Subcellular Localization and Activity of the Multidrug Resistance Protein 1

Multi-drug resistance or MDR is a major impediment to the successful administration of chemotherapy. Broadly defined, the term MDR applies to any mechanism the cell uses to counter the effects of chemotherapeutic agents, protecting the cell at once against the toxicity of many, structurally dissimilar compounds. There are several routes to MDR for a cell, and they include everything from decreasing intracellular drug concentrations to increasing rates drug metabolism. The study presented here focuses on the role of drug transporters in conferring drug resistance. MDR transporters are traditionally thought to extrude cytotoxins from the cell at the plasma membrane, and they thereby serve as a permeability barrier for drug entry into the cell. Several MDR transporters have been cloned to date, but this study focuses on the functional characterization of one of these proteins, the human multidrug resistance protein or MRP 1. MRP1, like other members of the MDR transporter family, is thought to be expressed at the plasma membrane and decrease the intracellular accumulation of many different chemotherapeutic compounds. Unlike other MDR transporters, however, MRP 1 is thought to require reduced glutathione to enable the transport of most of its chemotherapeutic substrates. Using a fluourescently tagged MRP1 protein, we make two novel demonstrations: that MRP1 can contribute to a drug resistance phenotype from intracellular membranes, as well as from the plasma membrane; and two, that MRP1 is active in the absence of glutathione

Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in lab-reared and field-collected Anopheles stephensi-an Asian malarial vector

<p>Abstract</p> <p>Background</p> <p>Mosquitoes are intermediate hosts for numerous disease causing organisms. Vector control is one of the most investigated strategy for the suppression of mosquito-borne diseases. <it>Anopheles stephensi </it>is one of the vectors of malaria parasite <it>Plasmodium vivax</it>. The parasite undergoes major developmental and maturation steps within the mosquito midgut and little is known about <it>Anopheles</it>-associated midgut microbiota. Identification and characterization of the mosquito midgut flora is likely to contribute towards better understanding of mosquito biology including longevity, reproduction and mosquito-pathogen interactions that are important to evolve strategies for vector control mechanisms.</p> <p>Results</p> <p>Lab-reared and field-collected <it>A. stephensi </it>male, female and larvae were screened by "culture-dependent and culture-independent" methods. Five 16S rRNA gene library were constructed form lab and field-caught <it>A. stephensi </it>mosquitoes and a total of 115 culturable isolates from both samples were analyzed further. Altogether, 68 genera were identified from midgut of adult and larval <it>A. stephensi</it>, 53 from field-caught and 15 from lab-reared mosquitoes. A total of 171 and 44 distinct phylotypes having 85 to 99% similarity with the closest database matches were detected among field and lab-reared <it>A. stephensi </it>midgut, respectively. These OTUs had a Shannon diversity index value of 1.74–2.14 for lab-reared and in the range of 2.75–3.49 for field-caught <it>A. stephensi </it>mosquitoes. The high species evenness values of 0.93 to 0.99 in field-collected adult and larvae midgut flora indicated the vastness of microbial diversity retrieved by these approaches. The dominant bacteria in field-caught adult male <it>A. stephensi </it>were uncultured <it>Paenibacillaceae </it>while in female and in larvae it was <it>Serratia marcescens</it>, on the other hand in lab-reared mosquitoes, <it>Serratia marcescens </it>and <it>Cryseobacterium meninqosepticum </it>bacteria were found to be abundant.</p> <p>Conclusion</p> <p>More than fifty percent of the phylotypes were related to uncultured class of bacteria. Interestingly, several of the bacteria identified are related to the known symbionts in other insects. Few of the isolates identified in our study are found to be novel species within the gammaproteobacteria which could not be phylogenetically placed within known classes. To the best of our knowledge, this is the first attempt to study the midgut microbiota of <it>A. stephensi </it>from lab-reared and field-collected adult and larvae using "culture-dependent and independent methods".</p

Incidence of gastro-intestinal strongylosis during monsoon seasons in native goat breeds of Kerala

Gastro-intestinal strongylosis is a major parasitic infection in caprine causing reduced performance, irreversible damage and death, which eventually leads to huge economic loss to the producers. The use of anthelmintics as a simple, effective and quick control method against the parasitism by the farmers has been rampant since decades. But its indiscriminate and undue usage invariably paved way to the development of anthelmintic resistance in parasites. It is high time that control strategies are designed so as to utilise chemotherapy appropriately at the time that coincide with heavy incidence of strongylosis. In the present study, a total of 109 goats, comprising of 58 Malabari and 51 Attappady Black goats from an organised farm were screened for the incidence for strongyle infection. The infection was found to be very high throughout the monsoon seasons in Kerala (June to October) with 94.86 ±1.47 per cent. Majority of the animals exhibited very heavy infection with faecal egg count of (FEC) >1500 during the study period. On coproculture, Haemonchus contortus was found to be the most predominant strongyle followed by Oesophagostomum spp. and Trichostrongylus spp

Molecular detection of Hepatozoon spp. in domestic cats of Kerala

Vector-borne diseases are an important cause of morbidity and mortality in domestic cat population and hepatozoonosis is one such infection. Incidence of hepatozoonosis has been described in a variety of animal species but information on cats is scarce. To investigate the occurrence of this pathogen in domestic cats, blood samples were collected from domestic cats from three districts of Kerala (Thrissur, Kannur and Wayanad). Field-stained blood smears were examined to detect the presence of gamonts of Hepatozoon spp. and the samples were subjected to molecular analysis by PCR amplification. Out of 122 blood samples screened, none of the sample revealed the presence of gamonts of Hepatozoon on microscopic examination. Polymerase chain reaction targeting 18S rRNA gene of Hepatozoon spp. demonstrated seven positive cases with a prevalence of 5.74 per cent. Results of this study indicate that hepatozoonosis is established within the domestic cats in Kerala and warrant the adoption of control measures

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Subcellular Localization and Activity of Multidrug Resistance Proteins

The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting