1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.
1,3-beta-D-Glucan synthase of Saccharomyces cerevisiae was solubilized and purified up to 700-fold by product entrapment. The specific activity of the partially purified enzyme was around 4 mumol glucose incorporated.min-1.mg protein-1. In SDS/PAGE, enrichment of a 200-kDa protein was clearly observed in parallel with the increase in specific activity. mAbs that could immunoprecipitate the 1,3-beta-D-glucan synthase activity were isolated, and some of them also recognized this 200-kDa protein in the Western blot. Internal amino acid sequences of this 200-kDa protein were determined after lysyl endopeptidase digestion. With the information of these amino acid sequences, we cloned two genes, GSC1 and GSC2 (glucan synthase of S. cerevisiae 1 and 2), which are very similar to each other (88% at the amino acid level); hydropathy profiles of both proteins suggest that these genes encode integral membrane proteins which can be assumed to have approximately 16 transmembrane domains. Disruption of each gene was not lethal, but disruption of both genes was lethal. The 1,3-beta-D-glucan synthase activities of membrane and partially purified enzyme of gsc1::URA3 cells were significantly lower than those of the wild-type and gsc2::LEU2 cells.
The cytocidal effect of HM-1 produced by Hansenula mrakii on yeast Saccharomyces cerevisiae cells was studied. The HM-1 strongly inhibited the growth of S. cerevisiae cells at a low concentration (IC50: 2.1 x 10(-8) M) by reducing the number of viable cells. The killer action of HM-1 was most efficient when cells were actively proliferating. Cells in a resting state were resistant, but they became HM-1-sensitive after about 90 min of culturing at 30 degrees C, concomitantly with the increment of budding index. In association with the reduction of viable cell number, ultraviolet light-absorbing cellular components were discharged from sensitive cells. HM-1 molecules appear to bind to susceptible cells rather loosely since cells incubated with HM-1 were able to proliferate after having been washed. By phase-contrast light microscopy and scanning electron microscopy, discharge of cell material was observed at the budding portions of HM-1-treated cells. Addition of sorbitol to make the culture medium isotonic partially reduced the cell death induced by HM-1. These results suggest that HM-1 acts on the budding region of proliferating yeast cells, resulting in pore formation, leakage of cell material and eventual cell death.
The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (approximately 4 degrees C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2 M acetic acid, 0.5 M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4 degrees C on the surface of COS cells, however, was susceptible to limited proteolysis at 37 degrees C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.
Purpose The standard of care in the neoadjuvant setting for human epidermal growth factor receptor 2 (HER2)-positive breast cancer is dual HER2-targeted therapy. However, a need to minimize treatment-related toxicity and improve pathological complete response (pCR) rates, particularly in luminal HER2-positive disease, exists. Methods Neopeaks, a randomized, phase 2 study, compared docetaxel + carboplatin + trastuzumab + pertuzumab (TCbHP; 6 cycles; group A), TCbHP (4 cycles) followed by trastuzumab emtansine + pertuzumab (T-DM1+P; 4 cycles; group B), and T-DM1+P (4 cycles; group C) regimens in HER2-positive primary breast cancer patients; concurrent hormone therapy with T-DM1+P was administered in case of estrogen receptor positivity (ER+). Based on tumor shrinkage, nonresponders in group C were switched to 5-fluorouracil + epirubicin + cyclophosphamide (FEC; 4 cycles). Primary endpoint was pCR (comprehensive pCR ypN0 [ypT0-TisypN0]). Results Of 236 patients enrolled, 204 were randomized to groups A (n = 51), B (n = 52), and C (n = 101). In group C, 80 (79%) patients continued T-DM1+P following favorable response, whereas 21 (21%) nonresponders switched to FEC. pCR rate was numerically higher with the TCbHP → T-DM1+P regimen (71%) versus the standard TCbHP (57%) and T-DM1+P (57%) regimens. The rate in group C was higher among responders continuing T-DM1+P (63%) versus nonresponders who switched to FEC (38%). pCR rates after initial 4 cycles of T-DM1+P (group C; 57%) and standard TCbHP regimen (57%) were equivalent. pCR rate in patients with ER+ was significantly higher in group B (69%) than groups A (43%) and C (51%), but was comparable in patients with ER− (67-76%). Compared with the T-DM1-based arm, the incidence of adverse events was higher in the taxane-based arms. Conclusion In the neoadjuvant setting, the pCR rate with the standard TCbHP → T-DM1+P regimen was numerically better than the TCbHP regimen alone and significantly better in patients with ER+. Personalization of the T-DM1+P regimen could serve as a reasonable approach to minimize toxicity while maintaining efficacy. Trial registration ID: UMIN-CTR: UMIN000014649.
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