Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.     

Dietary n-3 PUFAs affect the blood pressure rise and cardiac impairments in a hyperinsulinemia rat model in vivo

The cardiovascular consequences of eicosapentaenoic acid (EPA)- and docosahexaenoic acid (DHA)-specific intake were evaluated in vivo in a hyperinsulinemia (HI) model induced by dietary fructose intake. Wistar rats were fed a diet containing (or not for control) either EPA or DHA. The rise in blood pressure (BP), heart rate, and ECG were continuously monitored using an intra-abdominal telemetry system. The myocardial phospholipid fatty acid profile was significantly affected by DHA intake but less by EPA intake. The data indicated a reduced rise in BP in both DHA and EPA HI groups compared with controls. This result was confirmed by tail-cuff measurement after 5 wk [133.3 +/- 1.67 and 142.5 +/- 1.12 mmHg in n-3 polyunsaturated fatty acid (PUFA) and control groups, respectively], whereas n-3 PUFA did not affect BP in non-HI rats (116.3 +/- 3.33 mmHg). The heart rate was lower in the HI DHA group than in the other two dietary HI groups. Moreover, DHA induced a significantly shorter QT interval. It is concluded that the cardioactive component of fish oils is DHA through a mechanism that may involve the cardiac adrenergic system.     

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.     

M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis

The human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455-1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 microm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis.     

New approaches in hominoid taxonomy: morphometrics

We report here on new cranial data relevant to hominoid taxonomic analyses, based on a study of 438 skulls belonging to 13 nonhuman living hominoid taxa. Nineteen landmarks were selected to describe the overall shape of the maxillofacial complex, in order to investigate its discriminative power in taxonomic analyses. We used a geometric morphometrics approach to depict morphological variation from the genus down to the subspecific level, and we evaluated whether our morphologic criteria are relevant to discriminating species and subspecies among living hominoids. Considering previous genetic studies, we discuss whether our results can be extrapolated to the hominin fossil record, providing a reference for species and subspecies morphologic differentiation. Our results indicate that the relative warp method, as applied to facial landmarks, provides a powerful tool to discriminate taxa down to a subspecific level. Results show a noticeable divergence of P. t. verus compared to P. t. troglodytes and P. t. schweinfurthii. According to our data, the distance between eastern and western gorilla populations as well as between Bornean and Sumatran orangutan subspecies is as great as between the two species of Pan. In the same manner, differences between Hylobates and Symphalangus are similar to those between Pan and Gorilla genera. Congruence between the morphological distances computed in this study and previous morphological and genetical studies strongly supports their relevance for morphological species recognition in paleoanthropology. Our data provide an objective standard for assessing taxonomic differences among hominoids, and will enable us to define more precisely the significance of morphological differences in the fossil record.     

Characterisation of the BRCT domains of the breast cancer susceptibility gene product BRCA1

The breast cancer susceptibility gene product BRCA1 is a tumour suppressor but the biochemical and biological functions that underlie its role in carcinogenesis remain to be determined. Here, we characterise the solution properties of the highly conserved C terminus of BRCA1, consisting of a tandem repeat of the BRCT domain (BRCT-tan), that plays a critical role in BRCA1-mediated tumour suppression. The overall free energy of unfolding of BRCT-tan is high (14.2 kcal mol(-1) at 20 degrees C in water) but unfolding occurs via an aggregation-prone, partly folded intermediate. A representative set of cancer-associated sequence variants was constructed and the effects on protein stability were measured. All of the mutations were highly destabilising and they would be expected to cause loss of function for this reason. Over half could not be purified in a soluble form, indicating that these residues are critical for maintaining structural integrity. The remaining mutants exhibited much greater aggregation propensities than the wild-type, which is most likely a consequence of their reduced thermodynamic stability relative to the partly folded intermediate. The mutations characterised here are located at different sites in the BRCT-tan structure that do not explain fully their effects on the protein's stability. Thus, the results indicate an important role for biophysical studies in assessing the significance of sequence variants and in determining how they cause disease.     

RCAS1 is associated with ductal breast cancer progression

RCAS1/EBAG9 (receptor-binding cancer antigen expressed on SiSo cells/ estrogen receptor-binding fragment-associated gene 9), an estrogen-transcribed protein, has been shown to be expressed in a wide variety of cancers, including uterine, ovarian, and lung cancer cells. Soluble and membranous RCAS1 proteins may play a role in the immune escape of tumor cells by promoting T lymphocyte inhibition of growth and apoptosis. In the present report, the presence of RCAS1 was revealed in human ductal breast cancer biopsies by immunohistochemistry. Its cytoplasmic expression was exhibited in cancer cells obtained from tumor biopsies and in breast cancer cell lines. RCAS1 significantly correlated with tumor grade. In addition, RCAS1 was identified in MCF7 culture supernatants. Those observations suggest that RCAS1 is a new marker for breast cancer progression and a possible mechanism for breast cancer immune escape.