Hybridization histochemistry

Summary The location of gene expression by hybridization histochemistry is being applied in many areas of research and diagnosis. The aim of this technique is to detect specific mRNA in cells and tissues by hybridization with a complementary DNA or RNA probe. Requirements for optimal specificity, sensitivity, resolution and speed of detection may not all be encompassed in one simple technique suitable for all applications, thus appropriate procedures should be selected for specific objectives. With reference to published procedures and our own extensive experience, we have evaluated fixatives, probes, labels and other aspects of the technique critical to the preservation and hybridization in situ of mRNA and detection and quantition of hybrids.     

Molecular diagnosis of parasites

Summary New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene clonign and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Molecular diagnosis of parasites

New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene cloning and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Chymotrypsin gene expression during the intermolt cycle in the shrimpPenaeus vannamei (Crustacea; Decapoda)

InPenaeus vannamei, chymotrypsin is present as two isoenzymes in the hepatopancreas. The enzyme has been localized in F-cells by immunocytochemistry using a specific antibody. By in situ hybridization, with a 510 pb cDNA probe encoding for the first 170 amino acids of the shrimp chymotrypsin, mRNA was localized in the same cells. Gene expression was followed during the intermolt cycle by measuring changes in specific activity in crude extracts, and by the estimation of mRNA levels by Northern blots using the same probe. The increase in specific activity in premolt is preceded in early premolt by an increase in the amount of chymotrypsin mRNA. A second increase is observed in postmolt, suggesting a different mode of regulation of gene expression.     

In situ hybridization analysis of globin mRNAs in the primitive erythroid cells of the chick embryo

The possibility that the minor embryonic chick hemoglobins might be present in a particular subgroup of primitive erythroid cells has been investigated by in situ hybridization. Probe to detect the mRNA for the ^A globin chain of the minor embryonic hemoglobin was used, and the results of the hybridization were compared with those obtained using as probes the cDNAs for total globin mRNAs. All erythroid cells circulating in a 4-day-old chick embryo gave positive signals with both probes at an approximately constant ratio. This shows that all cells contain a similar assortment of hemoglobin types, excluding the possibility that a subgroup might contain the minor primitive hemoglobins exclusively. However, the cells are not homogeneous, since about 10% of them show a distinctly higher concentration of mRNA of all globin types.     

Molecular biological methods in the diagnosis of viral disease

Molecular biology allowed considerable improvements in diagnostic procedures by production of new and more specific sonds for the detection of traces of viruses, both on the nucleic acid and protein levels, and by determining the immune response of the host to specific antigens. Improvements in sensitivity and improved correlation to the stage of viral disease are already evident from several applications and strongly suggest a broad application of these approaches.     

Quantitation of proinsulin mRNA sequences in hamster insulinoma cells in culture by molecular hybridization

Summary The content of proinsulin mRNA sequences was measured in a cultured cell line established from a transplantable hamster islet cell tumor, by hybridization with proinsulin cDNA. The cells cultured in vitro were found to contain a significant amount of proinsulin mRNA sequences, compared with non-insulin-producing hamster tissues, and seem to be a useful system for the study of insulin gene expression.This work was supported in part by Grants-in-Aid Scientific Research (248129, 387065, 337013, 378052) and a Grant-in-Aid for Cancer Research (301538) from the Ministry of Education, Science and Culture, Japan.     

Specific radioimmunoprecipitation of histone H2A antigens by protein A conjugated sepharose

A modified radioimmunoprecipitation technique is described which allows the specific detection of histone H2A antigens. The technique circumvents unspecific binding of histones to the bacterial adsorbent.     

Specific radioimmunoprecipitation of histone H2A antigens by protein A conjugated sepharose

Summary A modified radioimmunoprecipitation technique is described which allows the specific detection of histone H2A antigens. The technique circumvents unspecific binding of histones to the bacterial adsorbent.     

Highly reactive oxygen species: detection, formation, and possible functions

The so-called reactive oxygen species (ROS) are defined as oxygen-containing species that are more reactive than O(2) itself, which include hydrogen peroxide and superoxide. Although these are quite stable, they may be converted in the presence of transition metal ions, such as Fe(II), to the highly reactive oxygen species (hROS). hROS may exist as free hydroxyl radicals (HO·), as bound ("crypto") radicals or as Fe(IV)-oxo (ferryl) species and the somewhat less reactive, non-radical species, singlet oxygen. This review outlines the processes by which hROS may be formed, their damaging potential, and the evidence that they might have signaling functions. Since our understanding of the formation and actions of hROS depends on reliable procedures for their detection, particular attention is given to procedures for hROS detection and quantitation and their applicability to in vivo studies.     

Intermittent demand forecasting in the Enterprise: Empirical verification

Forecasting methods are often valued by means of simulation studies. For intermittent demand items there are often very few non–zero observations, so it is hard to check any assumptions, because statistical information is often too weak to determine, for example, distribution of a variable. Therefore, it seems important to verify the forecasting methods on the basis of real data. The main aim of the article is an empirical verification of several forecasting methods applicable in case of intermittent demand. Some items are sold only in specific subperiods (in given month in each year, for example), but most forecasting methods (such as Croston's method) give non–zero forecasts for all periods. For example, summer work clothes should have non–zero forecasts only for summer months and many methods will usually provide non–zero forecasts for all months under consideration. This was the motivation for proposing and testing a new forecasting technique which can be applicable to seasonal items. In the article six methods were applied to construct separate forecasting systems: Croston's, SBA (Syntetos–Boylan Approximation), TSB (Teunter, Syntetos, Babai), MA (Moving Average), SES (Simple Exponential Smoothing) and SESAP (Simple Exponential Smoothing for Analogous subPeriods). The latter method (SESAP) is an author's proposal dedicated for companies facing the problem of seasonal items. By analogous subperiods the same subperiods in each year are understood, for example, the same months in each year. A data set from the real company was used to apply all the above forecasting procedures. That data set contained monthly time series for about nine thousand products. The forecasts accuracy was tested by means of both parametric and non–parametric measures. The scaled mean and the scaled root mean squared error were used to check biasedness and efficiency. Also, the mean absolute scaled error and the shares of best forecasts were estimated. The general conclusion is that in the analyzed company a forecasting system should be based on two forecasting methods: TSB and SESAP, but the latter method should be applied only to seasonal items (products sold only in specific subperiods). It also turned out that Croston's and SBA methods work worse than much simpler methods, such as SES or MA. The presented analysis might be helpful for enterprises facing the problem of forecasting intermittent items (and seasonal intermittent items as well).     

Methods for the identification of DNA hybridization groups in the genus Aeromonas.

Among the 269 substrates tested in assimilation tests we found some that may help in the identification of DNA hybridization groups in the genus Aeromonas. In addition, isoenzyme analysis and ribotyping seem to be accurate although not routine procedures that allow discrimination between genetic species.     

Deoxyribozymes: useful DNA catalysts in vitro and in vivo

Deoxyribozymes (DNA enzymes; DNAzymes) are catalytic DNA sequences. Using the technique of in vitro selection, individual deoxyribozymes have been identified that catalyze RNA cleavage, RNA ligation, and a growing range of other chemical reactions. DNA enzymes have been used in vitro for applications such as biochemical RNA manipulation and analytical assays for metal ions, small organic compounds, oligonucleotides, and proteins. Deoxyribozymes have also been utilized as in vivo therapeutic agents to destroy specific mRNA targets. Although many conceptual and practical challenges remain to be addressed, deoxyribozymes have substantial promise to contribute meaningfully for applications both in vitro and in vivo.     

Sialyl Lewisx-liposomes as vehicles for site-directed, E-selectin-mediated drug transfer into activated endothelial cells

E-selectin, exclusively expressed on activated endothelial cells, is a potential target for site-directed delivery of agents. We and others have shown that sialyl Lewis^x-liposomes (sLe^x-liposomes) are recognized by E-selectin. We now report an approach employing sLe^x-liposomes to deliver antisense oligonucleotides (AS-ODNs) directed against the adhesion molecule ICAM-1 to activated vascular endothelial cells. ICAM-1 expression was analyzed at the protein level by immunofluorescence and a cell surface ELISA, and at the RNA level by RT-PCR. We have investigated two different AS-ODNs complementary to the 3′ untranslated region and the AUG translation initiation codon of ICAM-1 mRNA. Both inhibited protein expression, but did not influence the mRNA level, pointing to a hybridization of AS-ODNs with the mRNA in the cytoplasm. Our results demonstrate the feasibility of a novel approach for the delivery of agents to activated endothelial cells by glycoliposomes targeted to E-selectin. Received 16 October 2000; revised 29 November 2000; accepted 29 November 2000     

PCR快速检测常见具有毒性质粒的沙门氏菌

针对常见具毒性质粒的沙门氏菌的spvR基因，设计出PCR特异引物spvRP35-P36，以便快速检测出具有毒性质粒的沙门氏菌。验证采用13株沙门氏菌和4株非沙门氏菌，其中6株常见具有毒性质粒的沙门氏菌均获得特异性扩增，7株不舍毒性质粒的沙门氏菌均未获得特异性扩增，4株非沙门氏茵检测结果为阴性。结果表明，本文中设计的引物具有高度的特异性，适用于常见具有毒性质粒沙门氏菌的快速检测，此引物已获国家发明专利。     

Quantification ofMycobacterium tuberculosis DNA using PCR and a simple dot blot-based DNA enzyme assay (DEA)

Conclusions Use of PCR for quantification of microorganisms has certain limitations. Methods currently under investigation, such as covalent binding of modified DNA onto the surface of microwells or coupling to magnetic beads with the aid of biotin-avidin, are hampered by problems concerning immobilization of DNA on a solid phase. Moreover, these strategies are laborious, including several washing steps and complicated detection systems (sandwich hybridization).We have developed an alternative method, exploiting the reliability of covalent DNA binding to positively charged nylon membranes enabling easy to handle direct hybridization. The method is adaptable for routine use in clinical laboratories. We have shown results of a quantitative assay to measure bacterial load of MTB. Quantification of MTB may have value in: (i) monitoring patients under anti-myobacterial therapy, and (ii) early in vitro drug susceptibility testing.