The COP9 signalosome is essential for development of Drosophila melanogaster.

The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants [1] [2]. It has also been identified in mammals, though its role remains obscure [3] [4] [5]. This complex is similar to the regulatory lid of the proteasome and eIF3 [5] [9] [10] [11] [12] and several of its subunits are known to be involved in kinase signaling pathways [4] [6] [7] [8]. No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation [13]. In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.

The COP9 signalosome: from light signaling to general developmental regulation and back.

The COP9 signalosome has eight core subunits that are highly conserved between plants and animals. Some of the subunits in Arabidopsis are found in forms that are independent of the complex. The COP9 complex is essential for animal development. The COP9 signalosome may have both an evolutionary and a physical relationship with both the regulatory lid of the proteasome and eIF3.

PCI complexes: pretty complex interactions in diverse signaling pathways.

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.

Molecular cloning and expression in Escherichia coli of a cyanobacterial gene coding for phytoene synthase, a carotenoid biosynthesis enzyme.

The first committed step in the biosynthetic pathway of carotenoids in plants and algae is the conversion of geranylgeranyl pyrophosphate (GGPP) to prephytoene pyrophosphate (PPPP), which is converted to phytoene. We have cloned the gene pys that encodes the enzyme phytoene synthase in the cyanobacterium Synechococcus PCC7942. The co-expression of pys in cells of Escherichia coli together with the gene crtE from Erwinia uredovora, which encodes geranylgeranyl pyrophosphate synthase, resulted in accumulation of phytoene. This result indicates that phytoene synthase is a single polypeptide enzyme that catalyzes the 2-step reaction from GGPP to phytoene. The deduced amino acid sequence of pys is highly conserved with that of pTOM5, a tomato cDNA that is differentially expressed during fruit ripening. These findings suggest that pTOM5 encodes phytoene synthase in tomato.

Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of beta-carotene.

Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.

The Arabidopsis homologue of an eIF3 complex subunit associates with the COP9 complex.

The Arabidopsis COP9 complex is a multi-subunit repressor of photomorphogenesis which is conserved among multicellular organisms. Approximately 12 proteins copurify with the COP9 complex. Seven of these proteins are orthologues of subunits of the recently published mammalian COP9 complex. Four of the proteins show amino acid similarity to various subunits of the COP9 complex, eIF3 complex and 19S cap of the proteasome. We have studied one of these proteins in order to determine if it is a component of the COP9 complex. Arabidopsis p105 is highly similar to the p110 subunit of the human elF3. The p105 gene is induced during photomorphogenesis, and RNA and protein analysis reveal different tissue accumulation patterns. p105 is found in a large protein complex. p105 interacts in yeast with both COP9 and FUS6, two known components of the COP9 complex. Our results indicate that p105 is not a component of the COP9 core complex, though it may interact with components of the complex.

Anterior operculum syndrome localized by SPECT.

UNLABELLED: The aim of this case report was to present a patient with complete anarthria and orofacial apraxia without other relevant neurological deficit. The clinical features are compatible with anterior operculum syndrome. METHODS: A regional brain perfusion scan was done using 99mTc-HMPAO and a SPECT gamma camera. A brain CT scan and an MRI were also performed. RESULTS: Brain CT and MRI were not diagnostic. On brain SPECT, hypoperfusion of the left inferior area of the frontal lobe was noted. CONCLUSION: The patient studied showed an uncommon case of anterior operculum syndrome of focal degenerative origin localized by SPECT. SPECT may be a useful and effective method for diagnosis of this unusual neurological deficit.

Splenogonadal fusion diagnosed by spleen scintigraphy.

Splenogonadal fusion (SGF) is a rare congenital malformation characterized by fusion of the spleen and a gonad (almost always the left one) frequently associated with orofacial and/or limb developmental abnormalities. Only 125 cases were reported between 1883 and 1994. This report concerns a case of SGF in a 20-yr-old woman with an accidental finding of a splenic space-occupying lesion protruding into the lower abdomen in ultrasound and CT. Radiocolloid spleen scintigraphy and SPECT proved to be the best procedure to establish the correct diagnosis of SGF. As SGF is often asymptomatic, more liberal use of splenic scintigraphy is suggested in patients with congenital limb and/or orofacial anomalies. SGF should be included among the differential diagnoses of left abdominal, pelvic or scrotal masses.

Distribution of cerebral flow using retrograde versus antegrade cerebral perfusion.

BACKGROUND: This study compared flow to the brain with retrograde and antegrade cerebral perfusion during circulatory arrest. METHODS: Twenty-four rabbits were injected with 5 mCi of technetium-99 macroaggregated albumin, a tracer trapped in the capillaries. Group I (n = 6) were maintained normothermic, and the tracer was injected into the ascending aorta. Group II (n = 6) were maintained normothermic, and underwent cannulation of the superior vena cava (SVC), exsanguination through the aorta, and injection of the tracer into the SVC, which was proximally occluded. In group III (n = 6), the animal was cooled to 25 degrees C. The animal was exsanguinated through the aorta and tracer was injected into the ascending aorta. In group IV (n = 6), animals were cooled to 25 degrees C. The animal was exsanguinated through the ascending aorta and tracer was injected into the SVC. Three animals (group V) were exsanguinated through the ascending aorta and a retrograde venogram of the SVC was performed. Scintigraphy of groups I to IV was carried out on a digital gamma camera. Brain trapping of tracer was graded from 0 to 5, with 0 being no tracer in the brain and 5 being dominant tracer trapping in the brain. RESULTS: Tracer trapping in the brain showed group I, 3.67+/-0.82; group II, 0; group III, 4.67+/-0.41; group IV, 0.17+/-0.41 (p<0.0001). Retrograde venogram of the SVC showed flow into the cerebral veins. CONCLUSIONS: Retrograde flow through the SVC reaches the cerebral venous system. Flow arriving in retrograde fashion does not go through the capillary system.

Nasal transit time in normal subjects and pathologic conditions.

A radionuclide method using Tc-99m was used to measure the nasal transit time in 43 patients, including normal smokers and nonsmokers and those with various nasal or paranasal pathologies. The technique described was slightly modified from that already in use in various centers in order to enable more precise measurement. Measurement of transit time of a droplet of Tc-99m phytate on the nasal mucosa appears to differentiate patients with immotile cilia syndrome from normal controls. A positive test, however, can occur also in smokers and in patients with other nasal and paranasal problems. The test is reproducible, cost-effective, and noninvasive, enabling the clinician to make a more appropriate selection of patients needing further investigation.

Cloning a gene coding for norflurazon resistance in cyanobacteria.

The herbicide norflurazon inhibits carotene biosynthesis in photosynthetic organisms by blocking the enzyme phytoene dehydrogenase (= phytoene desaturase). We have isolated norflurazon-resistant mutants of the cyanobacterium Synechococcus PCC7942. The herbicide-resistance gene from the mutant NFZ4 has been cloned by genetic complementation of the resistance trait in wild type cells. The experiment described here illustrates the usefulness of employing cyanobacteria to clone herbicide-resistance genes in a quick and simple way.

Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes.

Three different phytoene desaturase genes, from Rhodobacter capsulatus, Erwinia uredovora, and Synechococcus PCC 7942, have been functionally complemented with a gene construct from E. uredovora which encodes all enzymes responsible for formation of 15-cis phytoene in Escherichia coli. As indicated by the contrasting reaction products detected in the pigmented E. coli cells after co-transformation, a wide functional diversity of these three different types of phytoene desaturases can be concluded. The carotenes formed by the phytoene desaturase from R. capsulatus were trans-neurosporene with three additional double bonds and two cis isomers. Furthermore, small amounts of three zeta-carotene isomers (2 double bonds more than phytoene) and phytofluene (15-cis and all-trans with + 1 double bond) were detected as intermediates. When the subsequent genes from E. uredovora which encode for lycopene cyclase and beta-carotene hydroxylase were present, neurosporene, the phytoene desaturase product of R. capsulatus, was subsequently converted to the monocyclic beta-zeacarotene and its monohydroxylation product. The most abundant carotene resulting from phytoene desaturation by the E. uredovora enzyme was trans-lycopene together with a cis isomer. In addition, bisdehydrolycopene was also formed. The reaction products of Synechococcus phytoene desaturase were two cis isomers of zeta-carotene and only small amounts of trans-zeta-carotene including 15-cis. The I50 values for flurtamone and diphenylamine to inhibit phytoene desaturation were determined and differential inhibition was observed for diphenylamine.

[Well-differentiated thyroid carcinoma].

18 cases of well-differentiated thyroid carcinoma are described, of which 5 were papillary or mixed carcinomas and 3 were follicular. In 12 of 14 in whom radio-isotope scanning was performed, a cold or indeterminate nodule was found. Fine needle aspiration was positive and concordant with the operative histological findings in half the cases examined. In 4 hemithyroidectomy was performed, and in 14 total thyroidectomy. Thyroid remnants were found on scanning in all of the 14. These remnants were ablated with radioactive iodine (131I). An ablative dose of 30 millicurie was insufficient in almost all cases and a second dose was required. 3 cases with regional spread of the tumor were reoperated, and large ablative doses of 131I(150-170 mCi) were given. In 14 cases clinical remission was achieved. Followup included ultrasonography and total body 131I scans.

The molecular basis of resistance to the herbicide norflurazon.

We have cloned and sequenced a gene, pds, from the cyanobacterium Synechococcus PCC7942 that is responsible for resistance to the bleaching herbicide norflurazon. A point mutation in that gene, leading to an amino acid substitution from valine to glycine in its polypeptide product, was found to confer this resistance. Previous studies with herbicide-resistant mutants have indicated that this gene encodes phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids. A short amino acid sequence that is homologous to conserved motifs in the binding sites for NAD(H) and NADP(H) was identified in PDS, suggesting the involvement of these dinucleotides as cofactors in phytoene desaturation.

Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting step in carotenoid biosynthesis.

Mutant strains of the cyanobacterium Synechococcus sp. PCC 7942 that are resistant to the herbicides norflurazon and fluridone were analyzed. These herbicides inhibit phytoene desaturase, a key enzyme in the carotenoid biosynthetic pathway. In three mutants the phenotype was related to specific point mutations in pds, the gene encoding phytoene desaturase. Since the resistance was manifested in a cell-free carotenogenic assay, it is evident that the predicted amino acid changes in the target protein alter the enzyme-herbicide interactions. In addition, the mutations also reduced the in vitro activity of phytoene desaturase. Increased levels of the substrate phytoene, accompanied by a reduction in colored carotenoids, were detected in cells of each of the mutant strains. A correlation was established between the degree of increase in the steady-state levels of phytoene and the extent of reduction in total carotenoid content in the cells. These two phenomena in turn are correlated with the rate of enzymatic activity of phytoene desaturase that was measured in vitro. Hence we deduce that phytoene desaturation is a rate-limiting step in carotenogenesis in cyanobacteria. Support for this conclusion is obtained from analysis of cells of an additional mutant strain, which overexpress phytoene desaturase due to a deletion mutation in the promoter region of pds. Cells of this mutant contained more colored carotenoids than the wild-type and were resistant to herbicides that inhibit phytoene desaturase.

JAB1/CSN5 and the COP9 signalosome. A complex situation.

The Jun activating binding protein (JAB1) specifically stabilizes complexes of c-Jun or JunD with AP-1 sites, increasing the specificity of target gene activation by AP-1 proteins. JAB1 is also known as COP9 signalosome subunit 5 (CSN5), which is a component of the COP9 signalosome regulatory complex (CSN). Over the past year, JAB1/CSN5 has been implicated in numerous signaling pathways including those that regulate light signaling in plants, larval development in Drosophila, and integrin signaling, cell cycle control, and steroid hormone signaling in a number of systems. However, the general role of the CSN complex, and the specific role of JAB1/CSN5, still remain obscure. This review attempts to integrate the available data to help explain the role of JAB1/CSN5 and the COP9 signalosome in regulating multiple pathways (in this review, both JAB1 and CSN5 terminologies are used interchangeably, depending on the source material).

Dissection of the light signal transduction pathways regulating the two early light-induced protein genes in Arabidopsis.

The expression of light-regulated genes in plants is controlled by different classes of photoreceptors that act through a variety of signaling molecules. During photomorphogenesis, the early light-induced protein (Elip) genes are among the first to be induced. To understand the light signal transduction pathways that regulate Elip expression, the two Elip genes, Elip1 and Elip2, in Arabidopsis were studied, taking advantage of the genetic tools available for studying light signaling in Arabidopsis. Using two independent quantitative reverse transcriptase-PCR techniques, we found that red, far-red, and blue lights positively regulate expression of the Elip genes. Phytochrome A and phytochrome B are involved in this signaling. The cryptochrome or phototropin photoreceptors are not required for blue-light induction of either Elip gene, suggesting the involvement of an additional, unidentified, blue-light receptor. Although the COP9 signalosome, a downstream regulator, is involved in dark repression of both Elips, Elip1 and Elip2 show different expression patterns in the dark. The transcription factor HY5 promotes the light induction of Elip1, but not Elip2. A defect in photosystem II activity in greening of hy5 seedlings may result from the loss of Elip1. Heat shock positively controlled Elip1 and Elip2 in a light-independent fashion. This induction is independent of HY5, indicating that heat shock and light activate transcription of the Elip genes through independent pathways.