Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.     

Functional analysis of Arabidopsis thaliana isoforms of the Mg-chelatase CHLI subunit

The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.     

Regulation and evolution of chlorophyll metabolism

Chlorophylls are the most abundant tetrapyrrole molecules essential for photosynthesis in photosynthetic organisms. After many years of intensive research, most of the genes encoding the enzymes for the pathway have been identified, and recently the underlying molecular mechanisms have been elucidated. These studies revealed that the regulation of chlorophyll metabolism includes all levels of control to allow a balanced metabolic flow in response to external and endogenous factors and to ensure adaptation to varying needs of chlorophyll during plant development. Furthermore, identification of biosynthetic genes from various organisms and genetic analysis of functions of identified genes enables us to predict the evolutionary scenario of chlorophyll metabolism. In this review, based on recent findings, we discuss the regulation and evolution of chlorophyll metabolism.     

Hermaphroditism in fishes: an annotated list of species,phylogeny, and mating system

Fewer than 1% of vertebrate species are hermaphroditic, and essentially all of these are fishes. Four types of hermaphroditism are known in fishes: simultaneous (or synchronous) hermaphroditism (SH), protandry (male-to-female sex change; PA), protogyny (female-to-male sex change; PG), and bidirectional sex change (BS or reversed sex change in protogynous species). Here we present an annotated list of hermaphroditic fish species from a comprehensive review and careful re-examination of all primary literature. We confirmed functional hermaphroditism in more than 450 species in 41 families of 17 teleost orders. PG is the most abundant type (305 species of 20 families), and the others are much less abundant, BS in 66 species of seven families, SH in 55 species of 13 families, and PA in 54 species of 14 families. The recently proposed phylogenetic tree indicated that SH and PA have evolved several times in not-closely related lineages of Teleostei but that PG (and BS) has evolved only in four lineages of Percomorpha. Examination of the relation between hermaphroditism type and mating system in each species mostly supported the size-advantage model that predicts the evolution of sequential hermaphroditism. Finally, intraspecific variations in sexual pattern are discussed in relation to population density, which may cause variation in mating system.

     

Eligibility for Bevacizumab as an Independent Prognostic Factor for Patients with Advanced Non-Squamous Non-Small Cell Lung Cancer: A Retrospective Cohort Study

Background

Bevacizumab requires some unique eligibility criteria, such as absence of hemoptysis and major blood vessel invasion by the tumor. The prognostic impact of these bevacizumab-specific criteria has not been evaluated.

Methods

Patients with stage IIIB/IV, non-squamous non-small cell lung cancer who started chemotherapy before the approval of bevacizumab were reviewed. Patients with impaired organ function, poor performance status or untreated/symptomatic brain metastasis were excluded before the evaluation of bevacizumab eligibility. We compared overall survival and time to treatment failure among patients who were eligible (Group A) or ineligible (Group B) to receive bevacizumab.

Results

Among 283 patients with stage IIIB/IV non-squamous non-small cell lung cancer, eligibility for bevacizumab was evaluated in 154 patients. Fifty-seven patients were considered ineligible (Group B) based on one or more of a history of hemoptysis (n = 20), major blood vessel invasion (n = 43) and cardiovascular disease (n = 8). The remaining 97 patients were classified into Group A. Overall survival was significantly better in Group A (median, 14.6 months) than in Group B (median, 7.1 months; p<0.0001). Time to treatment failure was also significantly longer in Group A (median, 6.9 months) than in Group B (median, 3.0 months; p<0.0001). Adjusted hazard ratios of bevacizumab eligibility for overall survival and time to treatment failure were 0.48 and 0.38 (95% confidence intervals, 0.33–0.70 and 0.25–0.58), respectively.

Conclusion

Eligibility for bevacizumab itself represents a powerful prognostic factor for patients with non-squamous non-small cell lung cancer. The proportion of patients who underwent first-line chemotherapy without disease progression or unacceptable toxicity can also be biased by bevacizumab eligibility. Selection bias can be large in clinical trials of bevacizumab, so findings from such trials should be interpreted with extreme caution.     

Reversed Sex Change in The Haremic Protogynous Hawkfish Cirrhitichthys falco in Natural Conditions

Bi‐directional sex change has recently been reported in a range of reef fishes, including haremic species that were earlier thought to be protogynous (female to male). However, the occurrence of this phenomenon and the social conditions driving the reversion of males to females (reversed sex change) have been poorly documented under natural conditions. Reversed sex change is predicted to occur in low‐density populations where facultative monogamy is common. However, few studies have evaluated this over a long period in such populations. We documented the occurrence of bi‐directional sex change during a 3‐yr demographic survey of a population characterised by small harem sizes in haremic hawkfish Cirrhitichthys falco. New males were derived following a change in sex of functional females (secondary males; n = 3) and juveniles always matured first as females (n = 3). Thus, C. falco exhibited a typical protogynous sexual pattern, consistent with a range of haremic fish species. We observed reversed sex change in two males. In both cases, all the females disappeared from their harems and the neighbouring males expanded their territories to encompass the territories of the sex changers. However, bachelor males did not always revert to females. A dominant male experienced bachelor status twice but regained mating opportunities following the immigration of a female into his territory or by taking a female from a neighbouring harem. Thus, we conclude that bachelor males use reversed sex change as a facultative tactic to regain reproductive status in a haremic mating system. In addition, we discuss the influence of harem size upon occurrence of reversed sex change.     

Isolation of apoptosis- and differentiation-inducing substances toward human promyelocytic leukemia HL-60 cells from leaves of Juniperus taxifolia

A chloroform extract of the leaves of Juniperas taxifolia exhibited a marked antiproliferative effect on human promyelocytic leukemia HL-60 cells at a concentration of 2.5 microg/ml. Deoxypodophyllotoxin (4) was identified in the extract as an outstanding antiproliferative compound, and five diterpenes (1-3, 5, and 6) were isolated as known compounds with weak or no cytotoxicity. These compounds were examined for their respective apoptosis- and differentiation-inducing activities toward HL-60 cells by DNA fragmentation and NBT-reducing assays, respectively. Among them, 7alpha-hydroxysandaracopimaric acid (6) was found to have a potent differentiation-inducing activity in a dose-dependent manner at 0.125-2 microg/ml (0.39-6.29 microM), together with apoptosis-inducing activity at concentrations of more than 2.5 microg/ml (7.86 microM). Deoxypodophyllotoxin (4) that exerted cytotoxic and apoptosis-inducing activities at 2 ng/ml (5 nM) did not induce differentiation at the same concentration, and the other diterpenes (1-3 and 5) showed no effect on cell differentiation, even at 5 microg/ml. It was thus demonstrated for the first time that 7alpha-hydroxysandaracopimaric acid was an effective differentiation-inducing compound toward HL-60 cells.     

Establishment and characterization of a cell line derived from a human serous surface papillary carcinoma of the ovary

A novel serous surface papillary carcinoma of the ovary (SSPC) cell line, HYKSSPC, was established successfully. Carcinoma cells were obtained from ascitic fluid of a 60-year-old Japanese woman. The population doubling time was 51.4 h. A phase contrast micrograph showed a pavement stone-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy, and the mode was in 46-47. An immunocytochemical study showed that CA125, BerER4 and cytokeratin were positive and that CEA, calretinin and thrombomodulin were negative. This cell line preserved some characters of the adenocarcinoma while growing in vitro. A chemosensitivity test revealed that HYKSSPC cells were sensitive to CDDP (cis-platinum), 5-fluorouracil, mitomycin C, paclitaxel and irinotecan. To our knowledge, HYKSSPC is the first established cell line derived from SSPC, and it may offer some useful information for investigating this disease.