Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain DOR4 Toxic to Castor Semilooper <Emphasis Type="Italic">Achaea janata</Emphasis>: Proteolytic Processing and Binding of Toxins to Receptors

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.     

The ARF-p53 senescence pathway in mouse and human cells

Mouse and human cells have most frequently been used for studies that have led to the elucidation of various molecular pathways involved in senescence. The ARF-p53 pathway has been assigned as one of the major protagonists in these phenomena. ARF is an alternative reading frame protein encoded along with p16INK4A by the INK4a locus on human chromosome 9p21 and the corresponding locus on mouse chromosome 4. Whereas the mouse ARF (p19ARF) consists of 169 amino acids, the human ARF (p14ARF) consists of 132 amino acids, truncated at the C-terminus. Molecular studies on the regulation of ARF activity by its binding partners have revealed that mouse ARF protein, but not human ARF protein, interacts with a cytoplasmic protein, Pex19p. This interaction of mouse ARF with Pex19p results in its milder p53 activation function in mouse cells as compared to human cells and thus accounts, at least in part, for the weaker tumor surveillance and frequent immortalization of mouse cells.     

Bhattacharyya’s distance measure as a precursor of genetic distance measures

In Population Genetics, two populations are distinguished from each other on the basis of the differences in the distributions of the alleles at the locus or loci under consideration. These differences are measured by a “genetic distance” between the two populations (not to be confused with genetic distance between two loci, which is based on recombination fractions) and they play a major role in inferences at the population level. Several measures of genetic distance have been proposed by different authors (Sanghvi 1953; Cavalli-Sforza and Edwards 1967; Jukes and Cantor 1969; Nei 1972; Kimura 1980; Reynoldset al 1983; reviews in Felsenstein 1991; Nei and Kumar 2000). Most of these measures are actually dissimilarity measures and not mathematically true distance measures (B-Rao and Majumdar 1999). Independently, and much before the geneticists, statisticians too were concerned with the idea of distinguishing between two (statistical) populations. In order to discriminate between two populations on the basis of one or more characters, divergence measures like “Mahalanobis’D ² statistic” or “Mahalanobis’ generalized distance” (1936) and “Bhattacharyya’s distance” (1943, 1946), Kullback-Leibler’s divergence measure (1951) etc. have been proposed by statisticians. Mukherjee and Chattopadhyaya (1986) have mentioned measures based on distances, association between two attributes and discrimination function. There are similarities between the distance measures defined by applied scientists and by theoreticians. Felsenstein (1985) shows that three of the allele frequency-based genetic distance measures were anticipated by Bhattacharyya (1946). Nei and Takezaki (1994) have also studied the effectiveness of several genetic distance measures in the context of phylogenetic analysis, including Bhattacharyya’s distance measure.     

Optimization of an immunostaining protocol for the rapid intraoperative evaluation of melanoma sentinel lymph node imprint smears with the 'MCW melanoma cocktail'

BACKGROUND: In the management of cutaneous melanoma, it is desirable to complete the regional lymphadenectomy during the initial surgical procedure for wide excision of biopsy site and sentinel lymph node (SLN) biopsy. In this study, we optimized and evaluated a rapid 17 minutes immunostaining protocol. The discriminatory immunostaining pattern associated with the 'MCW Melanoma Cocktail' (mixture of Melan- A, MART- 1, and tyrosinase) facilitated the feasibility of intraoperative evaluation of imprint smears of SLNs for melanoma metastases. METHODS: Imprint smears of 51 lymph nodes from 25 cases (48 SLNs and 3 non-SLNs, 1 to 4 SLNs/case) of cutaneous melanoma were evaluated. RESULTS: Sixteen percent, 8/51 lymph nodes (28%, 7/25 cases) were positive for melanoma metastases in immunostained permanent sections with the 'MCW melanoma cocktail'. All of these melanoma metastases, except 1 SLN from 1 case, were also detected in rapidly immunostained wet-fixed and air-dried smears (rehydrated in saline and postfixed in alcoholic formalin). The cytomorphology was superior in air-dried smears, which were rehydrated in saline and postfixed in alcoholic formalin. Wet-fixed smears frequently showed air-drying artifacts, which lead to the focal loss of immunostaining. None of the 5 SLNs from 5 cases exhibiting capsular nevi showed a false positive result with immunostained imprint smears. CONCLUSIONS: Melanoma metastases can be detected intraoperatively in both air-dried smears and wet-fixed smears immunostained with the MCW Melanoma cocktail. Air-dried smears rehydrated in saline and postfixed in alcoholic formalin provide superior results and many practical benefits.     

Myosin-X provides a motor-based link between integrins and the cytoskeleton

Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.