The ABRF Edman Sequencing Research Group 2008 Study: investigation into homopolymeric amino acid N-terminal sequence tags and their effects on automated Edman degradation.

The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.

Effects of sevoflurane and propofol on left ventricular diastolic function in patients with pre-existing diastolic dysfunction.

BACKGROUND: The effects of anaesthetics on left ventricular (LV) diastolic function in patients with pre-existing diastolic dysfunction are not well known. We hypothesized that propofol but not sevoflurane will worsen the pre-existing LV diastolic dysfunction. METHODS: Of 24 randomized patients, 23 fulfilled the predefined echocardiographic criterion for diastolic dysfunction. They received general anaesthesia with sevoflurane 1 MAC (n=12) or propofol 4 mug ml(-1) (n=11). Echocardiographic examinations were performed at baseline and in anaesthetized patients under spontaneous breathing and under positive pressure ventilation. Analysis focused on peak early diastolic velocity of the mitral annulus (E(a)). RESULTS: During spontaneous breathing, E(a) was higher in the sevoflurane than in the propofol group [mean (95% CI) 7.0 (5.9-8.1) vs 5.5 (4.7-6.3) cm s(-1); P<0.05], reflecting an increase of E(a) from baseline only in the sevoflurane group (P<0.01). Haemodynamic findings were similar in both groups, but the end-tidal carbon dioxide content was more elevated in the propofol group (P<0.01). During positive pressure ventilation, E(a) was similarly low in the sevoflurane and propofol groups [5.3 (4.2-6.3) and 4.4 (3.6-5.2) cm s(-1), respectively]. CONCLUSIONS: During spontaneous breathing, early diastolic function improved in the sevoflurane but not in the propofol group. However, during positive pressure ventilation and balanced anaesthesia, there was no evidence of different effects caused by the two anaesthetics.

Unnecessary interruptions of cardiac massage during simulated cardiac arrests.

BACKGROUND AND OBJECTIVE: Cardiopulmonary resuscitation should not be interrupted until the return of spontaneous circulation or the decision to withhold further treatment. There are no data on how consistent in-hospital cardiopulmonary resuscitation is performed. Accordingly, the aim of the present study was to identify length and type of unnecessary interruptions in simulated cardiac arrests. METHODS: The study was carried out in a patient simulator. A scenario of cardiac arrest due to ventricular fibrillation was used. Resuscitation teams consisted of three nurses, a resident and a staff physician. Using videotapes recorded during simulations, the activities of the teams were coded in 5-s intervals. Unnecessary interruptions were defined as any interruptions in cardiac massage of 10 s or more that were not caused by defibrillation or endotracheal intubation. RESULTS: Twelve teams were studied. The total time of possible cardiac massage was 414 +/- 125 s. In each team at least one unnecessary interruption occurred (range 1-5). Interruptions mounted up to 65 +/- 40 s (range 20-155) or 16 +/- 10% (range 5-41) of the total time of possible cardiac massage. Failure to swiftly resume cardiac massage after an unsuccessful defibrillation accounted for 14 of 39 episodes and for 44 +/- 40% of the time of unnecessary interruptions. The debriefings revealed that participants had generally not noticed the unnecessary interruptions during the simulation. CONCLUSIONS: The present study identified a significant amount of unnecessary interruptions in cardiac massage. These interruptions were not noticed by the health-care workers involved.

Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1.

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1. METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress. CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.

Effects of halothane, sevoflurane and propofol on left ventricular diastolic function in humans during spontaneous and mechanical ventilation.

BACKGROUND: There is limited knowledge of the effects of anaesthetics on left ventricular (LV) diastolic function in humans. Our aim was to evaluate these effects in humans free from cardiovascular disease. METHODS: Sixty patients (aged 18-47 yr) who had no history or signs of cardiovascular disease were randomized to receive general anaesthesia with halothane, sevoflurane or propofol. Echocardiography was performed at baseline and during spontaneous respiration at 1 minimum alveolar concentration (MAC) of the inhalational agents or propofol 4 microg ml(-1) (step 1), and repeated during positive-pressure ventilation with 1 and 1.5 MAC of the inhalational agents or with propofol 4 and 6 microg ml(-1) (steps 2a and 2b). Analysis of echocardiographic measurements focused on heart rate corrected isovolumic relaxation time (IVRT(c)) and early diastolic peak velocity of the lateral mitral annulus (E(a)). RESULTS: IVRT(c) decreased from baseline to step 1 in the halothane group (82 [95% CI, 76-88] ms and 74 [95% CI, 68-80] ms respectively; P=0.02), remained stable in the sevoflurane group (78 [95% CI, 72-83] ms and 73 [95% CI, 67-81] ms; n.s.) and increased in the propofol group (80 [95% CI, 74-86] ms and 92 [95% CI, 84-102] ms; P=0.02). E(a) decreased in the propofol group only (18.8 [95% CI, 16.5-19.9] cm s(-1) and 16.0 [95% CI, 14.9-17.9] cm s(-1); P=0.003). From step 2a to step 2b, IVRT(c) increased further in the propofol group (109 [95% CI, 99-121] ms and 119 [95% CI, 99-135] ms; P=0.04) but remained stable in the other two groups. E(a) did not change from step 2a to step 2b. CONCLUSIONS: Halothane and sevoflurane did not impair LV relaxation, whereas propofol caused a mild impairment. However, the impairment by propofol was of a magnitude that is unlikely to cause clinical diastolic dysfunction.

Adverse cardiac events in ICU patients with presumptive antidepressant overdose.

BACKGROUND: Antidepressants account for most poison-related admissions to intensive care units. In selected patients with confirmed cyclic antidepressant intoxication a QRS interval <0.1 s in the ECG limb leads during the first six hours excludes adverse cardiac events. However, the incidence of cardiac events and the value of ECG criteria have never been assessed prospectively on patients with presumed antidepressant overdose. AIM: To assess ingested drugs, adverse cardiac events, and ECG findings in ICU patients with a presumptive diagnosis of antidepressant overdose. METHODS: 103 consecutive patients with a presumptive diagnosis of antidepressant overdose were enrolled and prospectively followed. Outcome criteria were arrhythmias, mortality, and duration of the ICU stay. RESULTS: Mixed intoxication was identified in 66 (64%) patients. Tricyclic antidepressants were found in 88 (85%), and serotonin-reuptake inhibitors in 25 (24%) patients. Mean APACHE II score was 9.5 (SD +/- 6.0). Arrhythmias affected 15 (15%) and cardiopulmonary resuscitation was performed on 4 (4%) patients. Three patients (3%) died in the ICU. Median duration of the ICU stay was 1 day (12 hours to 6 days). Adverse cardiac events affected patients with normal and prolonged QRS interval at study entry. CONCLUSIONS: Mixed intoxication is present in most ICU patients with suspected antidepressant overdose. There is a considerable risk for adverse cardiac events, even in the presence of normal ECG recordings within the first six hours after hospital admission.

[Diagnosis and risk stratification of acute coronary syndrome in general practice and in the hospital]. / Diagnostik und Risikostratifizierung des akuten Koronarsyndroms beim Hausarzt und im Spital.

Acute coronary syndromes usually present as acute chest pain but a manifestation with atypical symptoms or entirely without symptoms makes rapid diagnosis of this potentially lethal syndrome difficult and may lead to delay of the appropriate therapy. The role of the practitioner in this situation is complex since early exclusion of an acute coronary syndrome may be very difficult with the means available in a private practice; on the other hand the early hours which are critical for early therapy may be lost in a private practice. In the early phase of an acute coronary syndrome, diagnoses and risk stratification are based primarily on history, clinical presentation, ECG and biological markers. In the hospital, the time course of these parameters, functional tests for diagnosis and risk stratification as well as determination of left ventricular function and coronary angiography have additional relevance. Recent developments are the availability of highly sensitive and specific biological markers (Troponins), the new classification in acute coronary syndrome with and without ST-elevation and the identification of inflammatory processes in the coronary artery, which have added importantly to our understanding of the acute coronary syndrome. The availability of very early therapy and the possibilities of telemedicine have the potential to influence the pre-hospital diagnostic strategy and management of acute coronary syndromes.

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

Two metal-binding peptides from the insect Orchesella cincta (Collembola) as a result of metallothionein cleavage.

Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.

Regulation of human flap endonuclease-1 activity by acetylation through the transcriptional coactivator p300.

We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1's DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.

Interaction between GABA(A) receptor beta subunits and the multifunctional protein gC1q-R.

gamma-Aminobutyric acid type A (GABA(A)) receptors were immunopurified from bovine brain using a monoclonal antibody directed against the alpha1 subunit. Of the several proteins that copurified, a 34-kDa protein was analyzed further. After enrichment and tryptic proteolysis, the resulting fragments were sequenced, and the protein was identified as gC1q-R. Using anti-gC1q-R and anti-GABA(A) receptor antibodies, mutual coimmunoprecipitation could be demonstrated from solubilized rat brain membranes. The stability of this interaction was estimated to be very high. Using the yeast two-hybrid system, various GABA(A) receptor subunit intracellular loop constructs were tested for an interaction with gC1q-R. All beta subunits, but not alpha 1 and gamma 2 subunits, were found to bind to gC1q-R. NH(2)- and COOH-terminally truncated beta 2 subunit loops were used to find the region responsible for the interaction with gC1q-R. A stretch of 15 amino acids containing 7 positively charged residues was identified (amino acids 399--413). This region contains residue Ser-410, which is a protein kinase substrate, and it is known that phosphorylation of this residue leads to an alteration in receptor activity. Localization studies suggested a predominantly intracellular localization. Our observations therefore suggest a tight interaction between gC1q-R and the GABA(A) receptor which might be involved in receptor biosynthesis or modulation of the mature function.

Molecular evolution of the ocnus and janus genes in the Drosophila melanogaster species subgroup.

Genes involved in male fertility are potential targets for sexual selection, and their evolution may play a role in reproductive isolation and speciation. Here we describe a new Drosophila melanogaster gene, ocnus (ocn), that encodes a protein abundant in testes nuclear extracts. RT-PCR indicates that ocn transcription is limited to males and is specific to testes. ocn shares homology with another testis-specific gene, janusB (janB), and is located just distal to janB on chromosome 3. The two genes also share homology with the adjacent janusA (janA) gene, suggesting that multiple duplication events have occurred within this region of the genome. We cloned and sequenced these three genes from species of the D. melanogaster species subgroup. Phylogenetic analysis based on protein-encoding sequences predicts a duplication pattern of janA --> janA janB --> janA janB ocn, with the latter event occurring after the divergence of the D. melanogaster and Drosophila obscura species groups. We found significant heterogeneity in the rates of evolution among the three genes within the D. melanogaster species subgroup as measured by the ratio of nonsynonymous to synonymous substitutions, suggesting that diversification of gene function followed each duplication event and that each gene evolved under different selective constraints. All three genes showed faster rates of evolution than genes encoding proteins with metabolic function. These results are consistent with previous studies that have detected an increased rate of evolution in genes with reproductive function.

Isolation and characterization of four bactericidal domains in the bovine beta-lactoglobulin.

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.

Rapid identification of substrates for novel proteases using a combinatorial peptide library.

Fluorogenic substrates for assaying novel proteolytic enzymes could be rapidly identified using an easy, solid-phase combinatorial assay technology. The methodology was validated with leader peptidase of Escherichia coli using a subset of an intramolecularly quenched fluorogenic peptide library. The technique was extended toward the discovery of substrates for a new aspartic protease of pharmaceutical relevance (human napsin A). We demonstrated for the first time known to us that potent fluorogenic substrates can be discovered using extracts of cells expressing recombinant enzyme to screen the peptide library. The straightforward and rapid optimization of protease substrates greatly facilitates the drug discovery process by speeding up the development of high throughput screening assays and thus helps more effective exploitation of the enormous body of information and chemical structures emerging from genomics and combinatorial chemistry technologies.

Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava).

Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3' and 5' rapid amplification of cDNA ends. The sequence shows approximately 60-70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-beta-D-thiogalactopyranoside and delta-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was approximately 2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.

Transthoracic echocardiography for perioperative haemodynamic monitoring.

Transoesophageal echocardiography (TOE) is valuable for perioperative monitoring in patients at risk from haemodynamic disturbance. However, its use is not practicable in patients undergoing surgical procedures under regional anaesthesia. We describe two cases showing that transthoracic echocardiography (TTE) has the same advantages as TOE and thus may be valuable for monitoring awake patients. TTE should be considered when extended perioperative haemodynamic monitoring is needed but TOE is not possible.

Design of a new surgical approach for ventricular remodeling to relieve ischemic mitral regurgitation: insights from 3-dimensional echocardiography.

BACKGROUND: Mechanistic insights from 3D echocardiography (echo) can guide therapy. In particular, ischemic mitral regurgitation (MR) is difficult to repair, often persisting despite annular reduction. We hypothesized that (1) in a chronic infarct model of progressive MR, regurgitation parallels 3D changes in the geometry of mitral leaflet attachments, causing increased leaflet tethering and restricting closure; therefore, (2) MR can be reduced by restoring tethering geometry toward normal, using a new ventricular remodeling approach based on 3D echo findings. METHODS AND RESULTS: We studied 10 sheep by 3D echo just after circumflex marginal ligation and 8 weeks later. MR, at first absent, became moderate as the left ventricle (LV) dilated and the papillary muscles shifted posteriorly and mediolaterally, increasing the leaflet tethering distance from papillary muscle tips to the anterior mitral annulus (P<0.0001). To counteract these shifts, the LV was remodeled by plication of the infarct region to reduce myocardial bulging, without muscle excision or cardiopulmonary bypass. Immediately and up to 2 months after plication, MR was reduced to trace-to-mild as tethering distance was decreased (P<0.0001). LV ejection fraction, global LV end-systolic volume, and mitral annular area were relatively unchanged. By multiple regression, the only independent predictor of MR was tethering distance (r(2)=0.81). CONCLUSIONS: Ischemic MR in this model relates strongly to changes in 3D mitral leaflet attachment geometry. These insights from quantitative 3D echo allowed us to design an effective LV remodeling approach to reduce MR by relieving tethering.

Metallothioneins in terrestrial invertebrates: structural aspects, biological significance and implications for their use as biomarkers.

During the last few years the subject of metallothioneins (MTs) in terrestrial invertebrates has gained increasing attention. One reason for this may be that terrestrial invertebrates provide new insights into the biological diversity of MTs, with the potential of discovering alternative models of structural and functional relationships. Four groups of terrestrial invertebrates have been studied in detail, namely nematodes, insects, snails and earthworms, with the present article focusing on MTs from the latter two groups. Snails are interesting because they possess distinct MT isoforms involved in different metal-specific tasks. In the Roman snail (Helix pomatia), for example, one isoform is predominantly expressed in the midgut gland, accounting for the accumulation, binding and detoxification of cadmium. The second isoform, which is present in the snail's mantle, is substantially different regarding its primary structure. Furthermore, it binds nearly exclusively copper, and thus is probably involved in the homeostatic regulation of essential trace elements. Earthworm MTs merit our attention because of another peculiarity: they seem to be much more unstable than snail MTs, particularly under conventional conditions of preparation. The cDNA of the brandling worm (Eisenia foetida), for instance, codes for a putative MT, which is about twice the size of the actual protein. The isolated MT peptide binds four Cd2+ ions and represents a one-domain MT entity that is stable and functional in vitro. This strongly suggests that earthworm MTs are either posttranslationally modified, or subjected to enzymatic cleavage during preparation. Both snail and earthworm MTs are inducible by metal exposure, especially by cadmium, thus supporting the idea of using them as potential biomarkers for environmental metal pollution. Whilst snail MTs have already been tested in this respect with some success, the use of earthworm MTs as biomarkers still remains to be evaluated, especially in the light of the unknown significance of their posttranslational instability.

Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism.

Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.

The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase.

Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned. Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues. The H. pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies. Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges. HcpA is a beta-lactamase. It slowly hydrolyzes 6-aminopenicillinic acid and 7-aminocephalosporanic acid (ACA) derivatives. The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives. The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators. We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan. They might also be responsible for amoxicillin resistance of certain H. pylori strains.