SEXUAL DIFFERENTIATION OF GRIFFITHSIA (CERAMIALES, RHODOPHYTA): NUCLEAR PLOIDY LEVEL OF MIXED-PHASE PLANTS IN G. JAPONICA1

Mixed-phase plants of Griffithsia japonica Okamura spontaneously occurred in a laboratory culture. Four female plants produced tetrasporangia and spermatangia in addition to their normal female reproductive structures (bisexual/mixed-phase plants), and four male plants produced tetrasporangia as well as spermatangia (male/mixed-phase plants). To determine the nuclear ploidy level of these mixed-phase plants, relative nuclear sizes of male, female, tetrasporangial, and mixed-phase plants were measured using a microscopic image analysis system. Haploid gametophytes could be distinguished from diploid tetrasporophytes by relative nuclear sizes, with the later having nuclei twice the size of the former. Relative nuclear sizes of the mixed-phase plants were similar to those of the haploid plants. Thus, the mixed-phase plants were determined to be haploid. Haploid mixed-phase plants of G. japonica have a potential to produce male, female and tetrasporangial reproductive structures. Sex determination models are discussed to explain "haploid" mixed-phase phenomena in red algae .     

Role of Phe-99 and Trp-196 of sepiapterin reductase from Chlorobium tepidum in the production of L-threo-tetrahydrobiopterin

Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L -threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180° around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher K _m values than the wild type. Remarkably, the W196A mutation resulted in a higher K _m increase compared with the F99A mutation. Consistent with the results, the melting temperature ( T _m) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production.     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

Enhanced process monitoring of fed-batch penicillin cultivation using time-varying and multivariate statistical analysis

On-line monitoring of penicillin cultivation processes is crucial to the safe production of high-quality products. In the past, multiway principal component analysis (MPCA), a multivariate projection method, has been widely used to monitor batch and fed-batch processes. However, when MPCA is used for on-line batch monitoring, the future behavior of each new batch must be inferred up to the end of the batch operation at each time and the batch lengths must be equalized. This represents a major shortcoming because predicting the future observations without considering the dynamic relationships may distort the data information, leading to false alarms. In this paper, a new statistical batch monitoring approach based on variable-wise unfolding and time-varying score covariance structures is proposed in order to overcome the drawbacks of conventional MPCA and obtain better monitoring performance. The proposed method does not require prediction of the future values while the dynamic relations of data are preserved by using time-varying score covariance structures, and can be used to monitor batch processes in which the batch length varies. The proposed method was used to detect and identify faults in the fed-batch penicillin cultivation process, for four different fault scenarios. The simulation results clearly demonstrate the power and advantages of the proposed method in comparison to MPCA.     

A proteomic approach for dissecting H-Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells

To elucidate an understanding into H-Ras protein network, we have established various oncogene H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H-Ras, G12R H-Ras, and G12V/T35S H-Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF-MS analysis both commonly in oncogenic and partial loss of function H-Ras expression system. Thus, we tried to dissect H-Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H-Ras and a partial loss of function H-Ras in the H-Ras-expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2-DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT-PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H-Ras-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.     

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. Material and methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC. Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G₂/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21^waf1/cip1 was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21^waf1/cip1, followed by inhibition of cyclin B1 and cdc2, resulting in G₂/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.     

Sustained fixation induced changes in phoria and convergence peak velocity

Purpose

This study sought to investigate the influence of phoria adaptation on convergence peak velocity from responses located at different initial vergence positions.

Methods

Symmetrical 4° convergence step responses and near dissociated phoria (measured at 40 cm from the subject''s midline) were recorded from six subjects with normal binocular vision using an infrared limbus tracking system with a haploscope. Two different sustained fixations (1° and 16° convergent rotation along the subject''s midline) were used to study whether phoria had an influence on the peak velocity of convergence responses located at two initial vergence positions (1° or ‘far’ steps and 12° or ‘near’ steps).

Results

Phoria was significantly adapted after a sustained fixation task at near (16°) and far (1°) (p<0.002). A repeated measures ANOVA showed that convergence far steps were significantly faster than the near steps (p<0.03). When comparing convergence steps with the same initial vergence position, steps measured after near phoria adaptation were faster than responses after far adaptation (p<0.02). A regression analysis demonstrated that the change in phoria and the change in convergence peak velocity were significantly correlated for the far convergence steps (r = 0.97, p = 0.001). A weaker correlation was observed for the near convergence steps (r = 0.59, p = 0.20).

Conclusion

As a result of sustained fixation, phoria was adapted and the peak velocity of the near and far convergence steps was modified. This study has clinical considerations since prisms, which evoke phoria adaptation, can be prescribed to help alleviate visual discomfort. Future investigations should include a systematic study of how prisms may influence convergence and divergence eye movements for those prescribed with prisms within their spectacles.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Local and global dynamics of the G protein-coupled receptor CXCR1

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q = 0.1), only 13% of the expected number of backbone amide resonances is observed in (1)H/(15)N HSQC solution NMR spectra of uniformly (15)N-labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals is ascribed to mobile backbone sites and assigned to specific residues in the protein; 19 of the signals are from residues at the N-terminus and 25 from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the transmembrane helices or interhelical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing (13)C- and (15)N-labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 10(4) Hz time scale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.