all mRNA to be monitored. Because of its sequence independence, this technology can be used to monitor gene expression in both model and nonmodel systems lacking whole genome information. It can also be applied to separate and collect different cDNA species fingerprints to build a nonredundant EST library for microarray and other applications.