Interaction between proteins containing homeodomains associated to leucine zippers from sunflower

A strategy based on the use of PCR with one degenerate oligonucleotide deduced from conserved sequences and lambda gt10 primers was used to isolate homeobox containing sequences from sunflower stem and root cDNA libraries. Six different partial cDNAs coding for the first 48 amino acids of homeodomains and amino terminal sequences were analyzed and found to be members of the HD-Zip superfamily, which contain a homeobox linked to a leucine zipper coding region. A full-length cDNA clone, Hahb-10, was isolated and characterized. The leucine zipper portions of Hahb-10 and of the previously reported Hahb-1 have been utilized to construct fusions with the N-terminal domain of the lambda repressor. These fusions were tested for their ability to bind to lambda promoters in vivo. The expression of a protein containing an active dimerization domain, but not capable of DNA binding, exerts a dominant negative effect on the ability of repressor-zipper fusions to bind to its target DNA. From these experiments, it was concluded that Hahb-1 and -10, when co-expressed, form preferentially homodimers. Exchange of conserved threonines and leucines at positions a1 and d1 of both zippers reduces dimerization efficiency and allows the formation of heterodimers, suggesting that these residues are, among others, determinants of the specificity of interaction, most likely through changes in hydrophobic packing interactions at the dimer interface. The results imply that a great number of interacting molecular entities compose this protein superfamily which is presumably involved in regulating plant developmental responses.     

Ultrasonic vocalization behavior differs between lines of ethanol-preferring and nonpreferring rats

To further understand the relationship between emotional state and alcohol intake in rats, the tendency to emit ultrasonic vocalizations in response to an aversive, but nonpainful, air puff stimulus was tested in several rat lines. Included in this group were Maudsley Reactive (MR) and Non-Reactive (MNR) rats, and several lines of rats with either high ethanol preference or a low ethanol preference: Preferring, (P), Alko-Alcohol (AA), and Fawn-Hooded (FH) animals; and Non-Preferring (NP), Alko-Non-Alcohol (ANA), and Flinders Resistant Line (FRL). MR rats emitted fewer ultrasonic vocalizations (USVs) and showed less preference for ethanol than did MNR animals. An overall analysis that included the P, NP, FH, FRL, AA, and ANA groups demonstrated a significant negative correlation between the total number of USVs emitted and ethanol consumption. NP, FRL, and especially ANA rats (low ethanol-preferring) emitted the most USVs--to an extent similar to that typically found for normal rats. The duration of vocalizing was higher only in the NP and the FRL rats the relative to their P and FH comparison groups, respectively. In the ethanol-preferring and nonpreferring lines, the numbers of USVs emitted correlated positively with the duration of vocalizing, but not with the latency to vocalize, which in turn did not correlate strongly with ethanol intake. The latency to vocalize did not correlate significantly with ethanol intake across all drinking lines or MR or MNR rats, but was found to be higher in FH and AA rats relative to their nondrinking comparison groups. These associations suggest that the relationship between emotional state and ethanol drinking is complex and cannot be attributed to a simple elevated state of anxiety or emotionality. Further examination of the central nervous system mechanisms mediating the difference in USVs between paired lines of ethanol-preferring and nonpreferring rats may identify neurochemical factors that predict ethanol preference.     

The haematological management of patients with cyanotic congenital heart disease. A time for consensus?

AIMS: Recurrent venesection of patients with cyanotic congenital heart disease may be detrimental, with an increased risk of cerebrovascular events and symptomatic iron-deficiency. The aim of this study was to determine the venesection policies as practised in hospitals within a U.K. region and to determine if these policies followed current recommendations. METHODS AND RESULTS: Fifty-eight consultants (56% response rate) in cardiac specialties completed self-assessment questionnaires regarding the indications for and practice of venesection. Sixty-one percent of those responding were involved directly in the care of patients with cyanotic congenital heart disease and of these clinicians 97% used venesection. Indications for venesection varied, with 51% of those responding using an elevated haemoglobin per se (6.5-21.0 g. dl-1); 78% an elevated haematocrit (0.55-0.75) and 83% symptoms. Desired maintenance haemoglobin and haematocrit levels also varied greatly. Fifty percent of the consultants responding routinely screened their patients for iron deficiency and 23% felt there was no indication for investigating a low mean corpuscular volume. Only 18% of the policies described followed any evidence based principles. CONCLUSIONS: The practice of venesecting patients with congenital cyanotic heart disease varies greatly. Policies in many hospitals do not reflect the minimal benefits and considerable risks associated with recurrent venesection.     

The pig as a potential xenograft donor

Miniature swine have several advantages over other potential donor species as a xenograft donor for clinical use. Among these advantages are: (1) unlimited availability; (2) size (similar to human beings); (3) breeding characteristics; (4) physiologic and immunologic similarities to humans. Because of the genetic disparity between these two species, routine immunosuppression will probably not suffice for the long-term survival of pig to primate xenografts. Studies are therefore underway to induce tolerance across this species barrier, utilizing a mixed chimerism approach which has previously been successful for allogeneic and concordant xenogeneic combinations. Hyperacute rejection has been eliminated by an absorption technique and pig kidney xenograft survivals up to 13 days have been achieved.     

Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.     

Lesion volume measurement in multiple sclerosis: how important is accurate repositioning?

This study was designed to estimate the contribution of repositioning inaccuracies to measured intracranial lesion volumes in multiple sclerosis (MS). Five patients with MS were each scanned 10 times, using spin-echo imaging (2,000/34/90), contiguous 5-mm slices, and different scanning positions. The maximum displacements from baseline were +/-4 degrees (AP rotation) and 3 mm (slice offset). Lesion volume was measured twice for each scan using a semiautomated contour technique. Measured lesion volumes ranged from 4,328 mm3 to 164,831 mm3. The mean intrarater coefficient of variation (CV) calculated for individual patients ranged from 1.1 to 4% (median, 1.7%). Using analysis of variance, the overall variance and CV due to altered scan position were greater than that due to rater error (repositioning CV 4.0%, intrarater CV 3.5%). The worst-case difference between volumes in the same patient ranged from 8.9 to 32% (median, 9.9%). Both rater and repositioning errors were greater for smaller lesion volumes. The maximum potential error due to repositioning inaccuracies is of a similar magnitude to the 5 to 10% expected change in lesion volume over 1 year. This study justifies continued careful attention to accuracy in repositioning for serial MR studies in patients with MS.     

The effect of recombinant human erythropoietin on hemostasis, fibrinolysis, and blood rheology in autologous blood donors

We report three cases of Castleman's disease mimicking the features of collagen disease. Case 1: A 39-year-old woman presented with intermittent arthralgia and fever. Laboratory findings were positive results for antinuclear antibody (80x speckled type), the LE test, anti-SSA antibody, anti-RNP antibody, and Coombs test. The patient was suspected to have systemic lupus erythematosus (SLE) or Sj?gren syndrome, but a lymph node biopsy revealed the plasma cell type of Castleman's disease. Steroid treatment led to resolution of her symptoms. Case 2: A 60-year-old man with mixed type Castleman's disease had proteinuria with renal dysfunction, autoimmune thrombocytopenia, antinuclear antibody, anti-RNP antibody, anti-DNA antibody and anti-cardiolipin antibody. The patient was suspected to have SLE but cervical lymph node biopsy revealed the mixed type of Castleman's disease. Symptoms were not controlled with steroid therapy. He developed renal failure that required for hemodialysis and died of gastrointestinal bleeding due to severe thrombocytopenia. Case 3: A 46-year-old woman had Raynaud's phenomenon, sclerodactylia, and nail fold bleeding. Laboratory tests were revealed positive for antinuclear antibody, anti-ENA antibody, and LE cell preparation. Radiographic study showed multiple masses in the retroperitoneal spaces, which necessitated laparotomy. Firstly, the patient was suspected to have systemic sclerosis or mixed connective tissue disease (MCTD). A biopsy revealed the hyaline-vascular type of Castleman's disease. The serum level of IL-6 by ELISA was high in all of three cases. In case 1, symptoms improved and the IL-6 level normalized after steroid treatment.(ABSTRACT TRUNCATED AT 250 WORDS)