Functional complementation between the two homologous genes, ops and tps, during differentiation of Myxococcus xanthus

Summary Protein S is a development-specific protein of Myxococcus xanthus encoded by the tps gene. It has been shown that there are two extensively homologous genes (ops and tps) tandemly repeated in the same direction with a 1.4 kb spacer fragment between them (Inouye et al. 1983). Seven deletion mutants were constructed by removing the ops gene, the tps gene, segments of the spacer sequence or combinations of these regions. The deleted regions were replaced with DNA fragments carrying the Tn5 gene for kanamycin resistance.The effects of deleting different regions on morphological changes and on patterns of protein synthesis during fruiting body formation were examined. The process of fruiting body formation was severely delayed when both the ops and the tps genes were deleted. However, this delay could be suppressed by either the ops gene or the tps gene, individually, although in the latter case, a slight delay was still observed. These results indicate that the ops gene is expressed during fruiting body formation and plays a role in the normal program of M. xanthus differentiation. Furthermore, the role of the ops gene can be complemented by the tps gene. The deletion of the ops and/or tps genes had no effect on glycerol-spore formation.     

A soluble auxin-binding protein from mung bean hypocotyls has indole-3-acetaldehyde reductase activity

To clarify the roles of auxin-binding proteins (ABPs) in the action of auxin, soluble auxin-binding proteins were isolated from an extract of etiolated mung bean hypocotyls by affinity chromatography on 2,4-dichlorophenoxyacetic acid (2,4-D)-linked Sepharose 4B. A 39-kDa polypeptide was retained on the affinity column and eluted with a solution containing IAA or 2,4-D, but not with a solution containing benzoic acid. The protein was then purified by several column-chromatographic steps. The apparent molecular mass of the protein was estimated to be 77 kDa by gel filtration and 39 kDa by SDS-PAGE. We designated this protein ABP39. The partial amino acid sequences of ABP39, obtained after chemical cleavage by CNBr, revealed high homology with alcohol dehydrogenase (ADH; EC 1.2.1.1). While the ABP39 was not capable of oxidizing ethanol, it did catalyze the reduction of indole-3-acetaldehyde (IAAld) to indole-3-ethanol (IEt) with an apparent K_m of 22 μ M. The IAAld reductase (EC 1.2.3.1) is specific for NADPH as a cofactor. The ABP39 also catalyzed the reduction of other aldehydes, such as acetaldehyde, benzaldehyde, phenylacetaldehyde and propionealdehyde. Indole-3-aldehyde was a poor substrate. The enzyme activity was inhibited by both indole-3-acetic acid and 2,4-D in a competitive manner. Therefore, the enzyme is considered to be retained on the affinity column by recognition of auxin structure.     

Comparative study of eye defective worm 'menashi' and regenerating wild-type in planarian, Dugesia ryukyuensis

Inbreeding of the sexualized planarian, Dugesia ryukyuensis, produces eye-defective worms, menashi, in the F1 population. To study the effects of this mutation on the eye, we observed the eye-region of menashi using electron microscopy and compared it with the regenerating eye in wild-type worms. The intact eye of wild-type planarians consisted of a few pigment cells and a number of visual cells. Pigment cells containing spherically-shaped electron-dense melanosomes contacted each other and enclosed rhabdomes of visual cells. Rhabdomes had numerous tubular microvilli extending radially and touching the pigment cells. However, in menashi, various lengths of tubular microvilli were irregularly distributed near the pigment cells, which contained numerous electron-lucent premelanosomes, and no adhesive structures were found between the pigment cells. The premelanosomes of menashi were equal in size to those seen after 2 days of regeneration in wild-type planarians and were similar in maturation to those found after 3 days of regeneration in wild-type planarian. These results suggest that menashi is defective in the mechanism(s) of developing pigment granules and constructing visual cells. These findings also suggest that pigment cells in menashi are defective in the mechanism(s) involved with cell adhesion.     

Ectopic dermal ridge configurations on the interdigital webbings of Hammertoe mutant mice (Hm): another possible role of programmed cell death in limb development

BACKGROUND: The mechanism underlying the development of aberrant phalangeal pads and dermal ridge configurations in malformed limbs is not well understood. The forelimbs of Hammertoe (Hm) mutant mouse fetuses were examined sequentially to clarify the relationship between the occurrence of abnormal programmed cell death (PCD) and the formation of phalangeal pads and dermal ridge patterns. METHODS: Relevant morphological features, with special emphasis on pads and dermal ridge configurations, were inspected on the exposed dermal surface of the forelimbs of adult Hm mutant mice. The forelimbs of Hm mutant mouse fetuses (GD13-18) and newborns were examined histologically. The forelimbs of GD13 fetuses were subjected to Nile blue (NB) vital staining for in situ labeling of PCD. RESULTS: In the forelimbs of +/+ mice, the formation of dermal ridges was confined to pads, while in Hm/+ and Hm/Hm animals, which have interdigital webbing involving digits II-V, dermal ridges were formed also on the ventral side of the webbing, specifically on its lateral margins between the neighboring digits and on the medial margin of the webbing extending toward the palmar pad. PCD was decreased in the interdigital zones II-IV in GD13 Hm/+ and Hm/Hm fetuses. CONCLUSIONS: Reduced PCD interdigital tissue of Hm/+ and Hm/Hm fetuses may result in the failure of physiological elimination of interdigital cells and in the persistence of soft tissue webbing between digits. The failure of PCD to occur may also interrupt the interdigital surviving cells to reach the neighboring digits and the distal area of the palm, thereby producing ectopic dermal ridges. It seems that interdigital PCD contributes not only to digit separation but also to the development of digital and palmar pads.     

PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter

Neurotransmitter is released from nerve terminals by Ca2+-dependent exocytosis through many steps. SNARE proteins are key components at the priming and fusion steps, and the priming step is modulated by cAMP-dependent protein kinase (PKA), which causes synaptic plasticity. We show that the SNARE regulatory protein tomosyn is directly phosphorylated by PKA, which reduces its interaction with syntaxin-1 (a component of SNAREs) and enhances the formation of the SNARE complex. Electrophysiological studies using cultured superior cervical ganglion (SCG) neurons revealed that this enhanced formation of the SNARE complex by the PKA-catalyzed phosphorylation of tomosyn increased the fusion-competent readily releasable pool of synaptic vesicles and, thereby, enhanced neurotransmitter release. This mechanism was indeed involved in the facilitation of neurotransmitter release that was induced by a potent biological mediator, the pituitary adenylate cyclase-activating polypeptide, in SCG neurons. We describe the roles and modes of action of PKA and tomosyn in Ca2+-dependent neurotransmitter release.     

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

Chronic receptor-mediated activation of Gi/o proteins alters basal t-tubular and sarcolemmal L-type Ca²⁺ channel activity through phosphatases in heart failure

L-type Ca(2+) channels (LTCCs) play an essential role in the excitation-contraction coupling of ventricular myocytes. We previously found that t-tubular (TT) LTCC current density was halved by the activation of protein phosphatase (PP)1 and/or PP2A, whereas surface sarcolemmal (SS) LTCC current density was increased by the inhibition of PP1 and/or PP2A activity in failing ventricular myocytes of mice chronically treated with isoproterenol (ISO mice). In the present study, we examined the possible involvement of inhibitory heterotrimeric G proteins (G(i/o)) in these abnormalities by chronically administrating pertussis toxin (PTX) to ISO mice (ISO + PTX mice). Compared with ISO mice, ISO + PTX mice exhibited significantly higher fractional shortening of the left ventricle. The expression level of Gα(i2) proteins was not altered by the treatment of mice with ISO and/or PTX. ISO + PTX myocytes had normal TT and SS LTCC current densities because they had higher and lower availability and/or open probability of TT and SS LTCCs than ISO myocytes, respectively. A selective PKA inhibitor, H-89, did not affect LTCC current densities in ISO + PTX myocytes. A selective PP2A inhibitor, fostriecin, did not affect SS or TT current density in control or ISO + PTX myocytes but significantly increased TT but not SS LTCC current density in ISO myocytes. These results indicate that chronic receptor-mediated activation of G(i/o) in vivo decreases basal TT LTCC activity by activating PP2A and increases basal SS LTCC activity by inhibiting PP1 without modulating PKA in heart failure.