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.     

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.     

Remarkable synergies between baicalein and tetracycline, and baicalein and beta-lactams against methicillin-resistant Staphylococcus aureus

During the screening of compounds that potentiate the effect of antimicrobial agents against methicillin-resistant Staphylococcus aureus(MRSA), we found that an extract of thyme (Thymus vulgaris L) leaves greatly reduced the minimum inhibitory concentration (MIC) of tetracycline against MRSA. We isolated the effective compound and identified it as baicalein (5, 6, 7-trihydroxyflavone). One of the clinically isolated MRSA strains possessed tetK, a gene encoding active efflux pump for tetracycline. We examined the effect of baicalein on the efflux of tetracycline, using Escherichia coli KAM32/pTZ1252 carrying the tetK. The E. coli KAM32/pTZ1252 showed 8 to 16 times higher MIC than E. coli KAM32. We observed strong inhibition of transport of tetracycline by baicalein with membrane vesicles prepared from E. coli KAM32/pTZ1252. Baicalein also showed synergy with tetracycline in a MRSA strain that doesn't possess tetK, or with beta-lactams. Thus, mechanisms of the synergies seem to be versatile.     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

Techniques of SNP genotyping and remote sensing applied for elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype

We present a conceptual framework for applying techniques of SNP genotyping as a molecular biological approach and remote sensing as an ecological approach to elucidation of the contribution of polygene and environmental factors to inter-individual variation in skin pigmentation phenotype. Additionally, we discuss the obstacles that frustrate our efforts to identify how the human genome encodes the complex phenotype and suggest the use of computational methods designed for knowledge discovery within hereditary database.     

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.