Background
==========

Exposure of leaves to strong light sometimes causes the reduction of photosynthetic efficiency, a phenomenon referred to as photoinhibition \[[@B1]-[@B3]\]. The susceptibility of plants to photoinhibition depends on the species and growth light-environments \[[@B4]\]. In general, shade plants or low-light grown plants are more susceptible to photoinhibition than sun plants or high-light grown plants \[[@B4]\]. Since photoinhibition has a potential to lower productivity and plant growth, avoidance of photoinhibition is critical for the fitness and survival of plants in natural habitats \[[@B2],[@B5],[@B6]\].

It is now widely accepted that harmful Reactive Oxygen Species (ROS) produced upon illumination are involved in the mechanism of photoinhibition \[[@B7]\]. Singlet-excited oxygen (^1^O~2~) can be generated by the interaction of O~2~ with triplet-excited chlorophyll (^3^Chl) formed in the PSII reaction center \[[@B8]\]. Superoxide radical (O~2~^-^) is unavoidably produced by the Mehler reaction via electron transfer to O~2~ at photosystem I. Dismutation of O~2~^-^ results in the formation of hydrogen peroxide (H~2~O~2~) and the reaction between H~2~O~2~ and transition metal ions generates hydroxyl radical (•OH) which is the most reactive radical among ROS \[[@B7]\]. These ROS can oxidize molecules in chloroplasts including D1 protein in the PSII reaction center \[[@B9]\] and thiol enzymes in the Calvin-Benson cycle \[[@B10],[@B11]\] to inhibit partial reactions of photosynthesis eventually leading to photoinhibition. Recently, ROS have been shown to also bring about photoinhibition through the inhibition of *de novo* synthesis of D1 protein of PSII that is essential to recover from photoinhibition \[[@B12]\].

To protect photosynthetic machinery from ROS-mediated photoinhibition, chloroplasts contain a high amount of ascorbate in stroma at 20 to 300 mM \[[@B13]\]. In the stroma, ascorbate contributes to suppress the accumulation of photoproduced-H~2~O~2~ by acting as the electron donor for ascorbate peroxidase (APX) which detoxifies H~2~O~2~ to H~2~O \[[@B14]\]. Ascorbate can be also involved in the detoxification of ^1^O~2~, O~2~^-^ and •OH via nonenzymatic reduction \[[@B13]\]. In these reactions, ascorbate is univalently oxidized to monodehydroascorbate (MDA). To maintain ascorbate concentration in the chloroplasts, MDA should be promptly regenerated to ascorbate during illumination. MDA can be reduced to ascorbate by NAD(P)H in a reaction catalyzed by monodehydroascorbate reductase (MDAR) \[[@B15]\] or directly by reduced ferredoxin \[[@B16]\]. MDA, an organic radical, can undergo spontaneous dismutation to produce ascorbate and dehydroascorbate (DHA) in the absence of MDA reduction. DHA can be reduced by glutathione either through nonenzymatic reaction at a slow rate or enzymatic reaction at a much higher rate \[[@B17]\]. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) is considered to be involved in enzymatic DHA reduction in the chloroplasts \[[@B17]\]. Until now, however, direct *in vivo* evidence showing the physiological significance of DHAR has been yet not available.

We previously reported that leaves of *Ficus microcarpa* L. f. cv. Golden Leaves (GL), a tropical fig tree, lack heat-stable DHAR activity \[[@B18]\]. In GL, the canopy sun-leaves, which are always exposed to direct sunlight, show characteristic yellow whereas those in wild-type (WT, *Ficus microcarpa* L. f.) exhibit normal green even when exposed to direct sunlight. The mechanism for yellow leaves production in GL is unknown. In barley, it has been reported that leaves incubated with CO~2~-enriched air show yellow color due to photoinhibition of photosynthesis \[[@B19],[@B20]\], a phenomenon apparently similar to that observed in GL. We hypothesized that GL possess an incomplete machinery of photosynthesis which is susceptible to photoinhibition. The aim of this study was to directly examine the hypothesis. The side-by-side comparisons shown in this study demonstrate that GL is much more susceptible to photoinhibition of photosynthesis than WT.

Results
=======

Photoinhibition in the field
----------------------------

GL is a cultivar of the tropical fig tree *Ficus microcarpa* L. f. (WT) that is natively distributed in subtropical/tropical regions of Asia and Oceania. A significant characteristic of GL is the presence of yellow leaves that contain high amounts of flavonoids and negligible amounts of chlorophyll and carotenoids \[[@B21]\]. The reduced content of photosynthetic pigments in the yellow leaves can be increased by shading them from high-light. In shade conditions, therefore, GL exhibits normal green leaves which cannot be morphologically distinguished from WT leaves (Fig. [1](#F1){ref-type="fig"}). We have suggested that high susceptibility of GL to photoinhibition could explain the phenomenon \[[@B21]\]. However, there was no direct evidence available to show the susceptibility of GL to photoinhibition.

![Photograph of *Ficus microcarpa* L. f. (WT) and *Ficus microcarpa* L. f. cv. Golden Leaves (GL). Each branch was collected from canopy of mature trees grown in the field. Characteristic yellow leaves are observed only in sun-leaves of GL.](1471-2229-2-2-1){#F1}

Figure [2](#F2){ref-type="fig"} shows diurnal change of maximum PSII efficiency (*F*~v~/*F*~m~) in field grown attached sun-leaves collected from the canopy. Chlorophyll content of sun-leaves in GL (yellow) was 0.05 g m^-2^ and that in WT (green) was 0.44 g m^-2^. There was no significant difference between GL and WT in *F*~v~/*F*~m~ values measured early in the morning (6 a.m.). The value of *F*~v~/*F*~m~ in GL decreased from 0.77 (6 a.m.) to 0.46 (2 p.m.) and subsequently recovered to 0.74 in the evening (6 p.m.). In contrast to GL, *F*~v~/*F*~m~ values in WT were almost constant throughout a day. Shade-leaves of GL(green), collected from the inside of tree, did not show any decrease in the *F*~v~/*F*~m~ value (data not shown). These data obtained in the field conditions suggest that GL is highly susceptible to high-light.

![Photoinhibition of GL observed in the field-grown conditions. Top panel, a typical diurnal change in PPFD (▪) on the leaves and atmospheric temperature (□) in the field. Bottom panel, a diurnal change in maximum PSII efficiency of intact attached leaves in the field. (•, blue), WT; (Δ red), GL. *F*~v~/*F*~m~ value was measured as PSII efficiency with a portable chlorophyll *a* fluorometer (PAM-2000). Each data point is the mean of 10 separate measurements ± SD.](1471-2229-2-2-2){#F2}

Photosynthetic performance
--------------------------

We compared photosynthetic performance between GL and WT to confirm that the fundamental photosynthetic machinery of GL is normal. Shade-leaves of GL and WT, both of which are green, were used for this purpose. Figure [3](#F3){ref-type="fig"} shows the light response curves of CO~2~ assimilation rate (based on surface area) in detached shade-leaves. The chlorophyll content of shade-leaves in GL (green) was 0.58 g Chl m^-2^ and that in WT (green) was 0.70 g Chl m^-2^. GL showed a very similar light-response curve to WT and there was no substantial difference in the maximum activity of CO~2~ assimilation between them. These results clearly demonstrate that the photosynthetic capacity of GL is almost identical to that of WT, suggesting that photosynthetic machinery of GL is functionally not defective.

![Light-response curves for the rate of photosynthetic CO~2~ assimilation in WT and GL. Shade-leaves, which exhibited identical green coloration in both WT and GL, were used for measurements. Light intensities of irradiation were changed from high to low. (•, blue), WT; (Δ, red), GL. Points are the means of three (•) or four (Δ) separate measurements ± SD.](1471-2229-2-2-3){#F3}

Photoinhibition in shade-leaves of Golden Leaves
------------------------------------------------

Figure [4](#F4){ref-type="fig"} shows effects of high-light on several indicators of photoinhibition: D1 protein content, *F*~v~/*F*~m~ value, O~2~ evolution rate and CO~2~ assimilation rate. Before high-light exposure, there was no substantial difference between WT and GL shade-leaves in D1 protein content on protein basis (Fig. [4A](#F4){ref-type="fig"}) and *F*~v~/*F*~m~ value (Fig. [4C](#F4){ref-type="fig"}). GL showed a significant decrease in the D1 protein content and *F*~v~/*F*~m~ value upon high-light illumination (Fig. [4A,4B,4C](#F4){ref-type="fig"}). Similar to the indicators specific for PSII activity, those for net photosynthetic activity (i.e. O~2~ evolution and CO~2~ assimilation rates) also decreased upon high-light illumination (Fig. [4D,4E](#F4){ref-type="fig"}). In contrast to these responses observed in GL, WT showed only a small decrease in D1 protein content and *F*~v~/*F*~m~ value (Fig. [4A,4B,4C](#F4){ref-type="fig"}). The O~2~ evolution and CO~2~ assimilation rates in WT did not change even under high-light condition (Fig. [4D,4E](#F4){ref-type="fig"}).

![High-light induced photoinhibition of GL assessed by several parameters. Shade-leaves were exposed to high-light (2300 μmol m^-2^ s^-1^) at zero time after preexposure to medium-light (1000 μmol m^-2^ s^-1^) for 30 min. A schematic illustration shows the outline of photosynthesis including the electron transport process in thylakoid membranes. Letters (A-D) in filled circles, which correspond to those of the panels A-D, represent the sites where the photo synthetic parameters can be measured. **A,** Immuno blotting of the D1 protein contained in leaf-extract. **B,** Degradation of the *in vivo* D1 protein induced by high-light. Each point was plotted using a relative density of band on the gel as shown in A. **C,** Decline of *F*~v~/*F*~m~ induced by high-light. Note that the time indicated represents total illumination time. **D,** High-light induced inhibition of the activity of O~2~ evolution from intact detached leaves. **E,** High-light induced inhibition of the activity of CO~2~ assimilation in intact detached leaves. Points are taken by a separate measurement in different leaves (A, B, D) or a leaf (C, E). (•, blue), WT; (Δ, red), GL.](1471-2229-2-2-4){#F4}

Reduced NPQ capacity in Golden Leaves
-------------------------------------

When plants are exposed to high-light that exceeds the capacity of photosynthesis, excess absorbed light energy can be safely dissipated as heat \[[@B22]\]. This energy dissipation can be measured as the NPQ of PAM chlorophyll *a* fluorescence analysis. NPQ includes the quantum yield of PSII as an index of heat energy dissipation, photoinactivation of PSII and distribution of photon acceptance between PSII and PSI due to the state transition. It is known that the sate transition can be controlled by the redox state of Q~A~\[[@B23]\], which can be measured as 1-qP. Figure [5](#F5){ref-type="fig"} shows that there is no significant difference between GL and WT in yield and 1-qP values. Furthermore, measurements of the electron transport rate (ETR) showed essentially no difference in the sensitivity of PSII photoinactivation between GL and WT (data not shown). However, GL showed a much lower level of NPQ compared to WT (Fig. [5C,5F](#F5){ref-type="fig"}). These results suggest that low NPQ in GL can be attributed to dysfunction of the heat dissipation mechanism.

![Yield of PSII (A, D), 1-qp reflecting the reduction state of the q~a~ pool (B, E), NPQ (C, F) in shade-leaves of WT (•, blue) and GL (Δ, red). Each measurement was made after the exposure of leaves to actinic light for 3 min at different intensities (A-C). In D, E and F, high light (2300 μmol m^-2^ s^-1^) was used as actinic light to record time courses of Yield (D), 1-qP (E), and NPQ (F). Points are presented as the means of 5 (A-C) or 3 (D-F) separate measurements ± SEM.](1471-2229-2-2-5){#F5}

Discussion
==========

This study has demonstrated that photosynthetic activities of GL are highly susceptible to photoinhibition under high-light conditions (Figs. [2](#F2){ref-type="fig"}, [4](#F4){ref-type="fig"}). There was essentially no difference in photosynthetic efficiency between GL and WT at least up to 700 μmol m^-2^ s^-1^ for a short time (Fig. [3](#F3){ref-type="fig"}), but NPQ in GL was lower than that in WT at 2300 μmol m^-2^ s^-1^ (Fig. [5C,5F](#F5){ref-type="fig"}). It has been reported that a *npq1* mutant of *Arabidopsis thaliana* shows increased susceptibility to photoinhibition, but is unaffected in short-term photosynthetic O~2~ evolution and CO~2~ assimilation \[[@B24],[@B25]\]. For the operation of NPQ, special xanthophyll pigments in the light-harvesting complexes of PSII are known to have critical roles \[[@B26]\]. The extent of NPQ in plants is strongly correlated with the levels of zeaxanthin and antheraxanthin that are formed from violaxanthin by the enzyme violaxanthin deepoxidase (VDE) located on the luminal side of thylakoid membranes \[[@B22]\]. In excessive light conditions, VDE is activated and converts violaxanthin to zeaxanthin via antheraxanthin when acidification in thylakoid lumen reaches a critical threshold \[[@B27]\]. The activity of VDE is also regulated by the concentration of ascorbate in the lumen because VDE can utilize only ascorbate as a reductant \[[@B28]\]. It has been shown that decreases in ascorbate availability severely affect VDE activity *in vivo*\[[@B28]\]. Because thylakoid membranes do not have any active transport mechanisms for ascorbate, the concentration of ascorbate in the stroma determines that in the lumen by a passive diffusion mechanism \[[@B29]\]. In *Arabidopsis,* it has been clearly demonstrated that NPQ performance *in vivo* is reduced by a mutation that causes ascorbate deficiency \[[@B13]\]. We previously reported that the ascorbate content of GL is severally decreased in daytime under field conditions \[[@B30]\]. Thus, it is reasonable to assume that decrease of ascorbate content would be a cause of dysfunction of NPQ in GL.

Among antioxidant enzymes, we have shown that GL lacks heat-stable DHAR activity \[[@B18]\]. Plant cells possess several kinds of enzymes that exhibit DHAR activity: thioredoxin \[[@B31]\], glutaredoxin \[[@B32]\] and disulfide isomerase \[[@B33]\]. Recently, two distinct chloroplast proteins that exhibit DHAR activity have been isolated from spinach; one is *Kunitz*-type trypsin inhibitor \[[@B31]\] and the other is specific DHAR \[[@B34]\]. Because both chloroplastic DHARs show higher heat-stable activity than non-chloroplastic DHAR \[[@B31],[@B34]\], GL may lack chloroplastic DHAR(s) though molecular information is not available. In fact, heat-stable activity cannot be detected in nonphotosynthetic organs such as fruits, that do not include chloroplasts \[[@B18]\]. Although chloroplastic DHAR(s) have been presumed to function in the ascorbate regeneration system for maintaining the ascorbate concentration in the chloroplasts \[[@B7]\], the physiological significance of DHAR in chloroplasts is still controversial \[[@B35]-[@B38]\]. Since the activity of stromal MDAR is high, the spontaneous disproportionation from MDA to DHA and ascorbate may not occur under *in vivo* situations. Therefore, there is an argument that DHAR might be dispensable for the ascorbate-glutathione cycle to protect chloroplasts from high-light stress \[[@B36]\]. However, there is another location in chloroplasts where the stromal enzymes cannot directly regenerate ascorbate, namely, the lumen space.

In the lumen, oxidation of ascorbate by xanthophylls, α-tochopherol and PSII result in the production of DHA and all of these reactions could be promoted under high-light conditions \[[@B13],[@B35]\]. Therefore, stromal DHAR would be essential to reduce DHA produced in the lumen side. In this context, chloroplastic DHAR must be physiologically indispensable for photoprotection mechanisms particularly when leaves are exposed to high-light stress conditions. This is consistent with previous reports that DHAR activity in leaves of *A. thaliana* is increased by high-light conditions \[[@B39]\]. Thus, we consider that ascorbate availability of GL is decreased by high-light due to a lack of DHAR activity which eventually results in lowered performance of NPQ (Fig. [5C,5F](#F5){ref-type="fig"}). Ascorbate is involved in photoprotection not only through NPQ but also primarily through ROS scavenging \[[@B7]\]. It should be noted that high susceptibility of GL to photoinhibition could be also explained by a low ROS scavenging capacity which may result in photodamage of target molecules in chloroplast \[[@B7]\] and the inhibition of D1 *de novo* synthesis \[[@B12]\].

The mechanism for yellow leaf production is still unknown. Sun-leaves of GL contain low amounts of chlorophyll and carotenoids but shade-leaves show comparable amount of those to WT, implying that exhibition of yellow leaves in GL is associated with a long-termed irradiation of high-light \[[@B21]\]. When leaves of GL were exposed to high-light, they showed significant symptoms of photoinhibition (Figs. [2](#F2){ref-type="fig"}, [4](#F4){ref-type="fig"}). It has been suggested that long-termed photoinhibition (or chronic photoinhibition) can lead photobleaching of photo synthetic pigments such as chlorophyll and carotenoids \[[@B3]\]. The *npq1 Arabidopsis* mutant which is partially defective in NPQ has been reported to show the photobleaching of chlorophyll in high-light conditions \[[@B40]\], a phenomenon apparently very similar to that observed in GL. It is plausible that photoinhibition is involved in an early stage of the yellow leaf-producing mechanism. Unlike herbaceous plants, the leaf-yellowing phenomenon does not cause senescence or cell death \[[@B21]\]. It has been shown that flavonoids accumulated in leaves could protect tissues from oxidative damage by complementing the ascorbate-glutathione cycle \[[@B15],[@B41],[@B42]\]. In addition to the ROS scavenging function, flavonoids have long been known to be effective sunscreen pigments particularly against UV radiation \[[@B43]\]. Increased flavonoid pigments in GL may play roles including ROS scavenging function, UV-protection and light-attenuation for shadeleaves. The mechanisms of photoinhibition susceptibility and, in particular of high-light tolerance in GL leaves remain to be fully clarified. Although it is difficult to apply molecular biology techniques to GL, we consider that the tropical tree could provide a unique opportunity for examining the important determinants of survival in high-light environments.

Materials and Methods
=====================

Plant materials
---------------

Plant materials were *Ficus microcarpa* L. f. cv. Golden Leaves (GL) and *Ficus microcarpa* L. f. (WT) grown in the campus of the University of the Ryukyus on Okinawa island (26\'15\"N, 127\'45\"E) in Japan. The study period was from March to June in 1999--2000. The photo synthetic photon flux density (PPFD) on the plant canopy was c.a. 2500 μmol m^-2^ s^-1^ in full sunlight conditions during the period. There was no significant difference in growth conditions of PPFD between WT and GL. Green leaves, grown under light conditions of about 100 μmol m^-2^ s^-1^ as the maximum, were harvested and used for laboratory experiments as shade-leaves. To avoid the drought of leaves, leaf petioles were continuously kept in distilled water throughout the measurements. To activate the photosynthetic activity, detached leaves were exposed to an illumination of 1000 μmol m^-2^ s^-1^ for 30 min before experiments.

Illumination of leaves
----------------------

Leaves were illuminated with white light from three halogen lumps (400 W). Leaf temperature was maintained at 30°C with an electric fan or a thermostatically controlled water bath during illumination. Various light intensities were obtained by placing wire screens in front of the light source.

Measurements of chlorophyll fluorescence
----------------------------------------

Chlorophyll fluorescence was measured with a PAM-2000 chlorophyll fluorometer system under atmospheric conditions (Heinz Walz, Effeltrich, Germany). After a dark adaptation period of 15 min, minimum fluorescence (*F*~o~) was determined by a weak red light. Maximum fluorescence of dark-adapted leaf (*F*~m~) was measured during a subsequent saturating light pulse of white light (8000 μmol m^-2^ s^-1^ for 0.4 s). Maximum fluorescence (*F*~m~\') and steady-state fluorescence (Fs) of illuminated leaf were measured upon a subsequent saturating light pulse of white light (8000 μmol m^-2^ s^-1^, for 0.4 s) to determine NPQ (*F*~m~-*F*~m~\')/*F*~m~\'), yield of PSII \[(Fm\'-Fs)/Fm\] and qP (Fm\'-Fs)/Fm\'-Fo) \[[@B44],[@B45]\]. A 650 nm light-emitting-diode (LED) equipped with PAM-2000 was used for the illumination of leaf as actinic light.

Measurements of gas exchange
----------------------------

CO~2~ assimilation was determined by the difference in CO~2~ concentration between inlet and outlet of the assimilation chamber with a CO~2~ gas analyzer (LI-6251; LiCor, Inc., Lincoln, Nebraska) in 0.036% CO~2~ and 21% O~2~\[[@B46]\]. Measurements of CO~2~ assimilation rates were carried out under an illumination of 2300 μmol m^-2^ s^-1^, except for light response curve measurements. Approximately 25 cm^2^ of leaf were employed for an experiment. The air in the chamber was stirred rapidly to maintain a high boundary layer conductance for CO~2~ diffusion. O~2~ evolution was determined using a gas-phase Walker-type oxygen electrode system (Model LD-1; Hansatech, Norfolk, U.K.) in 5% CO~2~ and 21% O~2~\[[@B47]\]. Measurements of O~2~ evolution were carried out on 4 cm^2^ of leaf disc under an illumination of 600 μmol m^-2^ s^-1^.

Measurements of D1 protein content
----------------------------------

Leaf (0.5 g) was homogenized for 30 s at 0°C in a grinding medium containing 50 mM potassium phosphate (pH 7.0), 5 mM phenylmethylsulfonyl fluoride and 5% (w/v) polyvinylpolypyrrolidone. The homogenate (4 ml) was mixed with 4 ml of 125 mM Tris (pH 6.8) containing 5% (w/v) SDS, and then incubated at 80°C for 3 min to solubilize proteins. After cooling down the sample to room temperature, aggregates were removed by the centrifugation at 1,500 × g for 30 s. The supernatant (800 μl) was mixed with the same amount of a solution that contained 0.4 M sucrose, 8 M urea, 5 mM EDTA and 5% (w/v) 2-mercaptoethanol.

SDS-PAGE was carried out according to a previously reported method \[[@B48]\]. Polyacrylamide gels containing 6.0 M urea were used for the stacking gel (4.5 %) and separation gel (13 %). Samples that contained 8 μg protein were loaded in each lane. Immunoblot analysis was performed using specific polyclonal antibodies raised against the D1 protein \[[@B48]\]. The intensities of the protein bands were determined with NIH image system version 1.61 (NIH, USA).

List of abbreviations used
--------------------------

APX, ascorbate peroxidase; ^1^Chl, singlet excited chlorophyll; ^3^Chl, triplet excited chlorophyll; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; D1, the reaction center-forming protein of photosystem II; *F*~v~/*F*~m~, ratio of variable to maximum chlorophyll *a* fluorescence; GL, Golden Leaves; MDA, monodehydroascorbate; MDAR, monodehydroascorbate reductase; NPQ, nonphotochemical quenching; •OH, hydroxyl radical; ^1^O~2~, singlet excited O~2~; O~2~^-^, superoxide radical; PPFD, photosynthetic photon flux density; ROS, reactive oxygen species; WT, wild-type.

Acknowledgements
================

We are grateful to Dr. M. F. Cohen of University of the Ryukyus for critical reading of the manuscript. This work was supported by a Grant-in-Aid for Scientific Research (B) and (C) from Japan Society for the Promotion of Science to HY.


Introduction
============

Suspected acute appendicitis is the most frequent cause for emergency operations in visceral surgery worldwide. In the UK 37,289 patients had an emergency excision of the appendix in the year 2000 \[[@B1]\]. In approximately twenty percent of all cases however, the diagnosis is incorrect and patients undergo surgery without having acute appendicitis at all \[[@B2]-[@B5]\]. Operations of bland appendices may lead to morbidity in 4.6 percent \[[@B6]\] and to mortality in 0.14 percent \[[@B6]\] of cases. Despite the introduction of reports of highly accurate diagnostic procedures for the diagnosis of acute appendicitis a big retrospective cohort study \[[@B7]\] concluded that the rate of misdiagnosis (the false positive rate) has not changed over the last twenty years. One potential explanation of that finding might be, that studies reporting on test accuracy overestimate the true potential of correct classification due to inappropriate methodology and bias of reported results since primary research on evaluation of tests is generally poor in quality \[[@B8]-[@B10]\].

Online searches of the electronic databases revealed a number of broad reviews, commentaries and recommendations on tests for predicting acute appendicitis but there was a dearth of focused, rigorous diagnostic overviews of the available evidence. These publications showed that there are several prediction rules and tests or markers purported to be predictive of acute appendicitis. However, they offer only limited guidance for practice because traditional literature reviews evaluating tests for acute appendicitis have not applied the scientific strategies to assemble, appraise, and synthesize relevant evidence, which have been embodied in the criteria for high quality reviews.

Given the variation that exists in both practice and research, the uncertainty regarding the quality of the underlying evidence, and the importance of early prediction of acute appendicitis in view of the available effective treatments, there is a clear need for a comprehensive, systematic and quantitative overview of the diagnostic value of the various tests purported to be predictive of acute appendicitis.

At present there is a dearth of such reviews and in this commentary, we will describe how we are using such a systematic approach to collate and critically appraise the available literature in the diagnosis of acute appendicitis.

Methods
=======

Study identification
--------------------

Non-comprehensive search strategies can lead to significant bias in the retrieval of relevant literature. This weakens the strength of inferences from systematic reviews and poses a particular problem in reviews of diagnostic tests \[[@B11],[@B12]\]. Therefore we will identify literature via general bibliographic databases including MEDLINE and EMBASE, specialist computer databases such as DARE and MEDION (a database of diagnostic test reviews set up by Dutch and Belgian researchers), the Cochrane Database of Systematic Reviews, relevant specialist registers of the Cochrane Collaboration, conference proceedings and BIOSIS without language restrictions. In addition we will contact individual experts and those with an interest in this field to uncover grey literature and we will contact the manufacturers of tests. Hand-searching of selected specialist journals, checking of reference lists and SCISEARCH to identify frequently cited articles will complete our searches. In cases of duplicate publication, the most recent and complete versions will be selected. A comprehensive database of relevant articles will be constructed -- a preliminary search has been carried out in order to estimate the size of the relevant literature. MEDLINE Searches located 800 potentially relevant citations. Expanding search to other databases, hand searching, reference list searching and or contact with authors might add another 100% citations, so the total is likely to be 1600. Letters will be sent to major centres and the first author of each shortlisted selected paper published in the last five years, asking them whether they know of any published or unpublished relevant studies not included on our list. The search strategy used to identify articles in MEDLINE is shown in: [appendix.doc](#S1){ref-type="supplementary-material"}.

Study selection
---------------

Studies will be selected for inclusion in the review in a two-stage process using the selection criteria based on those shown in Table [1](#T1){ref-type="table"}. First, a comprehensive database of the literature search will be constructed. The citations will be scrutinised by two reviewers to obtain copies of full manuscripts of all citations that are likely to meet the selection criteria. Two reviewers will then independently select the studies, which meet predefined, and explicit criteria regarding populations, tests, outcomes and study design. These criteria will be pilot tested using a sample of papers and agreement between reviewers will be measured. When disagreements occur the two reviewers will meet. Experience suggests that often the cause of the disagreement is a simple oversight on the part of one of the reviewers. When this is not the case the issue will be resolved by consensus involving a third reviewer.

::: {#T1 .table-wrap}
::: {.caption}
######

Study Selection Criteria.
:::

  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **Population:** Patients suspicious to have acute appendicitis
  **Diagnostic tests:**
  Prediction rules
  Inflammatory markers (C-reactive protein, leucocytes count)
  Transabdominal ultrasound
  Computer Tomography (CT)
  Magnetic Resonance Imaging (MRI)
  Scintigraphy
  Diagnostic laparoscopy clinical history and physical examination
  **Outcome measures:** Test accuracy, morbidity and mortality from misdiagnosis
  **Study design:** Diagnostic test studies will be selected. They consist of observational studies (prospective or cross-sectional) of defined non-randomised populations in which the results of the diagnostic test of interest are compared with the results of a reference standard allowing generation of a 2 × 2 table to compute indices of diagnostic accuracy.
  ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::

Study validation
----------------

Papers meeting the selection criteria will be appraised to rate their methodological quality. In addition to using ratings of study quality as possible explanations for differences in results, the extent to which primary research met methodological standards is important per se for assessing the strength of any conclusions that are reached. There is an ongoing debate over what constitutes the best quality assessment tool for diagnostic test studies. We will evaluate elements of study design, which are likely to have a direct relationship to bias in a diagnostic test study \[[@B10]\]\[[@B13]\]\[[@B14]\]\[[@B15]\]. The items shown in Table [2](#T2){ref-type="table"} will be used for methodological quality assessment. Agreement for the quality assessments will be calculated, and disagreement resolved, in the same fashion as for the assessment of study selection. We will evaluate the agreement between the two reviewers using percentage agreement and weighted kappa statistics \[[@B16]\].

::: {#T2 .table-wrap}
::: {.caption}
######

Criteria for study validation.
:::

  ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **Population:** Consecutive recruitment of an appropriate spectrum of eligible patients will be considered ideal. Convenience sampling, i.e. arbitrary recruitment or non-consecutive recruitment will be deemed inadequate. In the absence of any explicit information on the method of recruitment, the article will be categorised as unclear reported population enrolment.
  **Diagnostic test:** The description of the diagnostic test will be considered ideal if the methodology is reported together with the measurement parameter and the cut-off level for an abnormal result. In the absence of any of the above information in the manuscript, then the diagnostic intervention will be considered as unclear reported.
  **Outcome measures:** Blinding will be considered ideal if it is clearly reported that the results of the various tests were not divulged. Information on the number of patients recruited into the study and those whose outcome data were known will also be sought from the manuscripts. Withdrawal of patients from the study, missing data and lack of outcome data outwith the study hospital will be categorised as lost to follow-up. In particular we will look for evidence of verification bias where the rates follow-up and confirmation of outcome are different in patients with positive test results compared to those with negative test results.
  Any available randomised trials will be assessed for validity separately to the diagnostic accuracy studies considering factors associated with bias in such trials, e.g. concealment of randomisation, sequence generation, blinding and follow-up.
  ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::

Data collection
---------------

The extraction of study\'s findings will be conducted in duplicate using a pre-designed and piloted data extraction form to avoid any errors. Given the extent of insufficient reporting in the medical literature, we propose to obtain missing information from investigators whenever possible. It is otherwise impossible to distinguish between what was done but not reported and what was not done. A template of data extraction form is shown in: [appendix.doc](#S1){ref-type="supplementary-material"}.

Analysis
--------

By analysis we mean synthesis of results from individual studies (meta-analysis), and exploration of variation in results from study to study (heterogeneity) and generation of the most useful combination of tests. We will conduct meta-analyses to generate summary estimates of sensitivities, specificities, predictive values, likelihood ratios (LRs) and receiver operating characteristic (ROC) curves where appropriate \[[@B13],[@B14],[@B17]\]. If meta-analysis is considered to be inappropriate, we will describe the identified evidence in the context of appraised quality. If a meta-analysis is considered appropriate, we will examine the correlation between true positive rates and false positive rates in individual studies. If the correlation is poor, we will use LR as the main accuracy measure. If we find a correlation then we will generate a summary ROC curve \[[@B18]\] in addition to pooling of LRs. Many authorities considered this the preferred method of pooling test results from primary studies \[[@B13],[@B14],[@B17]\]. The summary ROC plot provides a way of summarising the performance of a test from the results of several studies over a range of test thresholds. However, our preference for LRs is based on the published recommendations that LRs are more clinically meaningful as measures of diagnostic accuracy \[[@B15]\]. Our experience has been that the true positive rates and false positive rates in individual studies are poorly correlated in which case it is not feasible to generate a summary ROC curve. Moreover, when the outcome of a test is of binary nature (positive or negative) LRs are more clinically meaningful than ROC curves. One disadvantage of analysis using LR is that it generates two measures for each test, one for a positive result and another for a negative result. A ratio of LRs will be used to generate a single measure called diagnostic odds ratio, which is more suitable for statistical analysis. For the purpose of meta-analysis, we will weight the logLR from each study in inverse proportion to its variance in order to combine the LRs from each study. To demonstrate the practical application of the summary LRs generated, we will calculate posttest probabilities for acute appendicitis using Bayes\' theorem. An estimate of the pretest probability will be obtained by calculating the prevalence of the outcome event in the population studied. The following algorithm of equations will be used for calculating post-test probability:

pretest probability = prevalence of acute appendicitis

pretest odds = pretest probability / (1 -- pretest probability)

posttest odds = likelihood ratio × pretest odds

posttest probability = posttest odds / (1 + posttest odds)

In order to deal with the uncertainty of the estimate, we will generate 95% confidence intervals around the point estimate. Approximate variance for the posttest odds will be obtained by adding the variances of the combined LRs and pretest odds, enabling the calculation of its 95% confidence intervals. The 95% confidence intervals for the posttest probabilities will then be generated by converting the limits of the posttest odds to their respective probabilities.

Heterogeneity of results between different studies will be formally assessed using the Breslow-Day test which compares for each study the ratio of the odds of having the outcome of interest when the test result is positive to the odds of having the same outcome when the test result is negative\[[@B19]\]. To explore causes of heterogeneity in the estimates of diagnostic accuracy of the tests for acute appendicitis, we will conduct a sensitivity analysis. This will be carried out by subgroup analyses to see whether variations in population, intervention, outcomes and study quality will affect the estimate of diagnostic accuracy. Results of pooled analyses will be provided within cogent patient groups.

Discussion
==========

In summary, systematic reviews of diagnostic literature to predict acute appendicitis allow us to assess the quality of the available evidence and to identify specific tests (including history, physical examination and tests) that have diagnostic value. These reviews should lead to formulation of recommendations for current practice and future research. Just as an evidence-based culture in delivery of health care has been supported by systematic reviews of literature on therapeutic interventions, we can expect to see an extension of this approach in the area of care involving use of diagnostic and screening tests.

Competing interests
===================

none declared

Authors\' Contributions
=======================

LMB and JS initiated the project and wrote the protocol. DBB, SAB, MGB and FMO screened the pilot searches, all authors commented on earlier drafts and approved the final manuscript.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Supplementary Material
======================

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Acknowledgements
================

The authors would like to thank Gill Richie and Julie Glanville of the Centre for Reviews and Dissemination in York (UK) for searching the databases.


Background
==========

Attention Deficit Hyperactivity Disorder (ADHD), known as Hyperkinetic Disorder under the ICD-10 classificatory system, is a common disorder that affects between 1.5% and 6% of children \[[@B1],[@B2]\]. The disorder is an early onset condition that is associated with educational failure, social difficulties and an increased risk of antisocial behaviour and problems in adult life including substance misuse and criminality \[[@B1]\]. ADHD now represents the commonest reason for follow up in child and adolescent psychiatry clinics \[[@B2]\] and an average general practitioner can expect to have between two and four children receiving treatment for it on their list. Nevertheless there is evidence that most children who fulfil diagnostic criteria remain undetected in the general population \[[@B3]\].

The treatment of ADHD has been the focus of extensive review and several authoritative reports and recommendations have been recently circulated \[[@B4]-[@B8]\]. These recommendations support the use of stimulant medication (such as methylphenidate (Ritalin, Equasym) and dexamphetamine (dexedrine)) as first line treatment (either alone or in combination with non-pharmacological interventions including behaviour therapy) for children with accurately diagnosed ADHD \[[@B4]-[@B9]\]. Moreover the high level of under treatment has also been highlighted in the recent National Institute of Clinical Excellence (NICE) guidance on the use of methylphenidate in ADHD \[[@B4]\].

Discussion
==========

Current recommendations are that medication should be initiated by child and adolescent psychiatrists or paediatricians or learning disability specialists with particular expertise \[[@B4],[@B5]\]. Once the condition is stabilised children can then be followed up in primary care \[[@B4],[@B5]\] and NICE guidance recommends health authorities should draw up shared care protocols \[[@B4]\] . Is primary care in this country ready to follow these recommendations? Follow up of ADHD is often undertaken by primary care in the Unites States \[[@B8],[@B10]\]. Moreover adult ADHD is becoming increasingly recognised and some of the cohort of children and adolescents who are currently on stimulant medication will graduate to adult mental health services. These factors together with statements in the NICE guidance on the use of methylphenidate are likely to increase pressure on primary care to become more involved in the management of ADHD.

However there is a great need for caution and careful planning before implementing shared care protocols for ADHD and sharing follow up with primary care. Primary care teams are already having to assume responsibility for follow up of an increasing number of chronic conditions and although for some (such as diabetes) there is evidence this is successful \[[@B11]\], the monitoring of children on treatment for ADHD may pose different problems. Most general practitioners and nurses will have undergone no or very little training in child and adolescent psychiatry. Many general practitioners do not feel confident about their skills in recognising and undertaking follow-up management of ADHD and feel that education is essential \[[@B12]\]. Attitudes amongst members of the primary care team members may also represent a problem. The media represents an important source of knowledge of ADHD for many general practitioners \[[@B12]\]. Given that recent media features have highlighted problems with over diagnosis and over treatment this is likely to affect attitudes within the primary care team to the use of stimulant medication and heighten anxieties about management. Although there is some evidence that most general practitioners are willing to take on physical monitoring of children on medication for ADHD (e.g. height, weight and blood tests if necessary) the only available data suggest general practitioners feel clinical monitoring of these children is a specialist responsibility \[[@B12]\]. Therefore shared care protocols for ADHD may have limited impact in reducing specialist workload and will need to incorporate effective mechanisms for close integration and good communication between services to ensure that patients and their families receive appropriate care.

It is therefore important that a clear strategy for the follow up care of children with ADHD is devised. Planners need to ensure that roles and responsibilities of different healthcare sectors in monitoring children with ADHD on medication are clearly demarcated and acceptable to the professionals concerned. When these clear roles have been identified, it is then important to ensure that professionals possess the appropriate skills and knowledge to effectively undertake the assigned role. The resource implications of carrying out this monitoring also need to be addressed so that this can be done successfully (as has been achieved for monitoring of anticoagulant therapy). It is essential that patient needs are met in a coordinated and supported manner to avoid a situation of placing children and families into a \"no mans land\" of uncertain responsibility and inadequate skills,

Summary
=======

ADHD is likely to become increasingly important for primary care. To ensure that patient care is not compromised by the introduction of shared care protocols it is important that there is a clear dialogue between planners and healthcare professionals from both primary and secondary care. This is necessary to ensure mechanisms are in place for this care to be undertaken effectively and properly organised. Service delivery needs to be sensitive to patient need, acceptable to healthcare providers and tailored to their skills and finally must be adequately resourced.

Authors\' contributions
=======================

Both authors (AKT and AT) jointly initiated this paper, researched the topic and contributed to drafting and final preparation of this article.

Declaration of competing interests
==================================

AT (Anita Thapar) has organised academic meetings that have received sponsorship from Celltech (manufacturers of Equasym) and Janssen Cilag (manufacturers of Concerta).

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

We wish to thank Professor Eric Taylor for his helpful comments on this paper and Professors\' Richard Harrington and Martin Roland for reviewing the manuscript.


Background
==========

Proteorhodopsin is a 249-amino acid membrane protein native to several uncultured species of γ-proteobacteria, which are a component of marine plankton \[[@B1]\]. Addition of retinal to *E. coli* expressing pR was shown to cause a reddish coloration of the bacteria with an absorption maximum near 520 nm. The pR contained in the bacterial membranes was shown to act as a light-activated proton pump, but only when retinal is present. Time-resolved UV/vis studies at pH 8 also revealed that the protein undergoes a photocycle, similar to that of wild type bacteriorhodopsin, but with a predominance of the O intermediate instead of M.

The bR photocycle has been characterized by spectroscopic methods as having six principal photointermediates: bR, K, L, M, N and O. Each intermediate has a distinct absorbance maximum; the most studied are bR (570 nm), M (412 nm), and O (640 nm) since these are the ones that can be produced in the highest concentration at physiological pH values. Monitoring of the absorbance at individual wavelengths after photoexcitation is used to determine the relative concentrations and decay times of each of these photointermediates. The L → M transition in bR is characterized by the deprotonation of the Schiff base to Asp-85, producing the distinctive 412 nm absorbance maximum of M, and by so-called fast proton release, the ejection of a proton from a different (unknown) residue into the external medium on the \~10--100 μs time scale, depending on pH. Reprotonation of the Schiff base from Asp-96 occurs during the M → N transition with an absorbance maximum of 560 nm \[[@B2]\]. The N → O transition involves the reprotonation of the Asp-96 from the cytoplasmic space.

Like bR, pR consists of seven transmembrane α-helices that include in the membrane interior all of the residues conserved among archaeal rhodopsin proton pumps. In particular, analogues of Asp-85, Asp96, Arg-82, and Lys-216 of bR are present in pR. Conspicuously absent are analogues for Glu-194 and Glu-204 of bR. The latter, as well as Arg-82, have been implicated in fast proton release. In particular, mutagenesis of Glu-194 or Glu-204 in bR results in loss of fast proton release \[[@B3],[@B4]\]. The absence of homologs for these residues in pR leaves open the question of whether it carries out fast H^+^ release.

Experiments described here demonstrate that pR does indeed undergo fast H^+^ release, at least under elevated pH conditions that resemble somewhat those of the γ-proteobacteria\'s native open ocean environment. We also demonstrate that there is a post-translational modification of at least one of the three native cysteines when pR is expressed in *E. coli.* Both of these discoveries were made possible through purification methods for pR described herein.

Results
=======

Purification
------------

PR was obtained in 85% purity, assuming that values of ε~280~ and ε~546~ for pR are the same as for bR solubilized in DMPC/cholate/SDS mixtures at pH 8 (ε~280~= 7.85 × 10^4^ cm^-1^ M^-1^ and ε~551~ = 4.8 × 10^4^ cm^-1^ M^-1^) \[[@B5]\]. This assumption is actually expected to underestimate the purity of pR produced, by up to \~20%, since the pR we expressed has 10 tryptophan and 14 tyrosine residues, as compared to 8 tryptophans and 11 tyrosines in bR from H. salinarum. The absorbance of contaminant proteins was assumed to be 1.1 for a 1 mg/mL solution. By using these assumptions, the relative concentrations of pR and other proteins can be determined from the absorbance spectra of the various fractions (fig. [1](#F1){ref-type="fig"}). The resulting purity values correlate well with those Coomasie-stained SDS-PAGE gels (see below). The OG extract of cholate-washed membrane pellets starts out at a pR content of 7% total protein (w/w). The Phenylsepharose column increases the purity level to 24%, with approximately 5% loss. The final purification step by hydroxylapatite column chromatography produces pR with \~85% purity and a further loss of \~60%, i.e. the overall yield of the two column procedure was \~30%.

![**UV/visible absorption spectra of pR in octylglucoside solution (1--3%) at three stages of purification.** All three spectra were measured in the presence of octylglucoside at pH 8, and are normalized to the 280-nm protein peak. Spectrum A, the OG extract of cholate-washed *E. coli* membranes; spectrum B, pooled 546-nm absorbing fractions from Phenylsepharose column; spectrum C, same material after hydroxylapatite column.](1472-6793-2-5-1){#F1}

Polyacrylamide gel electrophoresis
----------------------------------

Relative to protein standards, the apparent molecular weight of bR is 25,000 while the apparent molecular weights of pR-wt and pR-TCM are 36,000 and 31,000, respectively (fig. [2](#F2){ref-type="fig"}, lanes E and C, respectively). SDS-PAGE (fig. [2](#F2){ref-type="fig"}) also confirms the estimates of purity level based on the assumed ε~280~/ε~546~ ratio identical with that of detergent solubilized bR. Interestingly, the pR appears to be a doublet band whose relative concentrations remain almost unchanged during purification. This doublet is also present in the less-purified sample of pR-TCM, with both bands shifted down by approximately the same amount (fig. [2](#F2){ref-type="fig"}, lane C).

![**SDS-PAGE of pR (wild type and pR-triple cysteine mutant).** Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. [1](#F1){ref-type="fig"}. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. [1](#F1){ref-type="fig"}.](1472-6793-2-5-2){#F2}

Subsequent SDS-PAGE analysis of pR samples that had been stored for periods of time up to several months indicate that after sitting for several weeks in octylglucoside solution at 4°C, the largest post-translational modification on wild-type pR is eliminated -- presumably hydrolysed off of the cysteine(s) -- leaving only a 31,000-MW band indistinguishable from that seen for pR-TCM (data not shown). Furthermore, after boiling for several min in gel loading solution, this cleaved wild-type protein, as well as the TCM, both give an extra artifactual band near 36,000 dalton. The latter band, a singlet, is coincidentally at almost the same apparent MW as the doublet from the uncleaved post-translationally-modified wild type pR (fig. [2](#F2){ref-type="fig"}, lane E). These potential artifacts should be taken into consideration in any attempt to reproduce the results in Fig. [2](#F2){ref-type="fig"}.

Photocycle kinetics and flash-induced proton concentration changes
------------------------------------------------------------------

Photocycle kinetics were measured at 400, 500. and 580 nm in the presence of the short-chain lipid DHPC. This lipid does not support the formation of closed bilayer vesicles, but rather forms micelles like a detergent. The time-resolved measurements showed no positive 400-nm absorbance signals at pH 8.0 or lower (Fig. [3](#F3){ref-type="fig"}). This is somewhat in disagreement with Béjà et al \[[@B1]\], who detected small 400-nm transient absorbance increases upon photolysis at pH 8.0. However, we observed a transient 400-nm absorbance increase at an elevated pH of 9.5 (fig. [3](#F3){ref-type="fig"}).

![**Dependence on pH of the M-like intermediate of pR.** Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 10^0^--10^7^ μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3--6 mJ.](1472-6793-2-5-3){#F3}

At pH 9.5 in the presence of DHPC, and observing transient changes at 500 nm (fig. [4](#F4){ref-type="fig"}), pR undergoes a 2-phase decay after the initial unresolved absorbance decrease. Multiexponential fits show that the first decay phase has a time constant of 4 μs, in good agreement with the 4-μs rise time of the 400 nm signal (Fig. [4](#F4){ref-type="fig"}). The amplitude of this decay represents about 80% of the initial absorbance depletion. The second phase of the 500 nm absorbance decay occurs with a substantially slower time constant of 0.5 s, returning the remaining 20% of initial absorbance change. The slowest decay components of the positive 400-nm signal and the negative 500-nm signal follow similar kinetics, although the amplitudes of these components differ by a factor of 3. At pH 9.5, the 580 nm trace has no significant positive values indicative of an O-like intermediate, although, in agreement with earlier measurements \[[@B1]\], at lower pH values a red-shifted transient is the predominant positive absorbance signal (data not shown).

![**Photocycle kinetics of pR at selected wavelengths at pH 9.5.** Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. [3](#F3){ref-type="fig"}. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. [3](#F3){ref-type="fig"}.](1472-6793-2-5-4){#F4}

Figure [5](#F5){ref-type="fig"} shows a different type of time-resolved measurement, probing not the pR chromophore, but rather pH changes in the protein environment. Proton concentration changes in the aqueous bulk phase were measured with the pH sensitive dye cresol red, which has a pK~a~ of 8.2--8.5. The bottom trace in Figure [5](#F5){ref-type="fig"} shows the absorbance change of the indicator during the pR photocycle. The negative signal is indicative of a pH decrease, corresponding to transient H^+^ release from the protein into the solution. The best-fit time constant for the release phase is 6 μs. The positive 400 nm trace in fig. [5](#F5){ref-type="fig"} (reproduced from fig. [3](#F3){ref-type="fig"}) shows that the proton release and uptake follow kinetics very similar to the apparent formation and decay of M, as is typically seen in bR near neutral pH \[[@B6],[@B7],[@B21]\]. However, no proton release signal could be observed for pR at pH 6 or 8 (data not shown).

![**Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake.** The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H^+^ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H^+^ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H^+^ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. [4](#F4){ref-type="fig"}.](1472-6793-2-5-5){#F5}

Discussion
==========

Purification of pR
------------------

Meaningful comparison of intrinsic physiological properties of pR and bR depends on the purification of pR. The *E. coli* expression system can easily be used to prepare pR at 85% purity with similar or less effort and time than required for bR (purple membrane) production from S9 *H. salinarum.*

The initial purity level of the OG-solubilized cholate-extracted membrane is about 7% pR by weight. Phenylsepharose™ column chromatography separates proteins on the basis of hydrophobicity, and has been used previously in hR purification \[[@B8]-[@B10]\]. As an initial purification step, the Phenylsepharose™ column achieved a substantial increase in protein purity to 25%, along with removal of most of the lipid. The final purification step, utilizing a hydroxylapatite column, has been previously used with rhodopsin \[[@B11]\]. This column proved to be more efficient as a final step in the purification than as a preliminary one, because large amounts of contaminant protein tended to slow the flow rate drastically. This step of the purification yielded an increase in protein purity to \>85%. The overall yield of pR from membrane through the column purifications is \~30%. Most of this loss, \~65%, occurs during the hydroxylapatite column.

Molecular weight differences
----------------------------

Béjà et al. reported a molecular weight for wild type pR of 27 kDa based on the predicted amino acid sequence of the protein \[[@B1]\]. Its 249 amino acids barely exceed the 248 of mature bR, which has a molecular weight of 26,000. However, we observed a significantly higher apparent molecular weight (\~36,000) for wild type pR on SDS-PAGE gels (Fig. [2](#F2){ref-type="fig"}, lanes D and E). Post-translational modification of pR must almost certainly account for some of the observed molecular weight difference between pR and bR. Lipids or sugars covalently bound to the protein surface would not be removed during the purification procedure and could cause a higher apparent molecular weight. Cysteines are frequently a site of lipid association with membrane proteins, (e.g. mammalian rhodopsin, which has two palmitoyl molecules attached to cysteine residues). Therefore we compared the SDS-PAGE gel mobility of wild-type pR to that of a cys-less mutant (pR-TCM). The elimination of the three possible sulfhydryl attachment sites lowers the apparent molecular weight of the pR-TCM by 5,000 (Fig. [2](#F2){ref-type="fig"}, lane C). From this, we conclude that at least one of the three cysteines in pR is probably modified post-translationally.

However, this by itself does not account fully for the anomalous mobility of pR on SDS-PAGE gels, because pR-TCM is still approximately 5,000 higher in molecular weight than bR according to SDS-PAGE (fig. [2](#F2){ref-type="fig"}, lanes A & C). Only \~2,600 of this can be accounted for by the V5 eiptope and poly-histidine tail that are appended to the C-terminus of pR by the pBAD-TOPO expression vector that we used \[[@B1]\]. There is undoubtedly a further post-translational modification of unknown nature.

Spectral comparisons
--------------------

Béjà et al. reported an absorbance maximum for pR of 520 nm in *E. coli* membranes, using a difference bleaching technique to remove interfering absorbance bands from other membrane components in the impure pR sample \[[@B1]\]. We confirmed this result using crude *E. coli* membranes without detergent present (data not shown). However, we observed an absorbance maximum of 546 ± 5 nm for pR in OG at pH 7 at all stages of purification (Fig. [1](#F1){ref-type="fig"}). Small blue shifts were observed for pR samples in OG when measured at pH 8 and 9.5 (8 and 16 nm, respectively; data not shown).

For the pR samples reconstituted in DHPC, which were used for flash photolysis experiments, the absorbance maxima were similar to those measured in OG (spectra not shown). A chromophore absorption maximum near 540 nm was also obtained by using difference spectroscopy of pR in crude *E. coli* membranes solubilized in OG (spectrum not shown). However, for pR measured directly in crude *E. coli* membranes, i.e. not solubilized in OG, we obtained the same value (520 nm) as reported previously \[[@B1]\].

Solubilization in detergent presumably leads to structural distortions of the native protein conformation, and therefore a change in the absorbance properties of the chromophore. However, the direction that λ~max~ for pR would have to shift upon solubilization in OG is inconsistent with the pattern for bR, whose λ~max~ decreases when it is solubilized in OG \[[@B12]\]. Furthermore, pR in OG showed resonance Raman spectra (D. Dunmire, R. A. Krebs and M. S. Braiman, unpublished data) indicative of a chromophore structure very close to native light-adapted (i.e. all-trans) bR in purple membrane. However, there is one major difference: pR in OG exhibits an upshifted, doublet C=N Schiff base band consisting of two components of nearly-equal intensity. These appear to correspond to the presence of two distinct subpopulations of pR, at least when expressed in *E. coli* and solubilized in OG micelles. The different values of λ~max~ for pR in membrane state \[[@B1]\] and OG solution might be related to the presence of these multiple subpopulations, but this connection remains unclear.

Principal photointermediates of pR
----------------------------------

Of the six principal photointermediates present in bR, four can be discerned from the time-dependent visible absorbance traces from pR in fig. [4](#F4){ref-type="fig"}, along with previously published time traces at 600 nm \[[@B1]\]: the resting state (pR), M, N and O. The resting state, with an absorbance maximum of 546 nm (see above), provides the baseline spectrum for the difference time courses reported. The M intermediate of bR has an unprotonated Schiff base group, giving rise to a blue-shifted λ~max~ (400 nm). Likewise in pR, an increase in the 400 nm absorbance should indicate formation of a deprotonated Schiff base, and therefore the presence of an M-like intermediate.

Interpretation of the 500 nm time course trace is more complicated. As in bR, it likely involves decay of M to N, as well as from N back to bR. The absorbance maximum of the N intermediate in bR is 560 nm, corresponding to a protonated Schiff base. This is not very different from λ~max~ for the resting state of pR (546 nm). The likely spectral overlap between pR and its own postulated N photoproduct complicates the determination of amounts of each that are present. The slow (\~500 ms) decay observed in both the 400 nm and 500 nm time courses indicates an equilibrium between the M and N intermediates that remains until pR returns to its initial resting state.

Béjà et al. reported a strong positive 580-nm transient absorbance increase in suspensions of membranes prepared from *E. coli* expressing pR \[[@B1]\]. This positive absorbance difference is indicative of O intermediate formation. We looked for its presence in partially-purified samples reconstituted in DHPC in the pH range 6--11. (Data are shown only at pH 9.5; see Fig. [4](#F4){ref-type="fig"}). Our observations at pH 9.5 do not show any evidence of O intermediate formation (Fig. [4](#F4){ref-type="fig"}). Only at lower pH values was a clear positive 580-nm absorption change observed (data not shown). This is in agreement with results on bR, for which O formation is also enhanced at lower pH values, and becomes small or nearly unobservable in the alkaline range.

Dependence of the M intermediate on pH
--------------------------------------

Deprotonation of the Schiff base linkage of the retinal and Lys-216 is dependent on its pK~a~ which changes between photointermediate states. The Schiff base readily undergoes deprotonation in the M intermediate. However, no M intermediate formation occurs below a pH of \~9 (figure [3](#F3){ref-type="fig"}); instead the O intermediate predominates \[[@B1]\]. Predominance of O at lower pH values is also observed in bR. However, in bR the M intermediate is detectable at low and high pHs, but has a longer lifetime at higher pH due to a long-lived equilibrium between M and another intermediate, N \[[@B13]\]. It seems likely that an N intermediate of pR is similarly in equilibrium with its M intermediate, based on the fact that the transient positive (400 nm) absorbance increase is smaller than the negative 500 nm bleach, and the time course at 500 nm shows a partial return to baseline on a timescale of \~50 μs.

The difference in pH dependence between the pR and bR photocycles can likely be attributed to differences in the microenvironment of the Schiff base, and is perhaps related to absence of Glu194/204 in pR. These adaptive differences presumably optimize the proteins to operate at maximum efficiency in the niches that their respective organisms occupy. In the case of the λ-proteobacteria, which in the open ocean (pH 7.8--8.0) occupy a signficantly more alkaline environment than halobacteria, perhaps the proton-release group is simply not under any evolutionary pressure to be capable of deprotonating in the M state at neutral pH. In this view, the principal role of E194 and E204 in bR may to modulate the pK~a~ of the H^+^-release group in the M state to a value lower than the pH of the organism\'s external environment.

Fast proton release in pR
-------------------------

Under the same conditions where M is observed (pH 9.5 and in 1% DHPC), pR undergoes fast proton release during its photocycle (Fig. [5](#F5){ref-type="fig"}). The pH indicator dye Cresol Red was used to detect pH changes in the bulk aqueous phase. These turn out to be similar to those observed for bR in the pH range 5.5--10. After photoexcitation, pR (like bR, presumably) ejects a H^+^ from a residue near its extracellular surface decreasing the pH of the solution. When the N → O transition takes place in bR, H^+^ is taken up from the medium, raising the pH once again. The H^+^ signals from pR measured with Cresol Red occur on a time scale similar to that assignable to M and N decay, returning to baseline about 1 s after photolysis.

There is a clear kinetic correlation between M (and/or N) intermediate formation and fast H^+^ release in pR. The linkage between these two phenomena is further supported by the observation that neither a transient 400-nm absorbance increase, nor fast H^+^ release, is shown to occur at pH 8.0 and below. Nor is either observed in the absence of a reconstituting lipid (DHPC in these experiments).

In bR, the ejected proton is thought to originate from a triad of amino acids, R82-E194-E204. However, in pR a homolog of only one of these three residues (the arginine) is present. This raises doubts about previous conclusions regarding the specific roles of these 3 residues in fast H^+^ proton release, in both pR and bR. In particular, the apparently obligatory roles of E204 and E194 in fast H^+^ release in bR are not matched in pR. Therefore, even in bR it is less likely that these groups themselves change protonation state between bR and M to provide the H^+^ released to the bulk medium. Instead, it now seems more likely that E204 and E194 merely help to lower the pK~a~ of the H^+^ release group from above 8, the apparent value for pR, into the vicinity of 6 for bR. It also seems very unlikely that the specific structural configuration of 2 carboxylic acid groups and arginine in bR could be conserved in pR, even if, as suggested previously \[[@B1]\], other surface carboxylic acids in pR could substitute in some ways for the roles of E194 and E204 in bR.

The only way that the H^+^ release mechanism can be strongly conserved between bR and pR is if arginine itself serves as the principal donor group for fast H^+^ release in both, with nearby residues (such as E194 and E204) merely modulating the pK~a~ of the arginine in the M intermediate. However, it remains unclear how the pK~a~ of Arg-82 in bR could be made sufficiently low in its M intermediate to serve as the H^+^ release group at pH values down to 6.0.

Alternatively, it is possible that the fast H^+^ release we observe from pR at pH 9.5 may differ from that in bR. One possibility is that in pR, the released proton could come directly from the chromophore counterion, Asp-97. This would be consistent with a proposed mechanism for fast H^+^ release that has been observed above pH 10 in the bR mutant E194Q \[[@B14]\]. In this mutant, Asp-85 was detected by low-temperature infrared difference spectroscopy to be deprotonated only in the N intermediate, and not in M \[[@B15]\]. It is not clear yet whether Asp-85 deprotonation in an N-like state could account for the proton-release kinetics of pR (Fig. [5](#F5){ref-type="fig"}), or whether the M intermediate itself might have a partially-deprotonated Asp-85.

The reason for the requirement of DHPC in M intermediate formation and fast proton release is unclear. Delipidated bR in octylglucoside is fully capable of M formation and presumably proton release, although with altered kinetics \[[@B12],[@B16]\]. The requirement for pR to be in lipid to show fast H^+^ release and M formation stems either from a protein/lipid interaction needed to establish a stable, active tertiary structure, or from the need for the phosphate group in DHPC to act as a proton release group. The latter seems unlikely due to the DHPC molecule being zwitterionic at pH 9.5, with no proton on the trimethyl-modified nitrogen of the choline. Hence, the DHPC most likely interacts with the protein to effect minor structural changes needed to place the active site residues in their functional configuration.

Conclusions
===========

A comparison of the primary sequences of pR and bR at first glance seems as to preclude fast H^+^ release as part of the proton-pumping mechanism of pR due to the absence of residues analogous to Glu194 and Glu 204 of bR. However, fast H^+^ release is indeed observed in pR under conditions where an M intermediate is formed. Glu194 and Glu 204 in bR play a role in fast H^+^ release that is apparently not required for the mechanism of the bR family of proton transporters. It is therefore necessary to conclude that either the H^+^-release groups in pR and bR are non-homologous surface carboxylic acid residues (as suggested previously \[[@B1]\]), or else that a conserved non-carboxylic acid residue, i.e. Arg82/94 or Asp85/97, is the H^+^-release group in pR. The higher pH requirement for the M intermediate of pR presumably corresponds with adaptation to the more alkaline oceanic environment in which the γ-proteobacteria are found.

The necessity of reconstituting pR with some lipid before it is capable of photocycling shows that the presence of lipids facilitates pR in assuming its fully active structure. *E.-coli*-expressed pR has post-translational modifications, including \~4000 daltons of substituents at one or more of its three cysteines. Such post-translational modifications might also play a role in explaining the different physiological properties of pR and bR.

Materials & methods
===================

Protein expression and detergent extraction
-------------------------------------------

Proteorhodopsin was expressed by *E. coli* strain UT5600 containing an additional plasmid encoding for the first-reported pR gene (accession \#AF279106, obtained from the uncultured proteobacterium EBAC31A08 clone BAC \[[@B1]\]) with an Ara promoter and ampicillin resistance (kindly provided by O. Béjà). Single colonies were selected and grown overnight in LB/amp media (200 ml, 37°C, 300 rpm). This culture was then diluted 10× into several 500-ml cultures. After a further 2 h incubation in the shaker bath, a stock solution of 20% L-arabinose was added, to give a final concentration of 0.2% L-arabinose. This culture was then incubated for 4 h (37°C, 300 rpm). The cells (\~20 ml wet volume) were then collected by centrifugation (6000 rpm × 30 min) and washed 3× with 100 mM HEPES, pH 7.1 (buffer A). The cells were then resuspended in buffer A and incubated at 4°C with 50 μg all-trans-retinal (added as a concentrated ethanol solution) for 3 h. The cells were collected by centrifugation (6000 rpm × 30 min), then resuspended in 60 mL buffer A containing 0.3 mg/mL lysozyme, and stirred for 4 h at room temperature. The cells were again collected by centrifugation (6000 rpm × 30 min), then lysed with 20 ml of 20% sodium cholate, pH 7.1 (30 min., 4°C). The cells were centrifuged again (6000 rpm × 30 min), and the supernatants collected. After extracting 3× more with the same cholate solution, the pooled supernatants were diluted 10× with buffer A and centrifuged at 180,000 g for 45 min to collect the membrane pellet. This cholate-washed membrane pellet was then further extracted 3× with 3.0% β-octyl-D-glucoside (OG) in buffer A (30 min, with stirring, 4°C). The pooled supernatants, containing OG-solubilized pR (\~15 mg), were then diluted 6× with buffer A.

Column purification
-------------------

The diluted OG-solubilized membrane extract (10 mg in 300 mL total volume of 0.5% OG) was loaded on a 25 × 1 cm column containing Phenylsepharose™ (6 fast flow high sub; Amersham Pharmacia Biotech). The column was eluted with a 0.5%-2.0% OG gradient in buffer A (300 mL total volume, 0.5 mL flow rate). The pR eluted at an OG concentration of 1.5--2.0%. Fractions having an A~280~/A~546~ ratio of 4.0 or lower were pooled (9.5 mg pR recovered in all) and concentrated using Vivaspin™ 20 concentrators having a 5000 MW cutoff (Vivascience, Westford, MA). A portion of the Phenylsepharose™-purified pR (1.5 mg) was diluted to an OG concentration of 0.5% with 0.5 M KCl, 100 mM acetate. It was then loaded on a 10 cm × 1 cm hydroxylapatite (BioGel HTP, BioRad) column and eluted under pressure with a 0--600 mM phosphate gradient (200 mL total volume, flow rate 0.5 ml/min). Fractions with an A~280~/A~546~ ratio of 2.5 or lower were pooled and concentrated for subsequent experiments (0.5 mg).

Mutagenesis
-----------

Methodology for the site-directed mutagenesis of pR is discussed in detail elsewhere (R. Parthasarathy, T. Caterino, R.A. Krebs, M.S. Braiman, manuscript in preparation). The triple cysteine mutant (pR-TCM) has all three of its native cysteines (Cys-107, Cys-156, and Cys-175) replaced with serines, and was prepared using the same *E. coli* expression system and purification methods as the wild type.

Polyacrylamide gel electrophoresis
----------------------------------

A 12% discontinuous SDS/polyacrylamide gel was used for molecular weight and purity analysis \[[@B17]\].

Flash photolysis
----------------

Time-resolved UV/vis spectroscopy methods were as described previously \[[@B18]\]. A Phenylsepharose™-purified pR sample was reconstituted into mixed micelles containing 1,2-diheptanoyl-SN-glycero-3-phosphocholine (DHPC), by adding a 1% solution of the short-chain lipid and then removing most of the detergent on a Sephadex G-25 column equilibrated with 1% DHPC in 100 mM NaCl. Proton release and uptake in the aqueous bulk medium were detected from the pR-containing micelles suspended in 1% DHPC, 100 mM NaCl, with 45 μM Cresol Red pH indicator dye. Flash-induced absorbance changes at 580 nm of samples with and without the Cresol Red were subtracted to determine the transient signals due to proton concentration changes. Photoexcitations were performed with 10-ns laser pulses of 3--6 mJ at 500 nm. The time courses in Figs. [3](#F3){ref-type="fig"},[4](#F4){ref-type="fig"} are an average of 40 cycles with the exception of the Cresol Red experiments averaging 100 cycles (Fig. [5](#F5){ref-type="fig"}, bottom trace) \[[@B19]-[@B21]\].

Abbreviations
=============

pR, proteorhodopsin; bR, bacteriorhodopsin; OG, β-octyl-D-glucoside; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis; pR-TCM, pR triple cysteine mutant \[C(107,156,175)S\]; DHPC, 1,2-diheptanoyl-SN-glycero-3-phosphocholine; DMPC, dimyristoylphosphatidylcholine; HEPES, N-2-hydroxyethylpiperazine-N\'-2-ethanesulfonic acid

Acknowledgements
================

This work was supported by Syracuse University, and by a grant of the Deutsche Forschungsgemeinschaft (Sfb 449-TPA5 to U. Alexiev and M. P. Heyn). A.-M. DeVita was supported by an NSF-REU award to Syracuse University Chemistry Department.


Background
==========

It is hypothesized that there are 1.5 million fungal species on earth, of which only about 70,000 have been described \[[@B1]\]. Thus nearly 1.43 million remain undescribed. It will be essential for the study of fungal evolution to determine the phylogenetic positions of undescribed fungi present in diverse environments. Many fungi have parasitic or symbiotic relationships to other organisms. This can make it difficult, if not impossible, to separate fungi from such organisms. On the other hand, PCR can be used to amplify the DNA fragments without isolation and cultivation. The genes used most widely in fungal phylogenetic studies are rRNA genes, elongation factor genes, tublin genes, and other universally conserved eukaryotic sequences. These genes are so conserved among all eukaryotes that they often result in artifacts when amplified by PCR. Therefore, we conclude that PCR primers that are unique to fungal genomes will be extremely useful for PCR-based phylogenetic study of fungi.

In addition, although the comparison of rDNA and other genes among species is a powerful tool to show the phylogenetic relationships among fungi, no individual gene can answer all questions about fungal evolutionary relationships \[[@B2]-[@B4]\]. Many genes are not useful for quantitative analysis because they are multi-copy and copy-number is different among various fungi.

Here we investigate the aminoadipate reductase gene. Most mycologists previously believed that lysine biosynthesis through the 2-aminoadipate pathway was a fungal-specific characteristic. However, some prokaryotes also synthesize lysine through the 2-aminoadipate pathway \[[@B5]-[@B7]\]. A comparison between the prokaryotic and fungal lysine biosynthetic pathways reveals that the synthesis of 2-aminoadipate from 2-oxoglutarate proceeds in the same way but the fungal process to synthesize lysine from 2-aminoadipate is different from the prokaryotic one \[[@B5],[@B8]\]. Therefore, prokaryotes have no aminoadipate reductase gene; on the other hand, the aminoadipate reductase is a key enzyme in the evolution of fungal lysine biosynthesis \[[@B8]\]. This enzyme consists of large and small subunits \[[@B9]\], and functions only in fungal cells. As far as we know, animals and plants have no homologue of this enzyme-coding gene.

In the lysine biosynthetic pathway of the ascomycete yeast *Saccharomyces cerevisiae*, a complex of LYS2 (1392 aa) and LYS5 (272 aa) serves as aminoadipate reductase, which converts 2-aminoadipate into 2-aminoadipate 6-semialdehyde via an adenosylated derivative (Fig. [1](#F1){ref-type="fig"}). Before this report, seven coding regions of the large subunit of the aminoadipate reductase were deposited in the international DNA/protein database \[[@B10]-[@B15]\]. The *Saccharomyces cerevisiae lys2* gene is a single-copy gene. Comparisons of single-copy genes of large size can provide good phylogenetic resolution and offer advantages over multi-copy genes \[[@B16]\]. According to the seven ascomycetous aminoadipate reductases, fungal the coding regions of *lys2* genes are more than 4,000 nucleotides-long.

![Reduction of 2-aminoadipate by aminoadipte reductase in fungi.](1471-2148-2-6-1){#F1}

Results and Discussion
======================

First we performed a homology search using BLAST \[[@B17]\] with the given parameter values on the DNA data bank of Japan (DDBJ). We searched the homologous sequence of the *S. cerevisiae lys2* gene for EST database of *Arabidopsis thaliana, Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Glycine max, Homo sapiens, Mus musculus, Oryza sativa, Xenopus laevis*, and *Zea mays*. As a result, we obtained no significant sequence. This result supports that the aminiadipate reductase gene is a fungal-specific gene.

We determined the DNA sequence of 1,058-bp from *S. complicata* and 1,079-bp from *A. awamori*. These sequences have been deposited in the DDBJ under accession numbers AB076076 for *S. complicata* and AB076077 for *A. awamori*. An alignment of the nine ascomycetes was created using the program CLUSTAL W \[[@B18]\]. The alignment in this study is available on request using e-mail. We considered 325 amino acid sites excluding indels and PCR primers-sites. A maximum parsimonious phylogenetic tree was constructed using a Branch-and-Bound algorithm method, in MEGA version 2.1 \[[@B19]\], with 1,000 bootstrap replicates. We obtained equally two most parsimonious trees.

The consensus parsimony tree (Fig. [2a](#F2){ref-type="fig"}) shows three major ascomycete lineages; the archiascomycete, the euascomycete, and the hemiascomycete. This is consistent with current ascomycete systematics based on other gene sequences. The bootstrap analysis indicated 96% and 90% support values for monophyletic lineages of euascomycetes and hemiascomycetes, respectively. However, it indicated only 57% support for the monophyly of the archiascomycetes.

![a) The bootstrap consensus tree of the two most parsimonious trees based on the amino acid sequences of the aminoadipate reductase. The most parsimonious trees using the Branch-and-Bound algorithm of MEGA version 2.1 \[[@B19]\] with 1,000 bootstrap analyses. b) The maximum likelihood phylogenetic relationships. This analysis was performed using PAML \[[@B20]\], version 3.1. The model of amino acid substitution by Whelan and Goldman \[[@B21]\] was used.](1471-2148-2-6-2){#F2}

Maximum likelihood analysis was performed using PAML \[[@B20]\], version 3.1. In this analysis, we used the model of amino acid substitution by Whelan and Goldman \[[@B21]\]. The best tree (lnL: -4292.79) is shown in Fig. [2b](#F2){ref-type="fig"}. The ML tree is largely consistent with the most parsimonious tree, and shows the archiascomycetes to be monophyletic.

The anamorphic yeast *Saitoella complicata* has unique morphological and chemotaxonomic characteristics, and its 18S rDNA shows affinity to those of archiascomycetes \[[@B3],[@B22]-[@B24]\]. As far as we know, this interesting and important yeast has not been analyzed in a molecular evolutionary study, aside from rDNA comparison. Here we determined the DNA fragment from the aminoadipate reductase gene and also show that the deduced amino acid sequence has an affinity for the archiascomycete *Schizosacchaormyces pombe* in phylogenetic analyses. The phylogenetic tree (Figs. [2a, 2b](#F2){ref-type="fig"}) clearly indicated that the budding yeast *S. complicata* was far from the other budding yeasts (the hemiascomycete lineage).

The phylogenetic position of the black-koji mold *Aspergillus awamori* suggests a close relationship to *Penicillium chrysogenum* (99% bootstrap support). This is an expected result. The sequence similarity between *A. awamori* and *P. chrysogenum* is 86% in amino acid comparison and 76% in DNA comparison. This sequence-difference in the aminoadipate reductase comparison is greater than that in shown from rDNA comparison.

Conclusions
===========

The PCR primers designed in this study were shown to be effective for amplifying the aminoadipate reductase gene from divergent ascomycetes. In addition, this region of the PCR product is useful for clarifying the ascomycete phylogeny. We believe that this region would be a powerful tool for fungal ecological and evolutionary studies.

Materials and Methods
=====================

In this study we used *Aspergillus awamori* IAM 2112, *Saitoella complicata* IAM 12964. The genomic DNAs were isolated using a DNeasy Plant Mini Kit (QIAGEN, Valencia, CA). Multiple alignment was created among the seven known ascomycetous aminoadipate reductases (*Acremonium chrysogenum*, AJ261064; *Candida albicans*, U58133; *Neurospora crassa*, AL389890; *Penicillium chrysogenum*, Y13967; *Pichia sorbitophila*, AJ288950; *Saccharomyces cerevisiae*, M36287; *Schizosaccharomyces pombe*, AL353014) using the program CLUSTAL W \[[@B17]\]. According to the multiple alignment, we found only two conserved regions for the PCR-primers. Based on the conserved amino acid sequences, two primers were designed 5\'-GGNATHGCNCAYGAYCCNRTNCA-3\' and 5\'-GGYTTRTCNAYYTTNCCRTTNGGRTT-3\'. The amplification was carried out under the following conditions: denaturation at 94°C for 5 min, 30 cycles of (94°C for 1 min, 57°C for 1 min, 72°C for 1 min), and a final extension at 72°C for 10 min. The PCR products were cloned using a PCR Cloning Kit (QIAGEN). Direct sequencing for the PCR products and sequencing for the several cloned plasmids were performed using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, CA).

Acknowledgements
================

We thank the two anonymous reviewers for their helpful comments. The Ministry of Education, Culture, Sports, Science, and Technology of Japan supported this work.


Background
==========

Publication bias represents an Achilles\' heel in meta-analyses. A particular form of publication bias is the so-called language bias, by which randomised controlled trials (RCTs) with greater estimates of effect size tend to be published in English rather than in the original authors\' native languages \[[@B1]\]. This bias is compounded by Anglophone databases and journals considerably under representing the totality of RCTs. For example, more than half of trials published in a Hungarian journal, identified through a cover-to-cover hand search, were not reported in MEDLINE \[[@B2]\]. Electronic searches of non-English databases may yet not suffice: a mono-lingual English searcher would be able to identify only 73% of the trials in the Spanish/Portuguese LILACS \[[@B3]\] and 70% of the German PSYNDEX \[[@B4]\].

Attempts are underway to remedy this situation. Two notable examples are the Medical Editors Trial Amnesty \[[@B5]\] and the Cochrane Controlled Trials Register (CCTR). The latter now contains more than 300,000 citations. Despite these enormous efforts we still are unsure if any database provides comprehensive coverage, particularly for non-English trials. We have recently established a comprehensive register of Japanese trials of psychotropic drugs. We therefore examined how successful the Cochrane psychiatry groups have been in identifying trials conducted in Japan and reported in Japanese by comparing the yields of the groups\' specialized registers against this recently created \"gold standard.\"

Methods
=======

Three of the authors (TI, AI, SN) have established a comprehensive register of Japanese RCTs of psychotropic drugs. First, we requested all the pharmaceutical companies marketing a psychotropic drug in Japan (n = 20) to provide them with the references concerning their RCTs with Japanese participants. Second, we carefully searched the on-line bibliographic database, JMEDICINE (Jan 1981--June 2000). This contains reports of medical literature in Japanese. Thirdly, we also collected references to RCTs through the \"reference search\" of the identified reports. Since on-line JMEDICINE is not available before 1980, and even the best electronic search is likely to miss a considerable proportion of reports of trials, these authors made considerable efforts to hand search psychiatric and medical journals likely to contain reports of psychotropics trials (n = 35). Abstracts of academic meetings have not been included; neither have RCTs undertaken in Japan but published in the English language journals.

We then asked two Cochrane groups in mental health, namely the Cochrane Schizophrenia Group and the Depression, Anxiety and Neurosis Group, to search their specialized registers for reports of trials possibly conducted in Japan. We employed two ways to identify such trials. The first strategy was to use the search term \"Japan\*\" (\*is a wild card). Because we were not sure if all candidates contained a word \"Japan^\*^,\" we employed the second strategy in which we searched for all the authors\' names appearing in the Japanese register.

Results
=======

The Japanese register contained 56 reports of RCTs of antidepressants for depressed people but the CCDAN register contained 18, with an overlap of only nine. It was possible to retrieve five more studies as the CCDAN register contained six English language duplicate publications of the five original Japanese RCTs. The remaining three studies found in the CCDAN register but not in the Japanese database were English reports of RCTs conducted in Japan but reported only in the English language. Searching the CCDAN register would therefore have identified a maximum of only 25% (=14/56, 95%CI: 15 to 38%) of all Japanese reports of relevant RCTs.

The CSG register contained 36 reports of RCTs of neuroleptics given to Japanese people with schizophrenia. The Japanese register contained 61. Of these, only six had been registered in the CSG register; 13 more studies were possible to retrieve as the CSG register contained 18 English language duplicate publications of original Japanese RCTs. The remaining 12 reports found in the CSG register but not in the Japanese register were reports of RCTs conducted in Japan but published only in the English journals. Searching the CSG register only would have identified 19 or only 31% ( = 19/61, 95%CI: 21 to 44%) of all relevant reports in the Japanese language.

Discussion and conclusion
=========================

The comprehensiveness of the collaborative databases is now deemed all the more important because doubts have been expressed as to effectiveness of traditional statistical tools to examine publication bias such as funnel plots \[[@B6]\]. However, searching the Cochrane group registers would have missed 69% to 75% of all Japanese reports of RCTs of antidepressants or neuroleptics. These results clearly demonstrated the limitations of the current Cochrane group registers, despite their extensive searching (information available from the CCDAN and CSG modules in the Cochrane Library). Similar, or worse, yields may be expected with RCTs conducted in other East Asian countries (Korea, China), and in other fields of medicine.

Many systematic reviews, at least in the field of mental health, are therefore likely to present results grossly underestimating research output from Asia. What evidence there is suggests that this will result in a systematic over estimate of effect \[[@B1]\]. Efforts must be expended for non-Anglophone countries to prepare their own register of RCTs for all health interventions in humans. The Japanese database of psychiatry trials has now been merged with the Cochrane group registers.

Competing interests
===================

None declared.

Authors\' contributions
=======================

TAF, TI and CEA participated in the design of the study and in its coordination. TI, CEA, HM, AI and SN participated in the collection of the datasets and in their analyses. TAF and CEA drafted the manuscript and all the authors participated in the rewriting. All authors read and approved the final manuscript.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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