Preparation and Characterization of Monoclonal Antibodies Specific for Lauroylated Isoform of Bovine Transducin α-Subunit: Immunohistochemical Analysis of Bovine Retinas

Abstract: The photoreceptor G protein transducin [α- and βγ-subunits (Tα/Tβγ)] plays a central role in the visual transduction process. The amino-terminus of bovine Tα is modified by one of four distinct fatty acids—laurate (C12:0), myristate (C14:0), C14:1 (5- cis ), and C14:2 (5- cis , 8- cis )—but the biological significance and the localization of the four isoforms of Tα are poorly understood. To investigate the cellular distribution of each isoform, we prepared monoclonal antibodies against a synthetic C12:0-, C14:0-, C14:1-, or C14:2-nonapeptide corresponding to the N-terminal region of Tα. Among several types of antibodies isolated, only one type, represented by LA4, reacted specifically with the C12:0-peptide as well as purified Tα but not with the other proteins in bovine retinal homogenate, including recoverin, indicating that the epitope comprises both C12:0 and the N-terminal amino acids of Tα. Immunohistochemical analyses of bovine retinal sections by LA4 showed the uniform distribution of C12:0-Tα in almost all the rod outer segments. Hence, it seemed unlikely that each isoform of Tα was localized in specific cells. This observation, together with evidence for a possible functional diversity among the isoforms, suggests that the four isoforms of Tα in a single rod cell may contribute simultaneously to a fine tuning of the photon-signal transduction process.     

Chromosaponin I stimulates the growth of lettuce roots

A γ-pyronyl triterpenoid saponin termed chromosaponin 1 (CSI), a conjugate of soyasaponin I and γ-pyrone, was found at 3 mM to stimulate the growth of lettuce root ( Lactuca sativa L. ev. Grand Rapids) to about 190% of the control. Since CSI is an amphipathic reductant, the stimulating effect of this saponin was compared with other reductants and other surface-active compounds. Trolox (2-carboxy-2.5.7.8-tetraamethyl-6-chromanol). another amphipathic reductant, also stimulated root growth, while other hydrophilic reductants including ascorbate. NADPH, NADH and glutathione did not. Some surfactants promoted root growth but their stimulating effects were smaller than the optimum effect of CSI. These results suggest a possible Function of CSI as an amphipathic reductant in root growth regulation.     

Inhibition of the Breakdown of Xyloglucans in Azuki Bean Epicotyls by Concanavalin A

Indole-3-acetic acid at 10 µM caused a 30% decrease inthe weight-average molecular mass of xyloglucans extracted with24% KOH from the cell walls of epicotyl segments of azuki bean(Vigna angularis Ohwi et Ohashi cv. Takara). Concanavalin A(Con A) at 2 g liter^–1 completely inhibited the IAA-inducedchange in the molecular mass of the xyloglucans. Con A alsosuppressed the autolysis of pectin-depleted cell walls, as wellas the breakdown of xyloglucans by a protein fraction that hadbeen extracted with 1 M NaCl from the cell walls of azuki beanepicotyls. These results indicate that Con A is a potent inhibitorof the breakdown of xyloglucans both in vivo and in vitro. Mostof the activity responsible for the decrease in staining byiodine and the increase in reducing power of solution of xyloglucansin the protein fraction from cell walls bound to a column ofCon A-Sepharose and was eluted by the specific hapten, methyl     

Gravitropic response inEichhornia cressipes (water hyacinth) I. process of gravitropic bending in the peduncle

In the last half of the anthokinetic cycle inEichhornia cressipes the peduncle showed a two-step bending response which started after the completion of the flowering: the primary bending in the upper region and the second one in the base of the peduncle. In the present study, only the primary bending response was examined and the following results were obtained.

1. Under the condition in the Western Japan, the anthokinetic cycle completed in 36–40 hr. The inflorescence started to grow during night, reaching the full flowering stage next morning, and the bending was initiated in the evening and completed next morning.
2. The bending was a positive gravitropism since the peduncle did not show bending when it was placed on a horizontal clinostat rotating at 2–3 rpm, or when the plant was placed up-side-down.
3. Before the end of flowering phase, the peduncle showed a normal negative gravitropic response but afterward it acquired the property to show a positive gravitropic response.
4. The bending was due to the extension of convex side of the peduncle, accompanied by a shrinkage of the concave side. The extension of the upper side was caused by cell extension. At the turning point from negative to positive gravitropic response, the extension of the peduncle ceased for several hours.

From the above results it is concluded that the primary bending response of the peduncle in water hyacinth was a positive gravitropism.     

Resolution ofFusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence homology comparison in protein data base

Proteins fromFusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data.     

Spin-Trapping and Direct Epr Investigations on the Hepatotoxic and Hepatocarcinogenic Actions of Luteoskyrin, An Anthraquinoid Mycotoxin Produced by Penicillium Islandicum Sopp. Generations of Superoxide Anion and Luteoskyrin Semiquinone Radical in the Redox Systems Consisted of Luteoskyrin and Liver Nadph- Or Nadh-Dependent Reductases

Luteoskyrin is a hepatotoxic and hepatocarcinogenic bisdihydroanthraquinone produced by Penicillium islandicum Sopp. By observing the EPR spectra of DMPO-spin adducts and luteoskyrin semiquinone radical, we investigated in vitro whether luteoskyrin is reduced to its semiquinone radical leading to the generation of active oxygen species in redox systems catalyzed by NADPH-dependent cytochrome reductases of the liver. We found (1) the formation of luteoskyrin semiquinone radical in the NADPH-cytochrome P-450 reductase system under anaerobic conditions, (2) the generation of O- in the systems composed of luteoskyrin, NAD(P)H, and either rat liver microsomal NADPH-cytochrome P-450 reductase or submitochondrial particles and (3) dicoumarol showed no effect on the O- generation in the case of submitochondrial particles. From these results we proposed that luteoskyrin liver injuries are induced by the active oxygen species generated in the process of autoxidation of luteoskyrin semiquinone radical which is produced in the one-electron redox systems catalyzed by the liver NAD(P)H-dependent cytochrome reductases.     

Experimental retracement of the origins of a protocell

Although Oparin used coacervate droplets from two or more types of polymer to model the first cell, he hypothesized homacervation from protein, consistent with Pasteur and Darwin. Herrera made two amino acids and numerous cell-like structures (sulfobes) in the laboratory, which probably arose from intermediate polymers. Our experiments have conformed with a homoacervation of thermal proteinoid, in which amino acid sequences are determined by the reacting amino acids themselves. All proteinoids that have been tested assemble themselves alone in water to protocells. The protocells have characteristics of life defined by Webster's Dictionary: metabolism, growth, reproduction and response to stimuli in the environment. The protocells are able also to evolve to more modern cells including the initiation of a nucleic acid coding system.Principal spinoffs from the results are revised evolutionary theory, models for protoneurons and networks thereof, and numerous industrial applications of thermal polyamino acids. Life itself has thus been reaffirmed to be rooted in protein, not in DNA nor RNA, which are however crucial to inheritance in modern life as instruction manual (Kornberg).Recognition of the advances have been considerably delayed by the deeply held assumption that life began by chance from random polymerization of amino acids, in contrast to the experimental findings. The concepts of DNA/RNA-first and protein-first are reconciled by a rise-and-fall progression as often seen in biochemical and biological evolution.The fact that amino acids order themselves explains in turn that thermal copolyamino acids are finding numerous applications. The entire sequence of processes in the proteinoid origins theory is now seen to be highly deterministic, in close accord with Einstein.     

The Role of DNase and EDTA on DNA Degradation in Formaldehyde Fixed Tissues

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.     

The restoration of the functions of serially passaged calf hepatocytes by spheroid formation

Summary A serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers—including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate hydrogenase, and ammonia-metabolizing activities—have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.     

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.