Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase

Rabbit antiserum against β-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with β-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of β-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-β-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that β-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of β-amylase bound to protein bodies in cereal seeds.     

Electron microscopy study of viral DNA packaging with lambda head mutants

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA^?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In $\text{λNu1}{}^{\mathrm{?}}\text{-}\text{infected}$ cells, however, most petit λ was not bound to DNA. In Fec^? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In $\text{D}{}^{\mathrm{?}}$ mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI^?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, $\text{p}\text{A}$ for the initiation of DNA packaging, $\text{p}\text{D}$ and $\text{p}\text{F}$I for the promotion of DNA packaging, and $\text{p}\text{D}$ for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.     

DNA regions essential for the function of a bacteriophage fd promoter.

The promoter for the major coat protein gene of bacteriophage fd contains a unique sequence. TATAAT, in the non-transcribed region corresponding to the Pribnow box. A R-Hha I cleavage site which destroys functions is located five pairs upstream from the TATAAT sequence (fifteen base pairs upstream from the RNA initiation site). The promoter was cleaved into two fragments by R-Hha I and each promoter fragment was joined to DNA fragments derived from other regions. Ligation of the TATAAT-containing fragment to any of the DNA fragments examined resulted in recovery of promoter function. The results suggest for this type of promoter that no unique sequence is necessary upstream from the R-Hha I cleavage site although a contiguous DNA chain must be present in this area.     

A sulfotransferase model: Covalent participation of a macrocyclic oxime in the hydrolysis of 2,4-dinitrophenyl sulfate

The liberation of 2,4-dinitrophenolate ion from 2,4-dinitrophenyl sulfate (DNPS) in aqueous organic solvent with 0.1 N sodium hydroxide was accelerated upon addition of an equimolar amount of Oxime-I (10-hydroxy-11-hydroxyimino[20]-paracyclophane) to the sulfate ester. Oxime-I was found to undergo covalent participation at the oxime group to afford oxime O-sulfonate. The rate acceleration with Oxime-I was larger than that with β-CD (cycloheptaamylose). The catalytic efficiency of Oxime-I has been ascribed primarily to the tighter inclusion of the substrate ester into the more hydrophobic Oxime-I cavity provided by the effective apolar paracyclophane skeleton, as well as to the greater nucleophilicity of the oxime group than of the hydroxyl group in β-CD. Consequently, Oxime-I may be considered as a conventional model for arylsulfatases and sulfotransferases, providing the effective binding process for the substrate.     

Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98.

Pyrolyzates of 25 amino acids and 5 indole derivatives were tested for mutagenicity in the histidine-requiring mutant Salmonella typhimurium TA 98. Significant mutagenic activity was detected with pyrolyzates of most of the amino acids. These pyrolyzates required a liver microsomal fraction, as representative of mammalian metabolism, to be detected as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of L-tryptophan. As little as 10 microgram of the pyrolyzate of L-tryptophan had detectable mutagenic activity toward TA 98. The optimal pyrolysis temperatures for the formation of mutagenic products were shown to be 500 degrees C for L-tryptophan and 600 degrees C for the other amino acids. The results from pyrolyses of some indole derivatives suggest that an amino group at the alpha-position to the carboxyl group of L-tryptophan plays an important role in the formation of mutagens.     

Effects of tryptic digestion on the enzymatic activites of chicken gizzard myosin.

Tryptic digestion of gizzard myosin resulted in the degradation of the 20K light chain (G1) to its 17K fragment, which could not be phosphorylated. The rapid loss of Ca2+-dependent activation of actomyosin ATPase activity accompanied the degradation of G1. Increase in the Ca2+-ATPase activity and decrease in the EDTA-ATPase activity of myosin accompanied the degradation of myosin heavy chain, but not the cleavage of G1.     

Characterization and molecular cloning of a putative binding protein for heparin-binding growth factors

A novel Mr 17,000 heparin-binding protein was purified from culture medium conditioned by A431 human epidermoid carcinoma cells. This protein, designated HBp17, was found to bind the heparin-binding peptide growth factors HBGF-1 and HBGF-2 in a noncovalent, reversible manner. In addition HBp17 was found to inhibit the biological activities of both HBGF-1 and HBGF-2. Both the binding and inactivation of HBGF-1 and HBGF-2 by HBp17 were abolished by heparin. Full-length 1163-base pair HBp17 cDNA was cloned and sequenced by using the polymerase chain reaction technique. The deduced primary structure of HBp17 consisted of 234 amino acids including each of five partial peptide sequences obtained from proteolytic fragments of purified HBp17. The encoded protein included a 33-residue N-terminal signal sequence for secretion and a single potential N-linked glycosylation site. No homology with any known protein was found for the deduced primary structure of HBp17. The expression of HBp17 mRNA was found to occur preferentially in normal human keratinocytes and in squamous cell carcinomas. This pattern of HBp17 gene expression suggests that this binding protein for HBGFs 1 and 2 has a physiological role in squamous epithelia.     

Alteration of pectoral fin nerves following ablation of fin buds and by ectopic fin buds in the Japanese medaka fish

The role of the pectoral fin bud for outgrowth by fin axons was assessed by ablation of pectoral fin buds and by transplantation of fin buds to ectopic sites in the embryos of the Japanese medaka fish (Oryzias latipes). Normally nerves from segments 1-4 (S1-4) and less frequently the S5 nerve converged at the base of the fin bud by extending toward the fin bud on the ventral surface of the axial muscles (H. Okamoto and J. Y. Kuwada, 1991, Dev. Biol. 146). Following ablation of the fin bud before motor growth cones have begun to extend laterally, nerves in S1-5 followed a trajectory down the middle of each segment parallel to the borders of the metamerically arranged axial muscles rather than converging. This trajectory was similar to that of more posterior segmental nerves which do not converge toward the fin bud. When fin buds were transplanted to more posterior segments, nerves from S1-5 often changed their trajectories and extended to the base of ectopic buds. Furthermore, motor nerves from segments posterior to S5, which normally do not innervate the fin bud, also extended to the ectopic fin bud. When faced with both the host and ectopic fin bud, motor nerves extended to either fin bud or branched and extended to both fin buds. These results demonstrate that the early fin bud is necessary for correct outgrowth of fin nerves and suggest that the fin bud normally attracts fin nerves to its base. One possible mechanism for the attraction of motor growth cones by the fin bud is a long distance cue emitted by the fin bud.