Quantification of microtubule stutters: dynamic instability behaviors that are strongly associated with catastrophe

Microtubules (MTs) are cytoskeletal fibers that undergo dynamic instability (DI), a remarkable process involving phases of growth and shortening separated by stochastic transitions called catastrophe and rescue. Dissecting DI mechanism(s) requires first characterizing and quantifying these dynamics, a subjective process that often ignores complexity in MT behavior. We present a Statistical Tool for Automated Dynamic Instability Analysis (STADIA) that identifies and quantifies not only growth and shortening, but also a category of intermediate behaviors that we term “stutters.” During stutters, the rate of MT length change tends to be smaller in magnitude than during typical growth or shortening phases. Quantifying stutters and other behaviors with STADIA demonstrates that stutters precede most catastrophes in our in vitro experiments and dimer-scale MT simulations, suggesting that stutters are mechanistically involved in catastrophes. Related to this idea, we show that the anticatastrophe factor CLASP2γ works by promoting the return of stuttering MTs to growth. STADIA enables more comprehensive and data-driven analysis of MT dynamics compared with previous methods. The treatment of stutters as distinct and quantifiable DI behaviors provides new opportunities for analyzing mechanisms of MT dynamics and their regulation by binding proteins.     

Influence of cytology development on frequency of pre-cancerous lesions and cervical cancer in east Croatia, 1978-2001

We analyzed the influence of cytology development to frequency of precancerosa (Cervical intraepithelial neoplasia grade 3 - CIN 3) and cervical cancer. The number of examined Pap smears increased significantly in the analyzed period. The calculated linear trend shows an average four-year increase of 17,283.35 smears. The number of detected CIN 3 increased accordingly. The calculated linear trend shows an average four-year increase by 45.03 and decrease of the rate of 0.07. A slight increase in cervical cancer was also noticed. The calculated linear trend shows an average four-year increase in cancer by 1.6 and decrease in the rate of 0.51. Analyses of cervical cancer by stages showed an increase in number of stage I and decrease of other stages. The calculated linear trend shows an average four-year increase of stage I by 4.94 or decrease in other stages of cervical cancer of 3.29 respectively. The rate of cervical cancer stage I decreased by 0.11 and of other stages by 0.39. Further analyses of the stage I showed that the linear trend of IA stage had an average four-year increase by 5.40 and decrease in stage IB of 0.48. The rate of the cervical cancer stage IA increased by 0.02, whereas the rate of IB decreased by 0.13. Cytology development in our country has resulted in detection of higher number of CIN 3 and the earliest stage (IA) of cervical cancer whereas the number of other stages (IB, II, III, IV) has decreased. However, a total number of cervical cancers haven't changed over the whole period. Thus, it is obvious that opportunistic program of detection, which has been using in Croatia, could not decrease frequency of cervical cancer. In order to achieve it, well-organized national program of detection is needed.     

How kinesin-2 forms a stalk

The heterotrimeric structure of kinesin-2 makes it a unique member of the kinesin superfamily; however, molecular details of the oligomer formation are largely unknown. Here we demonstrate that heterodimerization of the two distinct motor domains KLP11 and KLP20 of Caenorhabditis elegans kinesin-2 requires a dimerization seed of merely two heptads at the C terminus of the stalk. This heterodimeric seed is sufficient to promote dimerization along the entire length of the stalk, as shown by circular dichroism spectroscopy, Förster resonance energy transfer analysis, and electron microscopy. In addition to explaining the formation of the kinesin-2 stalk, the seed sequence identified here bears great potential for generating specific heterodimerization in other protein biochemical applications.     

Fulminant meningococcal sepsis in a young child--a case report

We are presenting a case of isolated fulminant meningococcal sepsis with two and a half year old child. Initial symptoms were obscure and common to many medical conditions, but also previously described as symptoms of meningococcal sepsis. Unrecognizing the seriousness of the condition child died at home, within few hours after examination and discharge from the hospital. Autopsy and microbiological findings unquestionably proved that the child died from septic shock caused by fulminant meningococcal sepsis.     

Image cytometric evaluation of nuclear texture features and DNA content of the reticular form of oral lichen planus

OBJECTIVE: To analyze image cytometric chromatin changes reflected in nuclear texture features and DNA ploidy of oral lichen planus in relation to the normal buccal mucosa and buccal mucosa expressing malignancy-associated changes in cancer patients. STUDY DESIGN: Twenty-eight patients with the reticular form of oral lichen planus, with a follow-up period of 25 years, 50 healthy controls and 50 lung cancer patients were included in the study. Scrapings of buccal mucosa were suspended in transport medium. Monolayer filter preparations were Feulgen-thionin stained. Image cytometric analysis was performed by Cyto-Savant. RESULTS: All oral lichen planus specimens in our study were diploid. In univariate analysis, differences between the normal buccal mucosa and oral lichen planus were found in several nuclear texture features, which gave an 80% correct classification rate in multivariate analysis. In the second part of the study, the classifier that recognizes malignancy-associated changes on the buccal mucosa of patients with lung cancer correctly recognized > 80% of oral lichen planus samples as normal buccal mucosa. CONCLUSION: Our results indicate that chromatin changes in oral lichen planus exist compared to normal cells; however, the chromatin structure of the reticular form of oral lichen planus does not express malignancy-associated changes and is more similar to normal squamous cells.     

Antioxidant and anti‐inflammatory effects of the monocarbonyl curcumin analogs B2BRBC and C66 in monocrotaline‐induced right ventricular hypertrophy

For 22 days after monocrotaline injection two groups of rats received either of the monocarbonyl curcumin analogs (2E,6E)‐2,6‐bis(2‐bromobenzylidene)cycloxehanone (B2BrBC) and (2E,6E)‐2,6‐bis([2‐tri?uoromethyl]benzylidene)cyclohexanone (C66), and their right ventricle parameters were compared to those from the control and the monocrotaline injected animals. B2BrBC and C66 treatments did not prevent the monocrotaline‐induced right ventricular hypertrophy but attenuated the changes in antioxidant enzyme activities and reduced inflammation. The level of thiol‐based nonenzymatic antioxidants did not change in the function of monocrotaline or curcumin analogs treatment. However, due to its stronger antioxidant properties, only B2BrBC treatment was effective in the reduction of monocrotaline‐associated lipid peroxidation. The obtained results suggest that increasing the levels of antioxidant enzymes may not be sufficient to reduce oxidative stress and chronic inflammation optimally and our current study supports the potential of compounds with more than one beneficial biological activity as a promising treatment against the progression of cardiac hypertrophy.     

Reaction of [Pt(Gly-Gly-N,N',O)I]- with the N-acetylated dipeptide L-methionyl-L-histidine: selective platination of the histidine side chain by intramolecular migration of the platinum(II) complex

The reaction of the monofunctional [Pt(Gly-Gly-N,N′,O)I]⁻ complex, in which Gly-Gly is the dipeptide glycyl-glycine coordinated through two nitrogen and oxygen atoms, with the N-acetylated dipeptide l-methionyl-l-histidine (MeCOMet-His) studied by ¹H NMR spectroscopy. All reactions were carried out in 50 mM phosphate buffer at pD 7.4 and at 25 °C. In the initial stage of the reaction, the platinum(II) complex forms the kinetically favored [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-S)]⁻ complex, with unidentate coordination of the MeCOMet-His dipeptide through the sulfur atom of the methionine residue. In the second stage of the reaction, complete intramolecular migration of the [Pt(Gly-Gly-N,N′,O)] unit from the sulfur to the N3 nitrogen atom of imidazole was observed and a new platinum(II)-peptide complex, [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-N3)]⁻ was formed. In comparison with previous results obtained for the reaction of [Pt(dien)Cl]⁺ with different methionine- and histidine-containing peptides, this migration reaction was sufficiently fast and strongly selective to the N3 atom of the imidazole ring of the histidine side chain. This study is an important step in the development of new platinum(II) complexes for selective covalent modification of peptides and proteins.     

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment ¹. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. Methods for fractionating leptospiral membranes ^2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids ^{6, 7}. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic β-sheets arranged in a barrel-like structure ^{8, 9}. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL54¹⁰ are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended ¹⁰. Download video file.^{(65M, mov)}