The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components.

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-kappa B in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-kappa B that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-kappa B activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-kappa B during infection. Our results argue that NF-kappa B activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-kappa B inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-kappa B in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

In Ayurvedic practice, neem is an immensely important medicinal plant that's been part of traditional medications dating back to ˂ 5000 years. Its botanical name being A. indica while it is known as Nimba in Sanskrit. Prior to written records, all the parts of neem were used to treat and cure various ailments, infections, fevers, etc. Such wide range of application has led to it being termed “the village pharmacy.” The current study reports for the first time extraction and biological activity evaluation of these AMPs and polypeptides derived from the seeds of A. indica.The present study intends to be the first to evaluate the antimicrobial and antitumoral properties of peptides and polypeptides on cancer cell lines from the total protein extracted from neem plant seeds by means of cell and molecular biology approaches. Protein extraction and precipitation was performed applying our optimized/standardized protocols. The antimicrobial activity was assayed efficiently using PAG-ADA method. The anticancer property of the extracted protein was detected by MTT assay, while MALDI-TOF/TOF and FTIR were performed to characterize the structure of these AMPs and polypeptides.Two standardized protein extraction procedures were employed and the protein obtained from these two procedures were 262.67 ± 6.12 and, 248.7 ± 9.49 mg/g of seeds, respectively. Our study reports two novel antimicrobial peptides, and four polypeptides extracted from the dried seeds of A. indica. Further, we confirmed the bactericidal activity of the peptides and polypeptides extracted from A. indica seeds using PAG-ADA techniques against six different human pathogens. Remarkably high zone of growth inhibition against MRSA (30.0 ± 1.52) mm was noted. On the other hand, the total protein extracted possessed excellent anticancer activity with low IC50 values of 33.46 ± 2.63 and 20.83 ± 1.60 μg/mL against HeLa cells and 6.94 ± 0.10 and 6.87 ± 0.90 μg/mL against BT549 cell lines at 24 and 48 h, respectively.AMPs and polypeptides extracted from seeds of the medicinal plant A. indica exhibited bactericidal activity against different bacterial pathogens. In addition, the extract possessed good anticancer activity against the cervical cancer and breast cancer cell lines. Therefore, these promising findings pave the way for identification of plant-based bioactives responsible for the anticancer and antimicrobial attributes.

The endolysosomal system comprises a unique environment for proteolysis, which is regulated in a manner that apparently does not involve protease inhibitors. The system comprises a series of membrane-bound intracellular compartments, within which endocytosed material and redundant cellular components are hydrolysed. Endocytosed material tends to flow vectorially through the system, proceeding through the early endosome, the endosome carrier vesicle, the late endosome and the lysosome. Phagocytosis and autophagy provide alternative entry points into the system. Late endosomes, lysosome/late endosome hybrid organelles, phagosomes and autophagosomes are the principal sites for proteolysis. In each case, hydrolytic competence is due to components of the endolysosomal system, i.e. proteases, lysosome-associated membrane proteins, H+-ATPases and possibly cysteine transporters. The view is emerging that lysosomes are organelles for the storage of hydrolases, perhaps in an inactivated form. Once a substrate has entered a proteolytically competent environment, the rate-limiting proteolytic steps are probably effected by cysteine endoproteinases. As these are affected by pH and possibly redox potential, they may be regulated by the organelle luminal environment. Regulation is probably also affected, among other factors, by organelle fusion reactions, whereby the meeting of enzyme and substrate may be controlled. Such systems would permit simultaneous regulation of a number of unrelated hydrolases.

Hyperexpression, alteration of trafficking and secretion of cathepsin D has been linked with tumour invasion and inflammation. To study these phenomena in a variety of cells large quantities of anti-cathepsin D antibodies and the appropriate immunogen are required. As the human immunogen for studies on human tissue is less easily accessed, antibodies to both human and porcine cathepsin D were raised in chickens, as high levels of antibody may be recovered from egg yolks, and the potential cross-reactivity of the anti-porcine cathepsin D IgY antibody was assessed. This preparation cross-reacted strongly with human cathepsin D, comparing favourably with the reactivity of the chicken antibody to the human immunogen. The necessity for isolating human immunogen can thus be circumvented. The cross-species-reacting chicken IgY was successfully used to localise cathepsin D in immunogold labelling of human tissues. To our knowledge, IgY antibodies have not previously been used by other researchers for this purpose. Application of the cross-reacting antibody to human splenic neutrophils (PMNs) has confirmed the presence of cathepsin D in some granules. Double labelling has shown these to be novel subpopulations of azurophil granules. Cathepsin D may, therefore, be relevant in the invasive and inflammatory activities of PMNs and a target for therapeutic strategies.

Of the cysteine proteases, cathepsin B has been most often implicated in tumor progression. Accumulating evidence indicates that the regulation of cathepsin B and other cysteine proteases is under both transcriptional and posttranscriptional control. The elucidation of such control mechanisms may be important for therapeutic strategies aimed at reducing cathepsin B overexpression in tumors and other pathologies. Transcription initiation at more than one initiation site and/or the induction of alternative splicing of mRNA may lead to the production of transcripts that differ in their rate of translation or their stability. Hence, the amount of protein produced may not be proportional to the amount of mRNA observed in tissues. Transcripts lacking exon 3 would encode a truncated procathepsin B, a form predicted to be trafficked to the cytoplasm due to the absence of its signal peptide. Evidence of alterations in trafficking of cathepsin B and distribution of organelles labeling for cathepsin B to more peripheral, basolateral or cell membrane localizations is seen in bladder, breast, colon, prostate and thyroid carcinomas and in gliomas. These may reflect alterations in the cytoskeleton as similar changes in localization of cathepsin B accompany the rearrangement of cytoskeletal elements and the acquisition of an invasive phenotype in MCF-10A breast epithelial cell lines after transfection with oncogenic c-Ha-ras. Alterations in trafficking of cathepsin B and other cysteine proteases may thus be brought about by changes in signal transduction, induced by transfection with an activated oncogene. Signal-transduction pathways as well as the enzymes directly involved in the invasive degradation of basement membranes may therefore both be targets for therapeutic intervention. The results of recent studies suggest that cathepsins B and L are more stable in the extracellular environment than previously believed and so could play a significant extracellular role in invasive cancer.

Cathepsins B and L are thought to function extracellularly in pathological conditions. pH-Activity profiles of cathepsin B, measured in phosphate and acetate-Mes-Tris buffers of constant ionic strength, indicated that cathepsin B is sensitive to specific buffer ions, as previously reported for cathepsin L. In assessing the activity of these enzymes in vitro the influence of the buffer must therefore be taken into account. In Hank's balanced salt solution, a buffer modeling the extracellular fluid, the half-life of activated human liver cathepsin B at 37 degrees C is 245 +/- 11.3 s, at pH 7.2, and 857 +/- 50.1 s, at pH 6.8 (the peritumor pH), indicating that cathepsin B is markedly stable under these conditions. The stability was increased by the additional presence of proteins. Without immediate activation, however, the stabilities of both cathepsins B and L were markedly decreased, a large proportion of their activity being lost before it could be measured. Enzymes injected into the extracellular space in the unactivated state would therefore survive for only a very short time in their native conformation. It is proposed that the active site thiolate-imidazolium ion pair contributes substantially to the stability of cathepsins B and L to extracellular ionic conditions.

Alterations in trafficking of cathepsins B and D have been reported in human and animal tumors. In MCF10 human breast epithelial cells, altered trafficking of cathepsin B occurs during their progression from a preneoplastic to neoplastic state. We now show that this is also the case for altered trafficking of cathepsin D. Nevertheless, the two cathepsins are not necessarily trafficked to the same vesicles. Perinuclear vesicles of immortal MCF10A cells label for both cathepsins B and D, yet the peripheral vesicles found in ras-transfected MCF10AneoT cells label for cathepsin B, cathepsin D or both enzymes. Studies at the electron microscopic level confirm these findings and show in addition surface labeling for both enzymes in the transfected cells. By immunofluorescence staining, cathepsin B can be localized on the outer surface of the cells. Similar patterns of peripheral intracellular and surface staining for cathepsin B are seen in the human breast carcinoma lines MCF7 and BT20. We suggest that the altered trafficking of cathepsins B and D may be of functional significance in malignant progression of human breast epithelial cells. Translocation of vesicles containing cathepsins B and D toward the cell periphery occurs in human breast epithelial cells that are at the point of transition between the pre-neoplastic and neoplastic state and remains part of the malignant phenotype of breast carcinoma cells.

SUMMARY This note describes further modifications to the LKB 7800 series KnifeMaker to improve the precision of scoring of glass strips and the consequent reproducibility of breaking of ‘cryo’ knives, by largely eliminating play in the mechanism, and by accurate adjustment of the cutting wheel position.

This note describes further modifications to the LKB 7800 series KnifeMaker to improve the precision of scoring of glass strips and the consequent reproducibility of breaking of 'cryo' knives, by largely eliminating play in the mechanism, and by accurate adjustment of the cutting wheel position.