Biomedical subjects

D Madan

Publications and source records attributed to D Madan.

4 recordsLinked to original sources

PubMed1998

An efficient, automatable template preparation for high throughput sequencing.

We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.

Automation↗

PubMed1997

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.

Amino Acid Sequence↗

PubMed1996

State-dependent modulation of mSlo, a cloned calcium-dependent potassium channel.

The mouse slopoke calcium-dependent potassium channel (mSlo) has been expressed heterologously in COS cells, and incorporated from COS cell membranes into artificial phospholipid bilayers. Under control conditions, the channel is not modulated by ATP. However, when mSlo is treated first with the calcium-dependent potassium channel opener NS004, which itself increases the open probability of the channel, subsequent addition of ATP causes a large further increase in channel open probability. An increase in channel activity is not by itself sufficient to confer sensitivity to ATP, because ATP does not modulate channels whose open probability has been increased by elevated calcium or depolarized voltage. The ATP analog AMP-PNP has only minimal effects on channel activity after treatment with NS004, suggesting that hydrolysis of the ATP is required for its action on mSlo. A peptide inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII) blocks the modulation of mSlo by ATP, whereas peptide inhibitors of other serine/threonine protein kinases are without effect. The results are consistent with a state-dependent modulation of mSlo by ATP, possibly via phosphorylation.

Adenosine Triphosphate↗

PubMed1990

Histopathological sinus node classification and its relation to coronary atherosclerosis using an atherometric system.

Studies were conducted into 100 consecutive subjects (autopsy material), 53 men and 43 women between 31 and 70 years old, who had died a natural death. The HUDSON-procedure was used for histopathological analysis of the cardiac conducting system (CCS). About 48 microscopic slides of each subject, including sinus node (SN) tissue, were inspected by 4 pathologists in a blind study. The findings were classified by 3 different groups according to histopathological data of the SN, viz. changes and distribution of P and T cells, stroma, artery, and innervation of SN. The 3 major epicardial branches of coronary arteries were prepared for morphometric studies by WHO standards. A digitizer coupled to a microcomputer LTEL (Cuba, IBM-compatible) was used for quantitative evaluation of atherosclerotic lesions (AL), and all data were subsequently processed on the same computer, using a statistical commercial package (Number Cruncher Statistical System) for results. An atherometric system was used and proved to be a properly standardized methodology, suitable to characterize AL in any vascular sector or group of patients. The most remarkable results were as follows: Three-group classification was useful in estimating the association between pathomorphological data of SN and coronary atherosclerosis. The atherometric system proved to be a powerful tool to study and characterize atherosclerotic lesions in coronary arteries.

Adult↗