Generation of Arabidopsis protein chips for antibody and serum screening.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.

[Molecular biological basis and diagnosis of hereditary defect of antithrombin III, protein c and protein S]. / Molekularbiologische Grundlagen und Diagnostik der hereditären Defekte von Antithrombin III, Protein C und Protein S.

Recent progress in molecular biology enabled the elucidation of the nucleotide sequences of the genes for antithrombin III (AT III), protein C (PROC) and protein S (PROS). Furthermore numerous mutations were identified causing genetic defects of the important inhibitors of blood coagulation. As the genes for AT III (13.8 kb) and PROC (11.2 kb) are small and easy to analyze a great number of molecular defects already are described in extensive databases (50, 73): 79 different mutations for AT III and 160 for PROC are included. The identification of mutations leading to AT III and PROC deficiency has given important information on the structure-function relationships of the proteins. In case of protein C deficiency the clinical relevance of DNA analyses is most important because the diagnosis at the protein level is often uncertain. The gene for PROS is not so easy to analyze like the other two genes. The PROS gene is large and also a PROS pseudogene exists. Although a number of mutations have been identified, there has not been published a database until now. The clinical relevance to identify gene defects in PROS deficiency is as important as for PROC deficiency. Presumably the elucidation of PROS gene defects will advance in the near future.

[Resistance to activated protein C by mutation of the factor V gene. Most frequent blood coagulation defect in venous thromboses]. / Resistenz gegen aktiviertes Protein C (APCR) durch Mutation des Faktor V-Gens. Häufigster Gerinnungsdefekt bei venösen Thrombosen.

Deep venous thromboses, in particular when recurrent, can be associated with chronic venous leg ulcers. Such complications are often seen in dermatology departments and frequently represent a therapeutic problem. Resistance to activated protein C (APCR) has recently been identified as the most frequent coagulation defect associated with an increased risk of venous thrombosis. In most cases, APCR is caused by a point mutation in the factor V gene which results in an impaired inactivation of activated factor V (Va). As a consequence of this, an important anti-coagulant mechanism in the physiological balance of the hemostatic system is abolished. This autosomal dominantly inherited genetic defects affects about 5% of the general population. In this article we draw attention to the existence of this recently identified, genetically determined risk factor for venous thrombosis, describe recent diagnostic developments and discuss therapeutic options.

The tandem repeat AGGGTAGGGT is, in the fission yeast, a proximal activation sequence and activates basal transcription mediated by the sequence TGTGACTG.

Ribosomal protein (rp) genes in the fission yeast Schizosaccharomyces pombe display two highly conserved sequence elements in the promoter region. The molecular dissection of these promoters revealed that basal transcription is not based on a TATA element. The sequence which promotes basal transcription is the conserved sequence CAGTCACA or the inverted form TGTGACTG, called the homol D box. Upstream of the homol D box a tandem repeat AGGGTAGGGT or the inverted form ACCCTACCCT appears in some promoters, called homol E. This element functions in the proximal arrangement with homol D as an activation sequence. A compilation of homol D and homol E sequences identified in other S.pombe promoters revealed that several putative polymerase II and polymerase III promoters display a homol D box or the homol E/homol D arrangement.

Rapid identification of gene defects in protein C deficiency by temperature gradient gel electrophoresis.

Protein C deficiency is an autosomally inherited disorder that is associated with a high risk of recurrent venous thrombosis. The authors have shown that temperature gradient gel electrophoresis (TGGE) is a simple and rapid screening method for the detection of mutations in the protein C gene. Samples from eleven patients with sequence defined point mutations in the promoter region of exon I, and in exons II, III, VII, VIII and IX were analysed by TGGE. In all cases the mutations were readily detected. The exons IV, V and VI were not submissive to TGGE analysis due to amplification difficulties. However, specific computer calculations predict a more general applicability of TGGE for the detection of any mutation in the protein C gene. The presented data establish the usefulness of TGGE as a simple and rapid screening method for the detection of hereditary mutations in the protein C gene.

Influence of N-acetylcysteine on indirect indicators of tissue oxygenation in septic shock patients: results from a prospective, randomized, double-blind study.

OBJECTIVES: Deactivation of endothelium-derived relaxing factor due to an increased oxygen radical load during sepsis may contribute to an impairment in microcirculatory blood flow. We investigated whether treatment with the sulfhydryl donor and oxygen radical scavenger, N-acetylcysteine, would improve whole-body oxygen consumption (VO2), gastric intramucosal pH, and veno-arterial CO2 gradient (veno-arterial PCO2) during septic shock. DESIGN: Prospective, randomized, double-blind study conducted over 2 yrs. SETTING: Septic shock patients admitted to the intensive care unit. PATIENTS: Fifty-eight patients requiring hemodynamic monitoring (radial and pulmonary artery catheters) due to septic shock, were included in this study. All patients were examined within 72 hrs after the onset of sepsis. They were optimally resuscitated by conventional means with volume and inotropic agents, and exhibited stable clinical conditions (hemodynamic values, body temperature, hemoglobin, FIO2). INTERVENTIONS: A gastric tonometer was inserted to measure the gastric intramucosal pH. Subjects randomly received either 150 mg/kg of intravenous N-acetylcysteine or placebo over a 15-min period, then a continuous infusion of 12.5 mg/hr of N-acetylcysteine or placebo over approximately 90 mins. MEASUREMENTS: Infusion measurements were begun 60 mins after the beginning of infusion and lasted approximately 30 mins. The infusion was then discontinued and 2 hrs later the final measurements were taken. MAIN RESULTS: Basic patient characteristics (age, sex, Acute Physiology and Chronic Health Evaluation [APACHE] II scores, Multiple Organ Failure scores) did not differ significantly, nor did pre- and 2-hr postinfusion measurements differ between any of the groups. Thirteen (45%) patients responded (i.e., showed an increase in VO2 > 10%, reaching a mean of 19%) to the N-acetylcysteine infusion. The N-acetylcysteine responders also showed an increase in gastric intramucosal pH, a decrease in veno-arterial PCO2, an increase in oxygen delivery, cardiac index, stroke index, and left ventricular stroke work index, as well as a significant decrease in systemic vascular resistance in comparison to baseline. The N-acetylcysteine nonresponders, as well as the patients in the placebo group, did not show any significant changes in any of these variables. The N-acetylcysteine responders had a higher survival rate (69%) than the non-responders (19%) and were studied earlier after onset of sepsis (37 hrs) than the nonresponders (61 hrs). The only significant difference between the entire N-acetylcysteine group (which included responders plus nonresponders) and the placebo group was an increased VO2 in the entire N-acetylcysteine group during infusion measurements. CONCLUSIONS: N-acetylcysteine provided a transient improvement in tissue oxygenation in about half of the septic shock patients, as indicated by an increase in VO2 and gastric intramucosal pH and a decrease in veno-arterial PCO2. The higher survival rate in the N-acetylcysteine responders and the fact that half of the patients receiving N-acetylcysteine did not respond, suggests that, in some patients, sepsis irreversibly damages the microvasculature to the extent that N-acetylcysteine has no effect. If analyzed by intention to treat, the N-acetylcysteine did not produce effects that were significantly different from the placebo. Whether the N-acetylcysteine challenge was merely diagnostic or whether N-acetylcysteine can be effective in the treatment of sepsis deserves further investigation.

A novel homozygous missense mutation (Val 325-->Ala) in the protein C gene causing neonatal purpura fulminans.

A novel homozygous GTG-->GCG (Val 325-->Ala) substitution was detected in the protein C gene of a newborn causing severe purpura fulminans post partum. In the consanguineous parents and two further infants a heterozygous type 1 protein C deficiency was found. Up to now the heterozygous individuals are clinically unaffected. The mutation co-segregates with the protein C deficiency state. It creates a restriction enzyme (Sac II) cleavage site.

Two genes encoding ribosomal protein L3 of Schizosaccharomyces pombe and their proximal promoter regions.

We have cloned and sequenced two genes, rpl3-1 and rpl3-2, encoding the ribosomal protein L3 of Schizosaccharomyces pombe. The two genes contain an open reading frame encoding 388 amino acids (aa) with a M(r) of 43,808. The aa sequences are identical, except at position 78, where Rpl3-1 displays a valine residue and Rpl3-2 contains isoleucine. The aa sequences show 75% identity to the RPL3 aa sequence from Saccharomyces cerevisiae. S1-nuclease protection analysis revealed that both genes are transcribed. The promoter sequences of the two rpl3 genes are significantly different, but both promoters contain the conserved homol-D element. Transcription starts between 40 and 50 nt downstream from this element.

The CAGTCACA box in the fission yeast Schizosaccharomyces pombe functions like a TATA element and binds a novel factor.

Fourteen ribosomal protein genes from the fission yeast Schizosaccharomyces pombe contain a highly conserved sequence, CAGTCACA, in the proximal promoter. This sequence, which was also conserved in its location, was found where the TATA element usually resides. Deletion and point mutations in the CAGTCACA box reduced the expression of these genes to almost zero and caused aberrant transcriptional start sites. Insertions between this box and the original transcriptional start sites led to new start sites which were the same distance from the CAGTCACA box as the original start sites. The results presented provide evidence that this box, like a TATA sequence, is involved in basal expression and fixing the transcriptional start sites of these genes. Furthermore, the CAGTCACA sequence is the target of a binding protein which appears to be different from the TATA-binding protein.

[Changes in blood coagulation in treatment with ALL-BFM-90 and NHL-BFM-90 protocols]. / Gerinnungsveränderungen bei Behandlung mit den Protokollen ALL-BFM-90 und NHL-BFM-90.

1. Treatment according to the ALL/NHL-BFM 90 protocol I (induction phase) caused multiple and severe coagulation changes in all 14 patients of our study. Glucocorticoids alone made Fibrinogen drop to 148 mg/dl, AT III and Protein C rise to 136% or even 179% respectively. After day 12, immediately following the start of therapy with Coli-Asparaginase (ASP), fibrinogen continued to drop to reach its lowest average value of 46 mg/dl on day 24. Anticoagulant factors like plasminogen (lowest average value: 36%), AT III (47%) and Protein C (93%) dropped abruptly. These alterations were reversed after discontinuation of Glucocorticoids and ASP. During consolidation (protocol II) similar alterations are observed as in protocol I when Glucocorticoids are applied alone. However, after Erwinia-ASP there is no fall in AT III, plasminogen, and Protein C as is observed in protocol I with Coli-ASP. 2. Severe hemorrhages or thromboses are uncommon as compared to the degree of coagulation changes which can be regularly observed. Complications occur more often in girls. Most of them are seen during the 2nd or 3rd week of simultaneous ASP-Glucocorticoid therapy. 3. To avoid twofold alteration of hemostasis it should be considered to apply Glucocorticoids and ASP separately and to replace Coli-ASP by Erwinia-ASP. The efficacy of prophylactic replacement of decreased coagulation factors has not yet been confirmed. Immunologic and infectious side effects have to be taken into consideration. 4. More definite recommendations can be given when each suspected bleeding and/or thrombosis is confirmed by imaging procedures, when it is documented and registered, and when coagulation studies are performed during the critical phase.

Fibrinogen Kiel: a congenital dysfibrinogenaemia with (A alpha-16 Arg----His) substitution characterized by HPLC without prior isolation of fibrinogen.

A congenital fibrinogen variant in a German family is described which has been identified as a substitution of His in position 16 of the A alpha-chain for Arg, manifested over three generations in heterozygous form. The characterization is based on the reaction of the variant fibrinogen with thrombin and reptilase, on the HPLC-chromatographic properties and the amino acid composition of the abnormal fibrinopeptide A. Clinical observations in the affected family members (neither haemorrhagic nor thrombophilic tendencies), the results of routine coagulation tests (normal global clotting tests, prolonged thrombin and thrombin-coagulase time, decreased fibrinogen concentration in functional as opposed to immunological tests), and the autosomal co-dominant modus of inheritance of the fibrinogen variant are all in complete agreement with other reports in the literature concerning the same amino acid exchange. The results of our experiments with fibrinogen Kiel allow no definite conclusion regarding the question of whether it consists of pure homodimers or as of a mixture of homo- and heterodimers.

Interleukin-6 and alpha-2-macroglobulin indicate an acute-phase state in Alzheimer's disease cortices.

Recent studies indicated that the formation of a major constituent of Alzheimer's disease (AD) senile plaques, called beta A4-peptide, does not result from normal processing of its precursor, amyloid precursor protein (APP). Since proteolytic cleavage of APP inside its beta A4 sequence was found to be part of APP processing the formation of the beta A4-peptide seems to be prevented under normal conditions. We considered whether in AD one of the endogenous proteinase inhibitors might interfere with APP processing. After we had recently found that cultured human neuronal cells synthesize the most potent of the known human proteinase inhibitors, alpha-2-macroglobulin (alpha 2M), upon stimulation with the inflammatory mediator interleukin-6 (IL-6) we now investigated whether alpha 2M and IL-6 could be detected in AD brains. Here we report that AD cortical senile plaques display strong alpha 2M and IL-6 immunoreactivity while no such immunoreactivity was found in age-matched control brains. Strong perinuclear alpha 2M immunoreactivity in hippocampal CA1 neurons of Alzheimer's disease brains indicates that neuronal cells are the site of alpha 2M synthesis in AD brains. We did not detect elevated IL-6 or alpha 2M levels in the cerebrospinal fluid of AD patients. Our data indicate that a sequence of immunological events which seem to be restricted to the local cortical environment is part of AD pathology.