The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.     

Focal cell contacts detected by ruthenium red, triton X100 and saponin in the granulosa cells of mouse ovary

Granulosa cells in growing follicles of mouse ovary, observed after treatment with ruthenium red (RR) as described by Luft (1971a, b), appeared to be covered by a continuous well-defined layer. On the contrary, treating granulosa cells with 1% Triton X100 (Vaccaro and Brody, 1981), followed by RR staining, resulted in the complete extraction of the plasma membrane coat (Triton does not affect the basement membrane and extracellular matrix proteoglycans). The use of 0.02% saponin together, with the RR stain, or 0.1% Triton X100 followed by RR staining, allows good visualization of follicular basement membrane and extracellular matrix proteoglycans without destroying cell morphology. Using this technique, we observed the extraction of the plasma membrane coat, but focal RR-stained condensations that were unaffected by saponin or 0.1% Triton X100 treatment were observed between plasma membranes of granulosa cells located around the periphery of large Graafian follicles. In some cases, RR condensations were located at the apex of plasmalemmal evaginations, in proximity to adjacent granulosa cells. Focal condensations of RR stain were never observed in secondary follicles. Present evidence suggests that focal cell contacts are mediated by transmembrane intercalated glycoproteins or proteoglycans and consequently play a role in cell adhesion. Their presence among granulosa cells of only very large Graafian follicles may be related to the maturation process of granulosa cells.     

Implications of disaggregation procedures on biological representation of human solid tumours

Abstract. This study was designed to define some biological aspects of cell suspensions, obtained by mechanical or enzymatic disaggregations, and to verify whether single cell suspensions are representative of original solid tumours. The study was performed on a series of 25 human solid tumours including breast carcinoma, ovarian carcinoma and malignant melanoma. A higher cell viability and a loss of aneuploid subpopulations, or a lower fraction of aneuploid cells, were observed in enzymatically-released samples than in samples obtained by the mechanical procedure. Moreover, the proliferative activity, which was generally similar for the cell suspensions obtained by the two disaggregation procedures, was always markedly lower in the cell suspensions than in solid samples from the same tumour. In conclusion, the results from this study indicate that many changes, such as selective release of cell populations from the tumour matrix, damage and destruction of aneuploid and proliferating cells can be induced to various extents by different disaggregation procedures.     

The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.     

Sequential 1H NMR assignment and secondary structure determination of salmon calcitonin in solution

Salmon calcitonin (sCT) has been investigated by NMR at 500 MHz in a 90% DMSOd6-10% 1H2O (v/v) mixture at 278 K. All backbone and side-chain resonances of the hormone have been assigned by using high-resolution phase-sensitive two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects, the NH-alpha CH spin-spin coupling constants, and the 1H/2H exchange kinetics measured in 80% DMSOd6-20% 2H2O (v/v) at 278 K enabled prediction of the secondary structure. Overall, an extended conformation is the dominant feature of the solution, but there are clear indications for a short double-stranded antiparallel beta sheet in the central region comprising residues 12-18, connected by a three-residue hairpin loop formed by residues 14-16. Two tight turns, made by residues 6-9 and 25-28, were also identified, but no evidence was found for the presence of a regular helical segment. The beta sheet favors an amphipathic distribution of the residues, orienting the predominantly hydrophilic Ser13, Glu15, and His17 side chains above the plane of the sheet, and the predominantly hydrophobic Leu12, Gln14, and Leu16 below it. This is interpreted as the "seed" of the amphipathic alpha helix postulated to be responsible for the interaction of sCT with lipids, a situation reminiscent of the folding mechanism of signal peptides in the interaction with membranes. The possible significance of the cis-trans Pro23 isomerism is discussed.     

The influence of the athymic mutation nude on the components of the circadian rhythm of activity in mice

The mutation known as nude brings about the lack of a thymus gland in mice. This immunodeficiency akes it possible to graft normally unaccepted, human cancerous tumors onto the mouse. Consequently, this animal is frequently used as a model for evaluating anti-cancer therapies. The effect of this mutation on biological rhythms constitutes a necessary step before using this model for cancer chronotherapy research. We evaluated the circadian and ultradian components of the rest-activity cycle in the following strains of mice: C57BL/6 with homozygous nu/nu, heterozygous nu/+, thymectomised +/+, and sham-operated +/+. The amount of activity was reduced in nu/nu as compared to the other groups. Nonetheless, neither the nude mutation nor thymectomy yielded any notable change in the circadian rhythm of activity.     

Intramolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing

Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.     

Mathematical analysis of haemopoietic grafted cell renewal and migration through the allogeneic or syngeneic mouse host

A mathematical analysis of results from kinetic studies of 125-iododeoxyuridine uptake and loss in almost all the lymphoid and non-lymphoid organs of mice is described. Applied to data gathered from a graft-versus-host reaction experiment, this analysis affords quantitative precision on the differential effects of organ alloantigens on the proliferating grafted cells. It is shown that, depending on the organ and the post-graft period, cell growth can be ascribed to alloantigen-driven cell renewal or to alloantigen-driven trapping or sequestration. Possible applications of the present approach in graft rejection monitoring are discussed.