GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis.

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.

Glucose repression of alpha-amylase in barley embryos is independent of GAMYB transcription.

The induction of alpha-amylase triggered by gibberellic acid in barley embryos is repressed by sugars. We investigated the effects of glucose on the gibberellin signal transduction pathway to localize the site of interaction of the sugar/hormone signalling pathways. Our results indicate that glucose represses gibberellin signalling late along this hormone transduction pathway, downstream of transcription of the gibberellin-modulated transcriptional activator (GAMYB) needed for alpha-amylase induction. This result suggests either that glucose repression is transduced by a pathway independent of gibberellin signalling or that repression occurs at the level of GAMYB translation.

Long-day up-regulation of a GAMYB gene during Lolium temulentum inflorescence formation.

Long-day exposure of the grass Lolium temulentum may regulate flowering via changes in gibberellin (GA) levels. Therefore, we have examined both GA levels and expression of a MYB transcription factor that is specific to the GA signal transduction pathway in monocots. This MYB gene from L. temulentum shows over 90% nucleotide identity with the barley and rice GAMYB genes, and, like them, gibberellic acid (GA3) up-regulates its expression in the seed. Furthermore, cDNAs of both the barley and L. temulentum GAMYB show the same simple patterns of hybridization with digests of L. temulentum genomic DNA. Compared with vegetative shoot apices of L. temulentum, the in situ mRNA expression of LtGAMYB does not change during the earliest steps of "floral" initiation at the apex. However, by 100 h (the double-ridge stage of flowering) its expression increased substantially and was highest in the terminal and lateral spikelet sites. Thereafter, expression declined overall but then increased within stamen primordia. Prior to increased LtGAMYB expression, long-day exposure sufficient to induce flowering led to increased (5- to 20-fold) levels of GA1 and GA4 in the leaf. Thus, increases first in GA level in the leaf followed by increased expression of LtGAMYB in the apex suggest important signaling and/or response roles in flowering.

Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone.

GAMYB is an MYB transcription factor which is expressed in cereal aleurone cells in response to gibberellin (GA). HvGAMYB binds to the TAACAAA box of a high-pl alpha-amylase gene promoter and transcriptionally activates its expression. In this study, we examined the role of HvGAMYB in activating expression of other GA-regulated genes encoding hydrolytic enzymes. In transient expression experiments, HvGAMYB transactivated expression of reporter genes fused to a low-pl alpha-amylase gene promoter, an EII (1-3, 1-4)-beta-glucanase gene promoter and a cathepsin B-like protease promoter. HvGAMYB DNA binding specificity was determined using a PCR-based random site selection using HvGAMYB fusion protein isolated from E. coli. The deduced consensus closely resembled gibberellin response elements in alpha-amylase promoters. Functional analysis of HvGAMYB by transient expression of C terminal HvGAMYB deletions in barley aleurone cells identified two transcriptional activation domains (TADs) which function in transcriptional regulation of both high- and low-pl alpha-amylase promoters. The same TADs were identified using a heterologous yeast expression system. Together, these results indicate that HvGAMYB has two TADs. These domains are C-terminal to its DNA-binding domain.

Cloning of a rice cDNA encoding a transcription factor homologous to barley GAMyb.

A cDNA clone, OsGAmyb, which encodes a homologue to the barley Myb-like transcription factor, HvGAMyb, was isolated from a rice endosperm cDNA library. The clone was used to show that expression of the OsGAmyb gene in aleurone cells was stimulated by gibberellic acid and the gene product was shown to transactivate an alpha-amylase gene promoter in transient expression analyses.

cGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone.

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.

The value of palpation, varicoscreen contact thermography and colour Doppler ultrasound in the diagnosis of varicocele.

Three non-invasive methods for the detection of a varicocele were evaluated in 63 men presenting with infertility. Physical examination, varicoscreen contact thermography and colour Doppler ultrasound were compared with spermatic venography as reference strategy. Physical examination had a sensitivity of 71%. Whether the non-palpable varicoceles are all subclinical is questionable since the specificity of physical examination was 69%. Varicoscreen proved be quick, easy and cheap but of no clinical value (sensitivity 97%, specificity 9%). Colour Doppler ultrasound using strict criteria was a good diagnostic tool (sensitivity 97%, specificity 94%). No imaging difference was seen with colour Doppler ultrasound among clinical and subclinical varicoceles. Since the debate on treating all degrees of varicocoeles is ongoing, we suggest that Doppler sonography should be a routine examination in infertile men.

Comparison of cystic rheumatoid arthritis and erosive rheumatoid arthritis.

OBJECTIVE: To test the hypothesis that cystic rheumatoid arthritis (RA), characterized as subchondral cysts as the only radiographic abnormality in hands and feet for 2 years after first abnormal radiograph, is a mild subset of RA. METHODS: Fifty-four patients with cystic RA were compared with 144 RA controls matched for age, sex, disease duration, and year of first visit. All patients were randomly selected from a database of 1580 patients with RA attending the clinic 1982-88. In 1994, data of 90% of the patients were collected by one investigator, blinded to the study groups. All available radiographs were scored for erosions and cysts by one radiologist. RESULTS: During 17 years of followup (range 2-48 yrs), the cystic RA group had less severe disease. There were fewer disease modifying antirheumatic drug prescriptions and fewer orthopedic operations in the group with cystic RA. The proportion of Rose-Waaler seropositives and the proportion of patients with extraarticular manifestations were the same for both groups. At final assessment, the median Health Assessment Questionnaire score was significantly lower for the group with cystic RA (0,88 vs 1,56; p <0.01). The final radiographic score was significantly lower for the cystic RA group (0,22 vs 0,58; p <0.01). The outcome differences remained after correcting for early radiographic score, rheumatoid factor, early erythrocyte sedimentation rate (ESR), and presence of comorbidity in a multiple regression model. Mortality was the same for both groups. CONCLUSION: Cystic RA is a relatively mild subset of RA.

Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI alpha-amylase gene promoter.

Functional analysis of a barley high-pI alpha-amylase gene promoter has identified a gibberellin (GA) response complex in the region between -174 and -108. The sequence of the central element, TAACAAA, is very similar to the c-Myb and v-Myb consensus binding site. We investigated the possibility that a GA-regulated Myb transactivates alpha-amylase gene expression in barley aleurone cells. A cDNA clone, GAmyb, which encodes a novel Myb, was isolated from a barley aleurone cDNA library. RNA blot analysis revealed that GAmyb expression in isolated barley aleurone layers is up-regulated by GA. The kinetics of GAmyb expression indicates that it is an early event in GA-regulated gene expression and precedes alpha-amylase gene expression. Cycloheximide blocked alpha-amylase gene expression but failed to block GAmyb gene expression, indicating that protein synthesis is not required for GAmyb gene expression. Gel mobility shift experiments with recombinant GAMyb showed that GAMyb binds specifically to the TAACAAA box in vitro. We demonstrated in transient expression experiments that GAMyb activates transcription of a high-pI alpha-amylase promoter fused to a beta-glucuronidase reporter gene in the absence of GA. Our results indicate that the GAMyb is the sole GA-regulated transcription factor required for transcriptional activation of the high-pI alpha-amylase promoter. We therefore postulate that GAMyb is a part of the GA-response pathway leading to alpha-amylase gene expression in aleurone cells.

Defaecation disorders in children, colonic transit time versus the Barr-score.

It is still unclear how to evaluate the existence of faecal retention or impaction in children with defaecation disorders. To objectivate the presence and degree of constipation we measured segmental and total colonic transit times (CTT) using radio-opaque markers in 211 constipated children. On clinical grounds, patients (median age 8 years (5-14 years)) could be divided into three groups; constipation, isolated encopresis/soiling and recurrent abdominal pain. Barr-scores, a method for assessment of stool retention using plain abdominal radiographs, were obtained in the first 101 patients, for comparison with CTT measurements as to the clinical outcome. Of the children with constipation, 48% showed significantly prolonged total and segmental CTT. Surprisingly, 91% and 91%, respectively, of the encopresis/soiling and recurrent abdominal pain children had a total CTT within normal limits, suggesting that no motility disorder was present. Prolonged CTT through all segments, known as colonic inertia, was found in the constipation group only. Based on significant differences in clinical presentation, CTT and colonic transit patterns, encopresis/soiling children formed a separate entity among children with defaecation disorders, compared to children with constipation. Recurrent abdominal pain in children was in the great majority, not related to constipation. Barr-scores were poorly reproducible, with low inter- and intra-observer reliability. This is the first study which shows that clinical differences in constipated children are associated with different colonic transit patterns. The usefulness of CTT measurements lies in the objectivation of complaints and the discrimination of certain transit patterns. Conclusion. Abdominal radiographs, even when assessed with the Barr-score proved unreliable in diagnosing constipation.(ABSTRACT TRUNCATED AT 250 WORDS)

Gadolinium-DTPA enhanced magnetic resonance imaging of bone cysts in patients with rheumatoid arthritis.

OBJECTIVES: To examine the contents of intraosseous cysts in patients with rheumatoid arthritis (RA) through the signal intensity characteristics on gadolinium-DTPA (Gd-DTPA) enhanced magnetic resonance imaging. METHODS: The hand or foot joints of nine patients with the cystic form of RA (where the initial radiological abnormality consisted of intraosseous cysts without erosions) were imaged before and after intravenous administration of Gd-DTPA. A 0.6 unit, T1 weighted spin echo and T2* weighted gradient echo were used to obtain images in at least two perpendicular planes. RESULTS: Most cysts showed a low signal intensity on the non-enhanced T1 weighted (spin echo) images and a high signal intensity on the T2* weighted (gradient echo) images, consistent with a fluid content. No cyst showed an enhancement of signal intensity on the T1 weighted images after intravenous administration of Gd-DTPA, whereas synovium hyperplasia at the site of bony erosions did show an increased signal intensity after Gd-DTPA. Magnetic resonance imaging detected more cysts (as small as 2 mm) than plain films, and the cysts were located truly intraosseously. In six patients no other joint abnormalities were identified by magnetic resonance imaging; the three other patients also showed, after Gd-DTPA administration, an enhanced synovium at the site of bony erosions. CONCLUSIONS: It is suggested that intraosseous bone cysts in patients with RA do not contain hyperaemic synovial proliferation. The bone cysts in patients with the cystic form of RA may be the only joint abnormality.

The value of radiographs and bone scintigraphy in suspected scaphoid fracture. A statistical analysis.

The role of radiography and bone scintigraphy in the diagnostic management of suspected scaphoid fracture is controversial. Two strategies were compared for patients with initial negative radiographs: repeated radiography versus selective bone scintigraphy. Using the known positive predictive value of scintigraphy, the sensitivity and specificity of both diagnostic strategies were evaluated in a series of 78 consecutive patients. The kappa value for initial radiographs was 0.76 but decreased to 0.5 for follow-up radiographs. Similarly, sensitivity decreased from 64% to 30% in follow-up radiographs. Specificity of the bone scan was 98%. The best diagnostic strategy in the management of clinically suspected scaphoid fractures consists of initial radiography followed by bone scintigraphy in patients with negative radiographs.

Gibberellin-responsive elements in the promoter of a barley high-pI alpha-amylase gene.

Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal alpha-amylase promoter (-41) conferred both GA- and ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pI alpha-amylase genes. The TAACAAA box also appears to be the site of action of ABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa.

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5-2 µm) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(ß-aminoethyl ether)N,N,N',N'-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.

Cystic rheumatoid arthritis: description of a nonerosive form.

In a study of patients with rheumatoid arthritis (RA), 9% (n = 70) were found to have a cystic form. At radiologic examination of these patients with cystic RA, the first abnormality seen consisted of periarticular intraosseous cysts without erosions. The cysts were distributed symmetrically, most often located at the proximal side of the joints and predominantly around the proximal interphalangeal, metacarpophalangeal, and wrist joints of the hands and the first interphalangeal and metatarsophalangeal joints of the feet. Computed tomographic scans showed the peripheral intraosseous location of the cysts. Magnetic resonance images showed that the cysts may contain fluid, inflamed synovia, or both. Cysts can be an important feature in the diagnosis of RA and a supplement to the criteria of the American Rheumatism Association. Osteoporosis, joint-space narrowing, and joint destruction occurred less frequently in patients with cystic RA than in patients with classic RA. Of the patients with cystic RA, 54% were male, and 50% were seronegative. This study is a supplement to and an enlargement on earlier descriptions of cyst predominance in RA.

The cystic form of rheumatoid arthritis.

A non-erosive form of rheumatoid arthritis (R.A.) was found in 62 patients out of 660 patients with R.A. These 62 patients exhibit slowly progressive cystic changes in about the same joints in which usually erosions develop in classic R.A. The E.S.R. is often low, half of the patients remained seronegative and there are 35 males and 27 females in the group. A smaller group of 15 out of these patients could be followed from a stage wherein the radiographs were normal to a stage of extensive cystic changes, over a period of at least 6 years. An attempt is made to delineate this group within the Rheumatoid arthritis disease entity.

The release of α-amylase through gibberellin-treated barley aleurone cell walls : An immunocytochemical study with Lowicryl K4M.

Localisation of α-amylase (EC 3.2.1.1.) in low-temperature-embedded isolated barley (Hordeum vulgare L.) aleurone has been achieved using rhodamine-labelled secondary antibodies and the protein A-gold technique. Treatment with gibberellic acid (GA3) resulted in an increase of immunofluorescence in the cytoplasm of aleurone cells and also its appearance in specific regions of the cell walls. Cytoplasmic label was neither perinuclear nor associated specifically with aleurone grains as had been found in earlier work, but was present throughout the cytoplasm of all cells. A relatively high level of labelling occurred in hydrolysed wall regions. Label was also associated with plasmodesmata in both hydrolysed and unhydrolysed wall regions. The pattern of labelling indicates that α-amylase is released from aleurone via digested wall channels and that, except for the inner wall layer, unhydrolysed regions are impermeable to the enzyme. It is suggested that the resistant wall tubes around plasmodesmata may facilitate enzyme release by providing a pathway for transfer, especially of wall hydrolases, into the more impermeable parts of the wall.

Temperature control in Lowicryl K4M and glycol methacrylate during polymerization: is there a low-temperature embedding method?

An apparatus for embedding tissues at resin temperatures down to 228 K is described. By placing thermocouples in the resin the temperature has been monitored during embedding at low temperature with glycol methacrylate (GMA) and Lowicryl K4M. Even in this apparatus with a liquid cooling bath the heat of polymerization is not dissipated and the resin temperature rises. This rise is directly proportional to the resin temperature at the onset of polymerization and is higher in Lowicryl K4M than GMA. The initial resin temperature also affects the time taken for polymerization. The time to the onset of the peak and its duration are both increased as the temperature is lowered. This effect is more pronounced with GMA than Lowicryl K4M and polymerization of GMA is inhibited at the lowest temperature used. When Lowicryl K4M, polymerized at low temperature, is warmed up to ambient a further exothermic reaction occurs, which causes the resin temperature to rise well above ambient. Both this temperature peak and that during polymerization are reduced, but not totally eliminated, by reducing the resin volume. Air-cooled systems are inefficient compared with the low-temperature apparatus used here and the resin temperature rise is consequently greater and, even with small resin volumes, it can be very high. It is therefore unlikely for published methods that the temperature specified has been maintained in the resin during polymerization. The implications of these findings are discussed in relation to enzyme and antigen survival. Recommendations include use of very small volumes of resin, refrigerated liquid-bath rather than air-cooled systems and contact with a heat sink when specimens are warmed up to ambient temperature. Examples of enzyme reaction, antigen survival and structural preservation obtained with the method are presented.

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells.

Localisation of α-amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of α-amylase protein from aleurone cells.