ResearchFusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca 2+ , leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca 2+ oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCζ) has been identified in mice. Mouse PLCζ triggers Ca 2+ oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCζ homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCζ protein sequences indicates a shorter X-Y linker region in human PLCζ and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCζ elicits Ca 2+ oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCζ elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCζ from monkeys and mice. These results are consistent with the proposal that sperm PLCζ is the molecular trigger for egg activation during fertilization and that the role and activity of PLCζ is highly conserved across mammalian species.

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Previous studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions (ϳ1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (<10%). To study the role of the sumoylation of TOP1, we mutated sites on green fluorescent protein (GFP)-TOP1 corresponding to the consensus sequence for protein sumoylation (⌿KXE, where ⌿ is a hydrophobic residue) and assayed the mutants for basal and camptothecin-induced sumoylation. Only one of the eight mutants, K117R, located in the highly charged NH 2 -terminal region, showed a substantial reduction (ϳ5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the ⌿KXE sequences flanking K117R additionally mutated (K103R/ K117R/K153R) showed little if any sumoylation, but was degraded like wild-type GFP-TOP1 during camptothecin treatment. However, K103R/K117R/K153R-GFP-TOP1 was markedly concentrated within nucleoli, depleted from the remainder of nucleus, and failed to be cleared from nucleoli in response to camptothecin treatment. These data are consistent with a model wherein basal transient sumoylation of the NH 2 -terminal, highly charged, disordered region prevents TOP1 binding to sites in nucleoli, thus driving it to bind in the nucleoplasm; and camptothecin treatment, which increases TOP1 sumoylation, further shifts the binding resulting in delocalization of TOP1 from nucleoli to nucleoplasm.

Objectives/Hypothesis Vocal fold atrophy, scar, and sulcus reduce the vibratory function of the vocal fold mucosa, which causes severe refractory dysphonia. We have reported encouraging preliminary results using an intracordal injection of basic fibroblast growth factor (bFGF) and showed improvement in phonatory parameters and voice. The present study summarizes our experience with 100 cases of stiffened vocal folds that were treated with bFGF injections. Study Design Retrospective chart review with Interstitial Review Board (IRB) approval. Methods Local injection of bFGF was performed in 100 cases of vocal fold pathology, which included 43 cases of vocal fold atrophy, 41 cases with scar, and 16 cases with sulcus. Ten micrograms of bFGF were injected into the vocal folds under topical anesthesia 4 times in each patient. Therapeutic outcomes were examined with maximum phonation time (MPT), voice handicap index‐10 (VHI‐10), and GRBAS scale. Results MPT, VHI‐10, and GRBAS scores significantly improved in all pathology groups. An improvement on the VHI‐10 greater than five points was observed in 82% of atrophy cases, 78% of scar cases, and 67% of sulcus cases. Improvement on the VHI‐10 was significantly better in the atrophy group than the scar or sulcus groups. The mild/moderate cases of scar and sulcus showed better improvement than severe cases. Conclusions The current large case series indicates positive effects of intracordal injection of bFGF for improvement of voice with no severe adverse events. The effects appeared best for cases of atrophy, while the treatment of severe scar and sulcus requires further improvement. Level of Evidence 4 Laryngoscope, 131:2059–2064, 2021

Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins.