Dissecting the complete lipoprotein biogenesis pathway in Streptomyces scabies

Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.     

Proteins encoded by the mre gene cluster in Streptomyces coelicolor A3(2) cooperate in spore wall synthesis

It is still an open question how an intracellular cytoskeleton directs the synthesis of the peptidoglycan exoskeleton. In contrast to MreB of rod-shaped bacteria, which is essential for lateral cell wall synthesis, MreB of Streptomyces coelicolor has a role in sporulation. To study the function of the S. coelicolor mre gene cluster consisting of mreB, mreC, mreD, pbp2 and sfr, we generated non-polar replacement mutants. The individual mutants were viable and growth of substrate mycelium was not affected. However, all mutants produced enlarged spores, which frequently germinated prematurely and were sensitive to heat, high osmolarity and cell wall damaging agents. Protein-protein interaction assays by bacterial two-hybrid analyses indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex, which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria. Screening of a genomic library identified several novel putative components of this complex. One of them (sco2097) was deleted. The Δsco2097 mutant formed sensitive spores with an aberrant morphology, demonstrating that SCO2097 is a new player in cell morphogenesis of Streptomyces. Our results suggest that all Mre proteins cooperate with the newly identified proteins in the synthesis of the thickened spore wall required to resist detrimental environmental conditions.     

Effect of environmental conditions on the α-glucosidase inhibitory activity of mulberry leaves

Mulberry leaves have been used as the sole food for silkworms in sericulture, and also as a traditional medicine for diabetes prevention. Mulberry leaf components, for example 1-deoxynojirimycin (1-DNJ), inhibit the activity of α-glucosidase and prevent increased blood glucose levels, and they are highly toxic to caterpillars other than silkworms. The α-glucosidase inhibitory activity of mulberry leaves changes with the season, but it is unknown which environmental conditions influence the α-glucosidase inhibitory activity. We investigated in this study the relationship between the α-glucosidase inhibitory activity and environmental conditions of temperature and photoperiod. The results demonstrate that low temperatures induced decreasing α-glucosidase inhibitory activity, while the induction of newly grown shoots by the scission of branches induced increasing α-glucosidase inhibitory activity. These results suggest that the α-glucosidase inhibitory activity was related to the defense mechanism of mulberry plants against insect herbivores.     

Chromosome analysis by spectral karyotyping of spermatozoa from an oligoasthenozoospermic carrier of a 10; 21 reciprocal translocation

Cytogenetic analysis of germ-line cells prior to intracytoplasmic sperm injection (ICSI) treatment is thought to be necessary for infertile males with an identified chromosomal abnormality. We analyzed the chromosomal karyotype of human spermatozoa from an oligoasthenozoospermic carrier of a reciprocal translocation t(10; 21). Cytogenetic analysis of 39 spermatozoa was performed by spectral karyotyping (SKY) and by ICSI into mouse oocytes. The motile morphologically normal spermatozoa were injected into mouse oocytes. Of these spermatozoa, 38 (97.4%) were activated. Twenty-one (53.8%) of the activated oocytes formed two pronuclei. Metaphase chromosome spreads from 13 spermatozoa were analyzed. Only one spermatozoon was normal and 2 spermatozoa exhibited balanced translocation. Nine and one spermatozoa showed abnormalities related and unrelated to the translocation, respectively. The numbers of normal/balanced spermatozoa were lower than those in previous reports analyzing reciprocal translocations using a previously described technique involving penetrated golden hamster oocytes. After genetic counseling with the carrier and his partner, ICSI treatment was performed. Healthy female and male infants were delivered at 37 weeks gestation via a Caesarean section. The female infant was a carrier of the reciprocal translocation and the male infant was confirmed normal on prenatal diagnosis at 16 weeks gestation. For genetic counseling prior to ICSI treatment, the incidence of unbalanced type spermatozoa after swim-up or Percoll gradient treatment should be investigated and discussed with couples having fertility problems related to oligozoospermia autosomal structural abnormalities.     

Probing the inhibitor selectivity pocket of human 20α-hydroxysteroid dehydrogenase (AKR1C1) with X-ray crystallography and site-directed mutagenesis

Human 20α-hydroxysteroid dehydrogenase (AKR1C1) is an important drug target due to its role in the development of lung and endometrial cancers, premature birth and neuronal disorders. We report the crystal structure of AKR1C1 complexed with the first structure-based designed inhibitor 3-chloro-5-phenylsalicylic acid (K_i = 0.86 nM) bound in the active site. The binding of 3-chloro-5-phenylsalicylic acid to AKR1C1 resulted in a conformational change in the side chain of Phe311 to accommodate the bulky phenyl ring substituent at the 5-position of the inhibitor. The contributions of the nonconserved residues Leu54, Leu306, Leu308 and Phe311 to the binding were further investigated by site-directed mutagenesis, and the effects of the mutations on the K_i value were determined. The Leu54Val and Leu306Ala mutations resulted in 6- and 81-fold increases, respectively, in K_i values compared to the wild-type enzyme, while the remaining mutations had little or no effects.     

Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans

Sleep-like states occur in the life of all animals carefully studied and are characterized by reduced behavioral and neural activity as well as reduced responsiveness to stimulation [1]. How is reduced responsiveness to stimulation generated? We used calcium imaging to investigate a sleep-like state in larvae of the nematode Caenorhabditis elegans. We found that overall spontaneous neural activity was reduced during the sleep-like state in many neurons, including the mechanosensory neuron ALM. Stimulus-evoked calcium transients and behavior were reduced in ALM during the sleep-like state. Thus, reduced activity of ALM may contribute to reduce responsiveness during a sleep-like state.     

Novel and potent calcium-sensing receptor antagonists: discovery of (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate (TAK-075) as an orally active bone anabolic agent

The calcium-sensing receptor antagonist (CaSR) has been recognized as a promising target of anabolic agents for treating osteoporosis. In the course of developing a new drug candidate for osteoporosis, we found tetrahydropyrazolopyrimidine derivative 1 to be an orally active CaSR antagonist that stimulated transient PTH secretion in rats. However, compound 1 showed poor physical and chemical stability. In order to work out this compound's chemical stability and further understand its in vivo efficacy, we focused on modifying the 2-position of the tetrahydropyrazolopyrimidine. As a result of chemical modification, we discovered (5R)-N-[1-ethyl-1-(4-ethylphenyl)propyl]-2,7,7-trimethyl-5-phenyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide monotosylate 10m (TAK-075), which showed improved solubility, chemical stability, and in vivo efficacy. Furthermore, we describe that evaluating the active metabolite is important during repeated treatment with short-acting CaSR antagonists.     

TNF-α -308 genotypes are associated with TNF-α and TGF-β₁ mRNA expression in blood leucocytes of humans

AimTumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the ?308 TNF-α genotypes.MethodsQuantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2^?ΔΔCT method. Detection of the ?308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.ResultsThe relative TNF-α mRNA expression revealed significant differences between the TNF-α ?308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α ?308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.ConclusionOur findings suggest that the G-allele of TNF-α ?308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.     

Finding of Torymus koreanus (Hymenoptera: Torymidae) attacking Dryocosmus kuriphilus (Hymenoptera: Cynipidae) in Japan

Torymus koreanus (Hymenoptera: Torymidae) was reared from galls of Dryocosmus kuriphilus (Hymenoptera: Cynipidae) for the first time in Japan. We here report morphological features and partial mtDNA sequencing data of T. koreanus. Torymus koreanus has several common characteristics with species of the cyaneus group defined by Zavada (2003 ). According to the key to species groups defined by Graham and Gijswijt (1998 ), it does not belong to any species group because of the entire posterior margin of metasomal tergum five.