scholarly journals Advances in the diagnosis and treatment of leprosy

2002 ◽  
Vol 4 (15) ◽  
pp. 1-14 ◽  
Author(s):  
Vishwa M. Katoch
Keyword(s):  
Direct Detection ◽  
Genomic Structure ◽  
In Situ Hybridisation ◽  
Technological Advances ◽  
New Information ◽  
Rna Targeting ◽  
Polymerase Chain ◽  
Monitoring Treatment ◽  
Effective Drugs

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nerves. Over recent years, many important advances have been made in developing molecular diagnostics, in identifying highly effective drugs and designing multidrug regimens for treatment, and in unravelling the genomic structure and functions of the leprosy bacillus. Using the new information about specific sequences of M. leprae, several gene probes and gene amplification systems for confirming diagnosis and monitoring treatment have been developed. Among these, polymerase chain reaction (PCR)-based methods have been useful in confirming the diagnosis in paucibacillary leprosy (where few bacilli are present). RNA-targeting systems for monitoring the progress of treatment, in situ hybridisation techniques for analysing specimens with nonspecific histological features, and molecular methods for direct detection of rifampicin/dapsone resistance are other major technological advances with immense applied value. Several effective regimens for the treatment of leprosy have been developed, which include rifampicin, clofazimine and dapsone as core drugs. Although these regimens are generally satisfactory, limitations in terms of persisting activity and late reactions/relapses in paucibacillary leprosy, and persistence of dead and/or live organisms in multibacillary forms of the disease, have been observed.

Microbial quantification in activated sludge: the hits and misses

2003 ◽  
Vol 48 (3) ◽  
pp. 121-126 ◽  
Author(s):  
S.J. Hall ◽  
J. Keller ◽  
L.L. Blackall
Keyword(s):  
Activated Sludge ◽  
Molecular Genetic ◽  
In Situ Hybridisation ◽  
Biological Nutrient Removal ◽  
Physical Parameters ◽  
Fluorescence In Situ Hybridisation ◽  
Batch Tests ◽  
Polymerase Chain ◽  
Key Microorganisms

Since the implementation of the activated sludge process for treating wastewater, there has been a reliance on chemical and physical parameters to monitor the system. However, in biological nutrient removal (BNR) processes, the microorganisms responsible for some of the transformations should be used to monitor the processes with the overall goal to achieve better treatment performance. The development of in situ identification and rapid quantification techniques for key microorganisms involved in BNR are required to achieve this goal. This study explored the quantification of Nitrospira, a key organism in the oxidation of nitrite to nitrate in BNR. Two molecular genetic microbial quantification techniques were evaluated: real-time polymerase chain reaction (PCR) and fluorescence in situ hybridisation (FISH) followed by digital image analysis. A correlation between the Nitrospira quantitative data and the nitrate production rate, determined in batch tests, was attempted. The disadvantages and advantages of both methods will be discussed.

Genome structure and evolution in the allohexaploid weed Avena fatua L. (Poaceae)

Genome ◽  
1999 ◽  
Vol 42 (3) ◽  
pp. 512-518 ◽  
Author(s):  
Q Yang ◽  
L Hanson ◽  
M D Bennett ◽  
I J Leitch
Keyword(s):  
Ribosomal Dna ◽  
Genomic Structure ◽  
In Situ Hybridisation ◽  
Avena Fatua ◽  
The Other ◽  
Morphological Data ◽  
Avena Species ◽  
Intergenomic Translocations ◽  
Genomic In Situ Hybridisation

Allohexaploid wild oat, Avena fatua L. (Poaceae; 2n = 6x = 42), is one of the world's worst weeds, yet unlike some of the other Avena hexaploids, its genomic structure has been relatively little researched. Consequently, in situ hybridisation was carried out on one accession of A. fatua using an 18S-25S ribosomal DNA (rDNA) sequence and genomic DNA fromA. strigosa (AA-genome diploid) and A. clauda (CC-genome diploid) as probes. Comparing these results with those for other hexaploids studied previously: (i) confirmed that the genomic composition of A. fatua was similar to the other hexaploid Avena taxa (i.e., AACCDD), (ii) identified major sites of rDNA on three pairs of A/D-genome chromosomes, in common with other Avena hexaploids, and (iii) revealed eight chromosome pairs carrying intergenomic translocations between the A/D- and C-genomes in the accession studied. Based on karyotype structure, the identity of some of these recombinant chromosomes was proposed, and this showed that some of these could be divided into two types, (i) those common to all hexaploid Avena species analysed (3 translocations) and (ii) one translocation in this A. fatua accession not previously observed in reports on other hexaploid Avena species. If this translocation is found to be unique to A. fatua, then this information, combined with more traditional morphological data, will add support to the view that A. fatua is genetically distinct from other hexaploid Avena species and thus should retain its full specific status.Key words: wild oats, Avena, genomic in situ hybridisation (GISH), intergenomic translocations, ribosomal DNA.

scholarly journals In SituReverse Transcriptase-Nested Polymerase Chain Reaction to Identify Intracellular Nucleic Acids without the Necessity of Dnase Pretreatment and Hybridisation

2001 ◽  
Vol 22 (3) ◽  
pp. 151-158 ◽  
Author(s):  
Mario Menschikowski ◽  
Margot Vogel ◽  
Rolf Eckey ◽  
Gerd Dinnebier ◽  
Werner Jaross
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acids ◽  
Nested Pcr ◽  
In Situ Hybridisation ◽  
Target Sequence ◽  
Chain Reaction ◽  
Multiple Control ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain

In the present study a protocol of in situ reverse transcriptase‐nested polymerase chain reaction (in situ RT‐nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by “DNA repair mechanisms” and “endogenous priming”, a two‐step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin‐labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5′‐tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT‐nested PCR, which in comparison to the method of in situ RT‐PCR‐in situ‐hybridisation is simpler and less time‐consuming, can be used as an alternative approach to identify intracellular nucleic acids.

Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

2005 ◽  
Vol 56 (8) ◽  
pp. 1127 ◽  
Author(s):  
D. G. Bourne ◽  
R. L. Blakeley ◽  
P. Riddles ◽  
G. J. Jones
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
In Situ Hybridisation ◽  
Cyanobacterial Blooms ◽  
Chain Reaction ◽  
Fluorescent In Situ Hybridisation ◽  
Cyanobacterial Toxin ◽  
Specific Amplification ◽  
Polymerase Chain

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

scholarly journals Cloning and regulation of the rat activin betaE subunit

2000 ◽  
Vol 24 (3) ◽  
pp. 409-418 ◽  
Author(s):  
MK O'Bryan ◽  
KL Sebire ◽  
O Gerdprasert ◽  
MP Hedger ◽  
MT Hearn ◽  
...  
Keyword(s):  
In Situ Hybridisation ◽  
Alternative Polyadenylation ◽  
Northern Blotting ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Amino Acid Residues ◽  
Mrna Transcripts ◽  
Polymerase Chain

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

scholarly journals Response of Nicotiana alata to Insertion of an Autoregulated Senescence- inhibition Gene

2001 ◽  
Vol 126 (5) ◽  
pp. 523-530 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart ◽  
Erik V. Nordheim
Keyword(s):  
Dry Weight ◽  
Biological Study ◽  
Nicotiana Alata ◽  
Isopentenyl Transferase ◽  
Shoot Dry Weight ◽  
New Information ◽  
Polymerase Chain ◽  
Selection For ◽  
Senesced Leaves

Nicotiana alata Link and Otto (Jasmine tobacco) was transformed with an autoregulated senescence-inhibition gene construct PSAG12-IPT encoding isopentenyl transferase via Agrobacterium-mediated transformation. Transformation was confirmed by polymerase chain reaction. Transgenic plants exhibited up to 2- to 4-fold fewer senesced leaves, 29% longer in situ flower life, 26% more shoot dry weight, and a 32% to 50% reduction in flowers per branch. Additionally, transgenics were 28% shorter and had up to 174% more branches, indicative of cytokinin overproduction and a lack of tight autoregulation of PSAG12-IPT. Variation among independent transgenics suggests selection for enhanced PSAG12-IPT is feasible. Our observations of increased branching and in situ flower longevity, as well as reduced plant height and flowers per branch provide new information on PSAG12-IPT and its potential value for biological study and horticultural application.

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