Purification, characterization, and amino acid sequences of pepsinogens and pepsins from the esophageal mucosa of bullfrog (Rana catesbeiana)

Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 32,000-34,000. The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.     

Autocatalytic processing of procathepsin E to cathepsin E and their structural differences

The processing of human gastric procathepsin E to its mature form, cathepsin E, was studied at pH 3.5. The results revealed the autocatalytic and apparently one-step conversion of procathepsin E to cathepsin E within 10 min of incubation at 14 degrees C under the conditions used. Analyses of the amino acid sequences of both procathepsin E and cathepsin E showed that cleavage occurred at the Met36-Ile37 bond to produce the mature form, cathepsin E. The NH2-terminal amino acid sequence of procathepsin E thus determined was identical with that predicted from the cDNA sequence by Azuma et al. except that the NH2-terminal glutamine residue in the latter was converted into a pyroglutamic acid residue in the former and that the glycine residue at position 2 in the latter sequence was deleted in the former. On the other hand, the NH2-terminal amino acid sequence of cathepsin E was identical with that reported previously by us.     

Structural evidence for two isozymic forms and the carbohydrate attachment site of human gastric cathepsin E

The amino acid sequences in the NH2-terminal region and some other parts of human gastric cathepsin E were investigated. The NH2-terminal sequencing revealed that the cathepsin E preparation which had been activated at pH 4.0 contained one major and one minor isozymes in an approximate molar ratio of 3:1. The NH2-terminal sequence of the former was very similar to but partly different from that predicted from cDNA sequencing by Azuma et al., whereas the latter had an NH2-terminal sequence identical with the predicted sequence. These results provide structural evidence for the presence of at least two isozymic forms in human gastric cathepsin E. In addition, the site of carbohydrate attachment was elucidated by isolation and analysis of a glycopeptide fraction from an enzymatic digest of cathepsin E. A single carbohydrate chain was deduced to be attached to the asparagine residue at position 34 in the major isozyme and to the corresponding asparagine residue in the minor isozyme.     

Clinical anatomy of the blood spaces and blood vessels surrounding the siphon of the internal carotid artery

The anterior blood space of the cavernous sinus is situated anterolateral to the carotid siphon in 70%, anterior to it in 15%, and lateral to it in 15%. Its height, depth, and mediolateral breadth were measured. The mean distance between the carotid siphon and the skin at the supraorbital foramen was measured with 63 (52.4-71.4) mm. The drainage of the orbital veins was studied and described as well as the area of origin and first course of the ophthalmic artery and its clinical importance.     

Physicochemical properties of pyocin F1.

The physiochemical properties of pyocin F1 were studied. Pyocin F1 consists of flexuous rod-like particles homogenous in size. Each particle was composed of rod and fiber parts. The rod part was 105.5 +/- 9.5 nm long and 10.0 +/- 1.4 nm wide, and showed regular striations amounting to 23 layers. The fiber part was composed of several filaments; the length of the longest filament was 43.0 +/- 12.0 nm. The amino acid composition, the partial specific volume (0.720 ml/g), the sedimentation coefficient (S020,W = 35.1S), and the translational diffusion constant (0.94 +/- 0.01 x 10(-7) cm2/s) were determined. The particle weight was calculated to be 3.23 x 10(6) daltons.     

Regulation of active and quiescent somatic stem cells by Notch signaling

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation-dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation-dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.