Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.     

CHROMOSOME DIFFERENTIATION IN SEVERAL SPECIES OF MELILOTUS

Shastry , Sishta V. S., William K. Smith , and Delmer C. Cooper . (U. Wisconsin, Madison.) Chromosome differentiation in several species of Melilotus. Amer. Jour. Bot. 47(8) : 613–621. Illus. 1960.—Two species of the section Eumelilotus (M. alba and M. officinalis), 2 of Micromelilotus (M. messanensis and M. segetalis), 2 F₁ hybrids (M. officinalis × M. alba and M. messanensis × M. segetalis), 2 autotetraploids (M. alba and M. officinalis), and 1 allotetraploid (M. officinalis × M. alba), were utilized during the course of this investigation. The 4 species and F₁ hybrids have 16 somatic chromosomes and the tetraploids have twice that number (32). The 2 Eumelilotus species are completely isolated because of seed failure after cross pollination. The F₁ hybrid (M. officinalis × M. alba), obtained elsewhere by the use of embryo-culture techniques, was intermediate between the parents in certain morphological characters and was ca. 75% pollenfertile whereas the parents approached complete fertility. No structural differences between the chromosomes were evident at pachytene. Disturbances which led to the reduced fertility occurred at later stages of meiosis. The Micromelilotus species are cross compatible, but the F₁ hybrid (M. messanensis × M. segetalis) is highly sterile. Despite chromosome structural differences of various types evident at pachytene, bivalents regularly occur at metaphase I. Irregular distribution of the chromosomes at later stages of meiosis leads to high sterility. Species which readily cross but produce a hybrid of very low fertility are likely to compound chromosomal structural differences, because of abnormalities in meiosis, in contrast with species that are completely incompatible.     

Posttransplant administration of donor leukocytes induces long-term acceptance of kidney or liver transplants by an activation-associated immune mechanism

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-gamma at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-gamma mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.     

Rapid mixing methods for exploring the kinetics of protein folding

Information on the time-dependence of molecular species is critical for elucidating reaction mechanisms in chemistry and biology. Rapid flow experiments involving turbulent mixing of two or more solutions continue to be the main source of kinetic information on protein folding and other biochemical processes, such as ligand binding and enzymatic reactions. Recent advances in mixer design and detection methods have opened a new window for exploring conformational changes in proteins on the microsecond time scale. These developments have been especially important for exploring early stages of protein folding.     

X-Linked juvenile retinoschisis associated with a 4-base pair insertion at codon 55 of the XLRS1 gene

X-linked juvenile retinoschisis (RS) is a bilateral vitreoretinal disorder with no known cure. The gene responsible for the disease was recently isolated by positional cloning methods and a spectrum of mutations has been described in families with RS pathology. In this report, we screened six sporadic cases of RS for mutations in the RS gene to understand the etiology of isolated cases. Our extensive studies revealed a novel 4 bp insertion in one family and the remaining families did not show mutations in the RS gene. This mutation altered the reading frame including codon 55 resulting in nine aberrant amino acid residues. The unaffected mother did not contain this mutation. Additionally, it was not found in 60 normal control chromosomes, suggesting that the insertion mutation is disease related in the family analyzed.     

Sordarin derivatives induce a novel conformation of the yeast ribosome translocation factor eEF2

The sordarins are fungal specific inhibitors of the translation factor eEF2, which catalyzes the translocation of tRNA and mRNA after peptide bond formation. We have determined the crystal structures of eEF2 in complex with two novel sordarin derivatives. In both structures, the three domains of eEF2 that form the ligand-binding pocket are oriented in a different manner relative to the rest of eEF2 compared with our previous structure of eEF2 in complex with the parent natural product sordarin. Yeast eEF2 is also shown to bind adenylic nucleotides, which can be displaced by sordarin, suggesting that ADP or ATP also bind to the three C-terminal domains of eEF2. Fusidic acid is a universal inhibitor of translation that targets EF-G or eEF2 and is widely used as an antibiotic against Gram-positive bacteria. Based on mutations conferring resistance to fusidic acid, cryo-EM reconstructions, and x-ray structures of eEF2, EF-G, and an EF-G homolog, we suggest that the conformation of EF-G stalled on the 70 S ribosome by fusidic acid is similar to that of eEF2 trapped on the 80 S ribosome by sordarin.     

Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI⁺] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN⁺] prions was less sensitive than [URE3], but more sensitive than [PSI⁺]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.