Infrared spectroscopy of proteins

This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.     

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.     

Local synthesis of the phosphatidylinositol-3,4-bisphosphate lipid drives focal adhesion turnover

1. Download : Download high-res image (191KB)
2. Download : Download full-size image

     

Regulation of the plane of cell division in vacuolated cells

Summary In small leaf explants fromNautilocalyx lynchii (Hook. f.) Sprague (Gesneriaceae) the vacuolated epidermal cells divide after 3–4 days. Most cells divide periclinally, but longitudinal and transverse divisions are also found. Before mitosis the cells form a phragmosome (PS), a cytoplasmic structure which contacts the cell cortex at the future division site. An experimental approach was used to find out at which time the plane of cell division becomes fixed: prior to or during the formation of a PS.When 3 day-old explants were divided into two parts by a longitudinal cut, a high percentage of the cells near the wound divided longitudinally. Cells which already had a PS at the time of wounding most often divided in the plane of the PS. Some of the cells with a non-longitudinal PS, however, formed a longitudinal cell wall after the replacement of the original PS by a longitudinal PS.The observations show that most cells which had not yet formed a PS could be induced to form a cell wall in a new direction. As soon as the formation of the PS had started, however, it became more difficult to induce a change in the plane of cell division. These results suggest that the division site is chosen during the formation of the PS.Abbreviations BMT band of microtubules - DIC differential interference contrast microscopy - l longitudinal - l-o longitudinal-oblique - MT microtubule - p periclinal - PM prometaphase - PPB preprophase band - PS phragmosome - t transverse - t-o transverse-oblique     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

用共聚焦显微镜观察ACh及ATP对豚鼠耳蜗外毛细胞Ca^2＋的调控

用激光扫描共聚焦显微镜研究了一般公认的耳蜗传出神经递质乙酰胆碱（ＡＣｈ）和三磷酸腺苷（ＡＴＰ）对豚鼠耳蜗外毛细胞（ＯＨＣｓ）胞内游离Ｃａ＾２＋浓度（Ｃａ＾２＋）的作用,ＯＨＣｓ用Ｃａ＾２＋敏感荧光染料Ｆｌｕｏ－３着色,胞内Ｃａ＾２＋的分布以细胞底部稍强。ＡＣｈ在ＯＨＣ底部引起Ｃａ＾２＋的缓慢上长并维持在一个较高水平。ＡＴＰ在整个ＯＨＣ引起一个急剧的Ｃａ＾２＋升高,升高幅度在ＯＨＣ顶部最大。随着ＡＴ     

The natural history of the helicoidal occlusal plane and its evolution in early Homo

In modern man the pitch of the occlusal plane may vary along the tooth-row. When anterior cheek-teeth show a plane sloping upward palatally, whilst that on posterior cheek-teeth slopes upward buccally, there results a twisted or helicoidal occlusal plane (Ackermann). Several hypotheses have been proposed for the structural basis of the helicoidal occlusal plane. Campbell's proposal ('25) has gained widest acceptance, namely that the helicoid results from anteroposterior differences in upper and lower alveolar arch width. In the early 1960s, while studying the Olduvai hominids assigned to Homo habilis, the author noted changing occlusal slopes along the tooth-row and a slight helicoid, although these featues had not been noted in other early hominids. Subsequently, Wallace showed a total absence of the helicoid from South African australopithecines, and its presence in Swartkrans Homo, SK 45 and SK 80. Recent studies confirm the presence of the helicoid in all available specimens of H. habilis, including Stw 53 found at Sterkfontein in 1976. Hence, this trait may distinguish between Australopithecus and early Homo. Measurements of the maxillary arch widths have shown that, whereas in Australopithecus arch widths increase to a maximum at M3, in early Homo maxillary arch widths are greatest at M2. The decline in posterior maxillary arch width is part of a general reduction of that region. Thus despite striking elongation of premolars and M1 in early Homo, M2 and M3 are mesiodistally abbreviated. It is hypothesized that the onset of posterior arch reduction, with the appearance of a helicoid, was a structural and functional concomitant of the transition from the presumed australopithecine ancestor to H. habilis.     

Potential identification of chemical carcinogens in a viral transformation system

Chemical carcinogens from several diverse chemical classes i.e.; aromatic amines, polycyclic hydrocarbons, nitrosamines, hormonal derivatives, metals and direct alkylating agents cause a 6.2–60.5-fold increase in the frequency of murine sarcoma virus (MSV)-induced transformation in a normal rat kidney (NRK) cell system. Exogenous metabolic activation with a rat liver S-9 homogenate is required for expression of this activity by procarcinogens. Non-carcinogenic analogs of these compounds fail to cause significant increases in the transformation frequency either with or without prior metabolic activation. Iododeoxyuridine, a mutagen also does not cause enhancement of transformation. This system may serve as the basis for a rapid and quantifiable means of identifying chemical carcinogens while introducing a new model for the understanding of the interactions between oncorna-viruses and chemical carcinogens.