Amyand's hernia with acute phlegmonous appendicitis: case report.

Any inguinal hernia containing the vermiform appendix is called Amyand's hernia. Amyand hernias are very rare and even rarer is the association of Amyand hernia with acute appendicitis. Due to the rarity of this entity, it constitutes a challenging case in terms of diagnosis and treatment. The surgical management is not yet standardized and there are no clear guidelines. There are some controversies regarding whether to perform an appendectomy if appendix appears normal or whether mesh can be used for the hernia repair if appendectomy is performed. We describe a case of Amyand hernia in a 90-year old man with acute appendicitis and we review current literature regarding surgical strategy.

Preoperative staging of colorectal cancer using virtual colonoscopy: correlation with surgical results.

OBJECTIVE: The aim of this study was to evaluate the clinical usefulness of computed tomography colonography (CTC) in the preoperative staging in patients with abdominal pain for occlusive colorectal cancer (CRC) and to compare the results of CTC with the surgical ones. PATIENTS AND METHODS: 127 patients with abdominal pain, iron deficiency anemia and occlusive CRC underwent a CTC examination in prone position without intravenous contrast agent and in prone position after administration of intravenous contrast medium. All the patients underwent surgery after CTC. Two radiologists with different experience analyzed the images first independently and then by consensus. They evaluated the location of the lesion, the depth of the invasion of the colon-rectal wall (T stage), lymph node involvement (N stage) and the presence or absence of distant metastasis (M stage). CTC findings were correlated with surgical outcomes. RESULTS: The overall accuracy values for tumour localization according to consensus reading of CTC examinations in comparison to surgical results were 100% (K = 1, p = 0.0001). The overall accuracy values of agreement for T staging of reader 1, reader 2 and consensus reading of CTC examinations in comparison to surgical results were respectively 95.5% (K = 0.876, p = 0.0035), 93.3% (K = 0.858, p = 0.0037) and 97.7% (K = 0.926, p = 0.0014) for ≤ T2; 91.3% (K = 0.839, p = 0.0027), 88.3% (K = 0.817, p = 0.0031), and 92.9% (K = 0.894, p = 0.0025) for T3; 89.6% (K = 0.825, p = 0.0037), 86.2% (K = 0.837, p = 0.0032) and 89.6% (K = 0.821, p = 0.0023) for T4. The overall accuracy values for N staging for reader 1, reader 2 and consensus reading was 90.2% (K = 0.865, p = 0.0029). The overall accuracy values for M staging of reader 1, reader 2 and consensus reading was 92% (K = 0.875, p = 0.0019). CONCLUSIONS: CTC with is a very useful tool for accurate pre-treatment staging and localization of occlusive CRC.

Static and dynamic MR imaging in the evaluation of temporomandibular disorders.

OBJECTIVE: The aim of this study is to prove if dynamic HASTE (half-Fourier acquisition single-shot turbo spin-echo) sequences can be used in the diagnosis of internal derangement disorders of temporomandibular joint (TMJ) as an alternative to static proton density (PD) weighted/turbo spin echo (TSE) T2-weighted sequences which are considered up to now as the gold standard in the evaluation of TMJ disorders (TMDs). PATIENTS AND METHODS: 194 patients for a total of 388 TMJs were examined with a 1.5 Tesla field strength superconducting magnet. Sagittal static PD-weighted/TSE T2-weighted and dynamic HASTE sequences have been used. Three experts in the field of oral radiology (specialist A, B and C) independently and blinded to clinical symptoms and any treatment, assessed the articular disc position in each TMJ (rated as normal or disc displacement with reduction or disc displacement without reduction). The agreement between static and dynamic images and between the three different specialists in the assessment of the articular disc position was evaluated using kappa statistic. RESULTS: The agreement between static and dynamic images is: for specialist A, K = 0.862; for specialist B, K = 0.870 and for specialist C, K = 0.862. CONCLUSIONS: Since there is no complete agreement between these two MR techniques, dynamic sequences can not be used as a reliable alternative to static sequences in the evaluation of internal derangement disorders of TMJ.

Primary structure and transcription analysis of a laccase-encoding gene from the basidiomycete Trametes trogii.

A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201. This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide. Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and Trametes C30 (98% similarity). The expression of lcc1 was analysed under different growth conditions; transcription of this gene was enhanced by the addition of organic nitrogen to the medium. The level of lcc1 transcription was higher when T. trogii was grown on synthetic medium supplemented with yeast extract rather than mycological peptone or tryptone. The transcription data were in agreement with total laccase activity measured in the supernatant and suggested that laccase production and lcc1 transcription are coordinately regulated in this organism. The lcc1 cDNA was expressed in the methylotrophic yeast Pichia pastoris and the detection of laccase activity indicated that this cDNA encodes a laccase.

Recombinant wheat antifungal PR4 proteins expressed in Escherichia coli.

PR proteins are soluble and host-coded molecules with antifungal activity induced by a variety of agents. Wheat contains several PR proteins and among them are those of the class 4 coded wheatwin1 and wheatwin2; the two native proteins have been isolated from wheat kernel and the coding cDNA clones have been recently characterized. Herein, we report the expression of recombinant wheatwin1 and wheatwin2 in Escherichia coli-insoluble fractions; a new protocol for the purification in high yields and correct processing of the two proteins was developed. The recombinant proteins have molecular weights identical to that of the native proteins, indicating that the removal of the N-terminal methionine and cyclization of glutamine to pyroglutamate was complete. Both recombinant proteins inhibited in vitro the growth of Fusarium culmorum exhibiting antifungal properties similar to those of the native proteins.

Isolation and amino acid sequence of two new PR-4 proteins from wheat.

We have purified and characterized two new pathogenesis-related (PR) proteins from wheat belonging to the PR-4 family. We named the proteins wheatwin3 and wheatwin4 in analogy with the previously characterized wheatwin1 and wheatwin2. Their isoelectric points were 7.1 and 8.4, respectively. We determined the complete amino acid sequence of both proteins by a rapid approach based on the knowledge of the primary structures of the homologous wheatwin1 and wheatwin2. Wheatwin3 differs from wheatwin1 in one substitution at position 88, while wheatwin4 differs from wheatwin2 in one substitution at position 78. The secondary structure and solvent accessibility of these residues were determined on the three-dimensional model of wheatwinl. Residue 88 was very accessible and was located in a flexible region. Preliminary results indicate that, like wheatwin1 and wheatwin2, wheatwin3 and wheatwin4 have antifungal activity.

A basic peroxidase from wheat kernel with antifungal activity.

A basic heme-peroxidase (WP1) was purified to homogeneity from wheat (Triticum aestivum) kernels. The protein was not glycosylated and exhibited a molecular mass of 36 kDa and a pI of 8.0. The N-terminal amino acid sequence revealed a very high similarity with a wheat flour peroxidase allergen associated with baker's asthma. WPI showed indole-3-acetic acid oxidase activity in the presence of Mn2+ and phenolic cofactors. Antifungal assays performed in vitro towards phytopathogenic fungi indicated that WP1 was active in inhibiting germ tube elongation. This first report on antifungal properties of a heme-peroxidase gives experimental support to the idea that peroxidases play a defensive role against invading pathogens.

Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii.

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXAlb) whose sequences are known.

Contactless optoelectronic technique for monitoring epoxy cure.

We describe a novel noninvasive optical technique to monitor the refractive-index variation in an epoxy-based resin that is due to the polymerization process. This kind of resin is widely used in polymer matrix composites. It is well known that the process of fabricating a thermoset-based composite involves mass and heat transfer coupled with irreversible chemical reactions that induce physical changes. To improve the quality and the reliability of these materials, monitoring the cure and optimization of the manufacturing process are of key importance. We discuss the basic operating principles of an optical system based on angle deflection measurements and present typical cure-monitoring results obtained from optical characterization. The method provides a flexible, high-sensitivity, material-independent, low-cost, noninvasive tool for monitoring real-time refractive-index variation.

Probing the modelled structure of wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures.

We set up a method to get rapid information on the three-dimensional structure of peptide and proteins of known sequence. Both native and alkylated polypeptide is hydrolyzed with a number of proteases at different digestion times and the resulting mixtures are compared by HPLC analysis to establish the differences in the hydrolysis pathways of the folded and unfolded molecule. Then, the unfractionated digestion mixtures of the native polypeptide are submitted to automatic sequence analysis to identify the hydrolysis sites. The sequence of each fragment present in the mixtures is reconstructed and its amount determined by quantitative data of the sequence analyses. We used this approach to determine the amino acid surface accessibility of wheatwin1, a pathogenesis-related protein from wheat, and constructed a predictive three-dimensional model based on the knowledge of the tertiary structure of barwin, a highly homologous protein from barley. The procedure allowed us to quickly identify and quantify the hydrolysis at the susceptible bonds which could be classified as exposed, partially hidden, or inaccessible. The results were useful to evidentiate and discuss concordances and differences between experimental and model predicted accessibilities of amino acid residues. Proteins 1999;36:192-204.

Isolation and characterisation of wheat cDNA clones encoding PR4 proteins.

Two cDNA clones encoding the previously characterised PR4 proteins wheatwin 1 and wheatwin2 from wheat (Triticum aestivum cv. S. Pastore) have been identified and named wPR4a and wPR4b, respectively. The clones have been isolated by screening a cDNA library with a specific cDNA probe obtained by RT-PCR. The wPR4a and wPR4b cDNAs contain open reading frames of 441 and 447 bp that encode for wheatwin1 and wheatwin2, respectively.

Laccase from the white-rot fungus Trametes trogii.

The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70 kDa, acidic isoelectric point and N-terminal sequence homologous to that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant molecules may be converted into substrates by introducing, in the correct position, o- or p-orienting ring-activating groups.

A computer program to compare sequence fingerprints of homologous proteins for the rapid assessment of their primary structure differences.

We have developed a computer program for the rapid assessment of the primary structure differences between a protein of unknown sequence and a homologous known protein. Both proteins are reduced, alkylated, and digested with the same hydrolytic agent. The unfractionated peptide mixtures are submitted to automatic sequence analysis. Based on the knowledge of the reference sequence, the program utilizes the analysis data to identify all the potential peptides present in the two mixtures, determining their primary structure, homology degree, and molecular weight calculated both as integer MH+ and average mass variables. These fingerprints allow the user to easily identify the structural differences between the two proteins and clarify possible doubts by a mass spectrometric analysis of the two mixtures. In order to verify the utility of the program, we provide an application example using the already reported data of two homologous proteins.

Assignment of protein disulphides by a computer method using mass spectrometric data.

We designed a computer program for the assignment of protein disulphides using mass spectrometric data. All the theoretical linear peptides containing from one to three cysteines are generated on the basis of the protein sequence. Their combination ways are determined in order to create all the possible disulphide-bridged fragments containing from two to six cysteines and to calculate their molecular weight. One, two and three S-S bridges per linked fragment were considered, taking into account the possibility that the fragments contain unabridged residues. The mass data obtained from the spectral analysis of peptide mixtures of the digested protein are then associated to the fitting structures of disulphide-bridged peptides, giving rise to the primary output. This output can then be screened by using information on the specificity of the proteolytic agent(s) used and/or any further mass data provided by Edman degradation and/or carboxypeptidase treatment of the peptide mixtures. The need for such a computer aid is discussed and examples of application are given.

Determination of the primary structure of homologous proteins by sequence analysis of peptide mixtures.

We propose a rapid method to determine the primary structure of a protein knowing the sequence of a homologous protein. The method consists in submitting both the reduced and alkylated proteins to an enzymatic or chemical hydrolysis and performing the sequence analysis of the peptide mixtures. The assessment of the unknown sequence and the degree of identify of the two proteins are reached by comparing the two sequence analyses. The sequences of all the possible peptides present in the two mixtures are reconstructed and the differences in the two sequences are determined. If necessary, the differences can be confirmed by performing a mass spectrometric analysis of the two mixtures. We used this procedure on two homologous proteins of known sequence to furnish an application example of the method.

An algorithm to analyse the hydrolysis pathway of peptides and proteins by sequence analyses of unfractionated digestion mixtures.

We have designed and implemented on a personal computer a program for identifying and quantifying the fragments present in a peptide mixture obtained by hydrolysing a polypeptide of known sequence using digesting agents. The qualitative data utilized by the main algorithm consist of the target sequence of the intact molecule and the amino acid residues identified at each step of the automatic sequence analysis of the unfractionated digestion mixture. In this way, the sequence of each fragment present in the mixture is quickly reconstructed. Furthermore, if the quantitative data of the amino acid residues identified at each step of the sequence analysis are utilized, the program will correlate the sequence of each fragment to its amount. We furnish an example of the application intended for the rapid identification and characterization of the extracellular proteinases produced by a basidiomycete fungus, utilizing the bovine insulin beta-chain as target substrate. A variety of uses for the method are discussed.

Characterization of extracellular proteases from Trametes trogii.

The peptidase activities excreted in culture broths of Trametes trogii mycelium have been identified by determining the digestion pathway of various peptides. Insulin beta-chain (30 residues), procasomorphin (10 residues) and two peptides of five residues (proctolin and thymosin alpha 1 fragment 23-27) were utilized as model substrates. Aminopeptidase, carboxypeptidase, endopeptidase and dipeptidyl aminopeptidase activities were revealed and information on their specificity was deduced. Preliminary data on the pH-dependent activity of the peptidases were also obtained by sequence analysis of the fragment mixtures produced at different pH values.

Structural and antifungal properties of a pathogenesis-related protein from wheat kernel.

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifugal activity toward the wide-host-range pathogen Botrytis cinerea and the wheat-specific pathogenic fungi of wheat Fusarium culmorum and Fusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.

An algorithm to determine protein sequence alignment by utilizing data obtained from a peptide mixture and individual peptides.

With the aim of limiting peptide purification steps and unambiguously ascertaining protein sequences, we have designed and implemented on a personal computer an algorithm to determine sequence alignment by utilizing data obtained from automatic Edman degradation performed on a single peptide mixture and individual peptides. The protein under study is digested by two different hydrolysis methods and fragments are just isolated from one mixture and sequenced, while the second mixture is submitted unfractionated to sequence analysis. The algorithm provides for the exact alignment of the individual peptides using the mixture data for the overlapping. We report an example of application of this approach by utilizing experimental data obtained from a protein of known sequence.

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm.

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion with Staphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.