Cytological studies on some species in genus Pennisetum

Summary Detailed microsporogenesis in 4 species of the genus Pennisetum namely P. typhoides (n = 7), P. longistylum (n = 18), P. polystachyon (n = 27) and P. pedicellatum (n = 27) was studied. Nature of chromosome pairing was critically studied and pairing was regular in diploid and allotetraploid species. Some multivalents formation occurred in segmental allopolyploids. They displayed numerous meiotic irregularities. Aberrant meiosis in the material is evaluated. The role of apomixis, hybridization and polyploidy in the evolution of the genus is discussed.

---

Zusammenfassung Es wird über eingehende Untersuchungen der Mikrosporogenese in 4 Species der Gattung Pennisetum, nämlich P. typhoides (n = 7), P. longistylum (n = 18), P. polystachyon (n = 27) und P. pedicellatum (n = 27) berichtet. Die Art der Chromosomenpaarung wurde kritisch geprüft, sie war bei den diploiden und allotetraploiden Species regulär. Multivalentbildung trat in einigen Fällen bei Segment-Allopolyploiden auf, sie zeigten zahlreiche meiotische Unregelmäßigkeiten. In dem Material aufgetretene aberrante Meiosen wurden ausgewertet. Die Rolle von Apomixis, Hybridisation und Polyploidie bei der Evolution der Gattung wird besprochen.

     

Proteomics indicates modulation of tubulin polymerization by L-menthol inhibiting human epithelial colorectal adenocarcinoma cell proliferation

Menthol is a naturally occurring cyclic monoterpene used in oral hygiene products, confectionary, pharmaceuticals, cosmetics, pesticides, and as a flavoring agent. In the present study, we analyzed the differentially expressing proteome in L-menthol-treated Caco-2 cell line as it was found to inhibit cell proliferation. Interestingly, free tubulin proteins were observed to be limited after menthol treatment. Semiquantitative RT-PCR with α-tubulin primers showed no change in the level of RNA expression in menthol-treated cell line. However, tubulin polymerization assay with menthol indicated a trend similar to taxol in promoting microtubule assembly. Further, physical counting of apoptotic nuclei and active caspase-3 assays confirmed onset of apoptosis though the rate was slower as compared with that of taxol treatment. This study is the first report of a monoterpene L-menthol modulating tubulin polymerization and apoptosis to inhibit cancer cell proliferation.     

Regulated hyperaccumulation of presenilin-1 and the "gamma-secretase" complex. Evidence for differential intramembranous processing of transmembrane subatrates

Intramembranous "gamma-secretase" processing of beta-amyloid precursor protein (APP) and other transmembrane proteins, including Notch, is mediated by a macromolecular complex consisting of presenilins (PSs), nicastrin (NCT), APH-1, and PEN-2. We now demonstrate that in cells coexpressing PS1, APH-1, and NCT, full-length PS1 accumulates to high levels and is fairly stable. Upon expression of PEN-2, the levels of PS1 holoprotein are significantly reduced, commensurate with an elevation in levels of PS1 fragments. These findings suggest that APH-1 and NCT are necessary for stabilization of full-length PS1 and that PEN-2 is critical for the proteolysis of stabilized PS1. In N2a and 293 cell lines that stably overexpress PS1, APH-1, NCT, and PEN-2, PS1 fragment levels are elevated by up to 10-fold over endogenous levels. In these cells, we find a marked accumulation of the APP-CTF gamma (AICD) fragment and a concomitant reduction in levels of both APP-CTF beta and CTF alpha. Moreover, the production of the gamma-secretase-generated Notch S3/NICD derivative is modestly elevated. However, we failed to observe a corresponding increase in levels of secreted A beta peptides in the medium of these cells. These results lead us to conclude that, although the PS1, APH-1, NCT, and PEN-2 are essential for gamma-secretase activity, the proteolysis of APP-CTF and Notch S2/NEXT are differentially regulated and require the activity of additional cofactors that promote production of AICD, NICD, and A beta.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

Model based dynamics analysis in live cell microtubule images

Background

The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data.

Results

In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior.

Conclusion

Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior.

     

Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members

The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

Secretion of the beta/A4 amyloid precursor protein. Identification of a cleavage site in cultured mammalian cells

Alzheimer's disease, a progressive neurodegenerative disorder, affects greater than 10% of the population of individuals greater than 65 years of age. A principal neuropathological feature of this disease is the senile plaque, a fibrillar extracellular deposit primarily composed of a approximately 4-kDa peptide, beta/A4, derived from the amyloid precursor protein (APP). Studies in cultured cells have documented that APP matures through a constitutive secretory pathway and is cleaved at or near the cell surface to release a large ectodomain into the extracellular space. To define the APP cleavage site, we constructed a Chinese hamster ovary cell line, which constitutively overexpresses human APP-770, and analyzed the COOH termini of secreted APP-770-related molecules. Using plasma desorption mass spectrometry and chemical microsequencing, we document that an APP cleavage site in Chinese hamster ovary cells leading to secretion occurs immediately COOH-terminal to lysine residue 687, which lies adjacent to the hydrophobic membrane-spanning domain.     

Structure-based design of eugenol analogs as potential estrogen receptor antagonists

Eugenol is an essential oil mainly found in the buds and leaves of clove (Syzygium aromaticum (L.) Merrill and Perry), which has been reported to have activity on inhibition of cell proliferation and apoptosis induction in human MCF-7 breast cancer cells. This biological activity is correlated to its activity as an estrogen receptor antagonist. In this article, we present the construction and validation of structure-based virtual screening (SBVS) protocols to identify the potent estrogen receptor α (ER) antagonists. The selected protocol, which gave acceptable enrichment factors as a virtual screening protocol, subsequently used to virtually screen eugenol, its analogs and their dimers. Based on the virtual screening results, dimer eugenol of 4-[4-hydroxy-3-(prop-2-en-1- yl)phenyl]-2-(prop-2-en-1-yl)phenol is recommended to be developed further in order to discover novel and potent ER antagonists.