Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

Scanning electron microscopy of macrophages in the tail musculature of the metamorphosing anuran tadpole,Rana japonica

Summary Scanning electron microscopy was used to investigate the morphological changes of the tail musculature of the metamorphosing anuran tadpole, attention being focused on phagocytosis by macrophages. Muscle fibers were stained en bloc with silver and freeze-fractured during dehydration, or torn after drying. Samples were sputter-coated with gold-palladium and observed in both secondary electron- and back-scattered electron modes with a scanning electron microscope.Various cells were identified by the methods of secondary electron- and back-scattered electron images. Some macrophages lying between muscle fibers at prometamorphic stages possessed numerous finger-like projections and well-developed ruffles. During degeneration of muscle fibers macrophages collected in the degenerating region and invaded the space between the disordering myofibrils. In advanced stages the numbers of macrophages clearly increased on or around the degenerating muscle fibers. At the climactic stage fragmented muscles were entrapped and then engulfed by the macrophages. With the completion of phagocytosis, the macrophages became globular with reduction of the ridge-like ruffles. Macrophages may play a role not only in scavenging the fragmented muscle fibers, but also using their long processes in active formation of the fragments.     

Farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthetases during Cucurbita pepo germination

Although the geranylgeranyl pyrophosphate synthetase activity was very low compared with farnesyl pyrophosphate synthetase activity on imbibition of pumpkin seed, the former increased markedly and the latter decreased as germination proceeded.     

Biochemical study of the adepidermal granules of Urodela and Anura—Especially on lipids

Summary The lipids of the adepidermal granules of Hynobius tokyoensis and Rana japonica were analysed qualitatively. To extract the lipids from the adepidermal granules, the epidermis of the larvae of both animals were removed from the body. Then the granules were extracted from the surface of the basal lamina by means of wiping with the cotton balls containing the solvent. It was ascertained with the electron microscope whether the adepidermal granules were extracted by the cotton balls or not.By the analysis which was made with thin layer chromatography, the same kind of phospholipids or neutral lipids were detected from the extracts of both R. Japonica and H. tokyoensis. Moreover, four spots which seemed to contain some sialic acids were detected from the both extracts.     

NMR analysis of structure and function of snake neurotoxins

The 270-MHz proton-nmr spectra of short neurotoxins (erabutoxins from Laticauda semifasciata and cobrotoxin from Naja naja atra) and long neurotoxins (toxin B from Naja naja and α-bungarotoxin from Bungarus multicinctus) have been analyzed. The conformation of erabutoxin b in solution is largely consistent with the x-ray crystal analysis, although the environment of His-7 in solution is definitely different from that in the crystal. The pH-dependent transition has been found for toxin B, indicating that the conformation in neutral solution is different from that in the crystal as grown from acidic solution. The deuterium-exchange rates of the amide protons for the four neurotoxins have been measured. The order of structural rigidity is the same as the order of the irreversibility of neuromuscular block by neurotoxins.     

State of functionally essential Trp-29 in snake venom neurotoxins: A proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (NMR) spectra have been recorded of various neurotoxins from snake venoms.pH dependence of the chemical shifts and resonance intensity has been followed for the functionally essential Trp-29. The indole N-1 proton of Trp-29 in -bungarotoxin, toxin B, and cobrotoxin exhibits appreciably large upfield shifts as thepH is lowered and the suppressed exchange with the solvent hydrogen atpH 3–4, but not inNaja haje annulifera 10 where Asp-31 is replaced with Gly-31. This observation strongly suggests the presence of a hydrogen bond between Trp-29 and Asp-31 that is probably important in stabilizing the arrangement of the functionally essential residues to form a distinct binding region for the receptor.     

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.     

Production of hydrogen peroxide by polyphenols and polyphenol-rich beverages under quasi-physiological conditions

To investigate the ability of the production of H(2)O(2) by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37 degrees C), and then measured the formation of H(2)O(2) by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H(2)O(2). We also examined the effects of a metal chelator, pH, and O(2) on the H(2)O(2)-generating property, and the generation of H(2)O(2) by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H(2)O(2)-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H(2)O(2) in beverages. From the results, the possible significances of the H(2)O(2)-generating property of polyphenols for biological systems are discussed.     

Oxidative deamination by hydrogen peroxide in the presence of metals

Various amines, including lysine residue of bovine serum albumin, were oxidatively deaminated to form the corresponding aldehydes by a H 2 O 2 /Cu 2+ oxidation system at physiological pH and temperature. The resulting aldehydes were measured by high-performance liquid chromatography. We investigated the effects of metal ions, pH, inhibitors, and O 2 on the oxidative deamination of benzylamine by H 2 O 2 . The formation of benzaldehyde was the greatest with Cu 2+ , and catalysis occurred with Co 2+ , VO 2+ , and Fe 3+ . The reaction was greatly accelerated as the pH value rose and was markedly inhibited by EDTA and catalase. Dimethyl sulfoxide and thiourea, which are hydroxyl radical scavengers, were also effective in inhibiting the generation of benzaldehyde, indicating that the reaction is a hydroxyl radical-mediated reaction. Superoxide dismutase greatly stimulated the reaction, probably due to the formation of hydroxyl radicals. O 2 was not required in the oxidation, and instead slightly inhibited the reaction. We also examined several oxidation systems. Ascorbic acid/O 2 /Cu 2+ and hemoglobin/H 2 O 2 systems also converted benzylamine to benzaldehyde. The proposed mechanism of the oxidative deamination by H 2 O 2 /Cu 2+ system is discussed.