Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.     

Functional Complementation of Nontoxic Mutant Binary Toxins of Bacillus sphaericus 1593M Generated by Site-Directed Mutagenesis

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.     

Genetic Analyses of Casuarinas Using ISSR and FISSR Markers

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT),, repeat motifs amplified relatively higher number of bands with an average of 6.0+/-3.5 and 6.3+/-1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.     

Effect of KATP channel openers on myogenic and neurogenic responses in goat urinary bladder

Isolated goat detrusor muscle exhibited spontaneous contractility with an irregular amplitude and frequency. The spontaneity of detrusor muscle exhibited a mean amplitude as 11.99 +/- 0.83 mm and frequency as 1.37 +/- 0.16/min. KATP-channel openers namely, cromakalim or pinacidil (10(-7) - 10(-4) M) added cumulatively, elicited a concentration-related inhibition of both amplitude and rate of spontaneous contractions. The mean IC50 values for both amplitude and frequency for cromakalim were 3.3 x 10(-6) M and 2.9 x 10(-6) M, respectively; and for pinacidil were 2.0 x 10(-5) M and 1.5 x 10(-5) M, respectively. Glibenclamide, a KATP-channel blocker inhibited the cromakalim-induced concentration-related relaxation of spontaneous contractions with a significant increase in its mean IC50. ACh-induced concentration-related contractile response was inhibited in the presence of either cromakalim (10(-4) M) or pinacidil (10(-4) M). The mean EC50 value of ACh, in the presence of cromakalim (2.5 x 10(-3) M) was significantly increased as compared to the control (1.2 x 10(-6) M). In the presence of glibenclamide (10(-5) M) the inhibitory effect of cromakalim was significantly reduced with consequent decrease in the EC50 value (1.9 x 10(-5) M). Application of EFS (30 V and 5 ms) on goat urinary bladder strips at 1, 2, 5, 10, 20 and 30 Hz elicited frequency-related contractile responses. Both cromakalim and pinacidil caused a rightward shift in the frequency-related contractile response curve with significant increase in the mean EF25 and EF50 values, respectively. In the presence of glibenclamide (10(-4) M), the frequency-related inhibitory response curve was shifted to left with significant (P < 0.001) increase in the mean EF25, EF50 and EF75. The present results suggest that in the goat detrusor muscle, agonist and EFS-induced contractile responses were more potently inhibited by cromakalim than pinacidil with activation of glibenclamide sensitive KATP channels.     

Co-expression of binary and 100 kDa mosquito larvicidal toxins of Bacillus sphaericus in Escherichia coli

The binary (51 and 42 kDa) and 100 kDa mosquito larvicidal toxins of Bacillus sphaericus are expressed at different growth stages of Bacillus. The genes encoding the binary toxin were expressed using T7 expression system of E. coli. In addition, a PCR amplified product containing the coding sequences of the 100 kDa toxin was cloned upstream to the binary toxin genes, and both the toxins were co-expressed in E. coli. Expression studies with these constructs in different E. coli hosts showed that when these two toxins were co-expressed, there was no augmentation of toxicity in comparison to the construct expressing the binary toxin alone. This result apparently indicates that there is no synergism between these two toxins. © Rapid Science Ltd. 1998     

Clinical evaluation of mtp40 polymerase chain reaction for the diagnosis of extra pulmonary tuberculosis

Rapid and sensitive detection of Mycobacterium tuberculosis from patient samples is vital for clinical diagnosis and treatment. The emergence of M. tuberculosis strains with either no copies or only a single copy of IS6110 in Asian countries makes the standard PCR based diagnosis of M. tuberculosis using IS6110 not reliable. We studied the diagnostic efficacy of the in-house PCR amplification of the candidate gene mtp40 as an alternative to IS6110 element based diagnosis. Clinical samples included pulmonary and extra-pulmonary specimens from TB suspected patients residing in Puducherry, South India and were analyzed using in-house PCR procedures targeting IS6110 element and mtp40 genes. Out of 317 clinical specimens analyzed, 132 (41.6 %) and 114 (36 %) were found positive for mtp40 PCR and IS6110 PCR, respectively. However, 18 specimens that were found to negative for IS6110 PCR were found positive for mtp40 PCR, which was further confirmed by DNA sequencing method. PCR amplification of mtp40 gene for the diagnosis of M. tuberculosis in clinical samples is fast, sensitive, and further identified clinical strains that lack IS6110 element in this region. It is clearly demonstrated that there is a significant difference between the two PCR procedures and the sensitivity and specificity levels of mtp40 PCR were found to be higher when compared with DNA sequencing method. Thus, mtp40 based PCR technique will be beneficial in diagnosis of TB where M. tuberculosis strains lack of IS6110 element is predominant.     

Mitochondrial polymorphisms in rat genetic models of hypertension

Hypertension is a complex trait that has been studied extensively for genetic contributions of the nuclear genome. We examined mitochondrial genomes of the hypertensive strains: the Dahl Salt-Sensitive (S) rat, the Spontaneously Hypertensive Rat (SHR), and the Albino Surgery (AS) rat, and the relatively normotensive strains: the Dahl Salt-Resistant (R) rat, the Milan Normotensive Strain (MNS), and the Lewis rat (LEW). These strains were used previously for linkage analysis for blood pressure (BP) in our laboratory. The results provide evidence to suggest that variations in the mitochondrial genome do not account for observed differences in blood pressure between the S and R rats. However, variants were detected among the mitochondrial genomes of the various hypertensive strains, S, SHR, and AS, and also among the normotensive strains R, MNS, and LEW. A total of 115, 114, 106, 106, and 16 variations in mtDNA were observed between the comparisons S versus LEW, S versus MNS, S versus SHR, S versus AS, and SHR versus AS, respectively. Among the 13 genes coding for proteins of the electron transport chain, 8 genes had nonsynonymous variations between S, LEW, MNS, SHR, and AS. The lack of any sequence variants between the mitochondrial genomes of S and R rats provides conclusive evidence that divergence in blood pressure between these two inbred strains is exclusively programmed through their nuclear genomes. The variations detected among the various hypertensive strains provides the basis to construct conplastic strains and further evaluate the effects of these variants on hypertension and associated phenotypes.     

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Background

Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ ^null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct.

Methods

Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically.

Results

Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34⁺CD117⁺ subpopulation, CD34⁺CD117^? subpopulation can acquire CD34⁺CD117⁺ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML.

Conclusions

Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.