Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase.

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.

Lessons from hereditary pancreatitis.

For decades there has been slow progress in understanding pancreatic diseases, particularly acute and chronic pancreatitis. As a result, there were no significant advances in the management of these patients. Treatment was mostly directed towards symptomatic relief and management of complications. A simple clinical observation that multiple members of a large family are affected by acute and chronic pancreatitis, some at very young age and in the absence of any alcohol use, led physician-scientists of the Midwest Multicenter Pancreatic Study Group (investigators from the University of Cincinnati, University of Kentucky, and University of Pittsburgh) to investigate the genetic basis of hereditary pancreatitis. Using information from the human genome project, the hereditary pancreatitis gene was identified as the cationic trypsinogen gene (protease serine 1, PRSS1). This discovery has led to the identification of a number of other genes and their products playing role in the pathogenesis of acute and chronic pancreatitis. In the emerging picture of pathogenesis of acute and chronic pancreatitis, trypsin appears to play a central role. This newly acquired knowledge is setting the stage for new preventive and management strategies for hereditary and sporadic acute and chronic pancreatitis.

Recurrent acute pancreatitis: an algorithmic approach to identification and elimination of inciting factors.

Recurrent acute pancreatitis represents a challenging clinical problem associated with significant morbidity, impairment in quality of life, and expense. If unchecked, recurrent episodes of acute pancreatitis may lead to chronic pancreatitis. In this work we have combined the opinion of experts in pancreatology and an extensive review of the literature to develop a logical algorithm that facilitates the stepwise identification and elimination of inciting factors using current technology. The approach taken in recurrent acute pancreatitis is clearly dependent on adequate and appropriate evaluation and treatment of the patient with an initial episode of acute pancreatitis. Future advances in the treatment of these patients will almost certainly depend on improved imaging modalities, prospective clinical trials assessing the efficacy of endoscopic and surgical intervention, a better understanding of mutations and pathophysiologic mechanisms responsible for recurrent acute pancreatitis, and the development of novel, effective preventive and therapeutic strategies.

Circular dichroism of host-guest complexes of achiral pyridino- and phenazino-18-crown-6 ligands with the enantiomers of chiral aralkyl ammonium salts.

Circular dichroism (CD) spectroscopy was used for distinguishing different types of chiral interactions in host-guest complexes of achiral pyridino- and phenazino-18-crown-6 ligands with chiral aralkyl ammonium salts. The general feature of the CD spectra of many homochiral (e.g., (R,R)-host and (R)-guest) and heterochiral (e.g., (R,R)-host and (S)-guest) alpha-(1-naphthyl)ethylamine hydrogenperchlorate salt (NEA) complexes with chiral pyridino- and phenazino-18-crown-6 hosts is exciton interaction. The most interesting example is the coupling of the transitions of the chiral guest NEA with the energetically close transitions of the achiral phenazino-18-crown-6 host 6. The CD spectrum of the complex is predominated by exciton coupling between the (1)B(b) transition of the chiral guest and the (1)B(b) transition of the achiral host. The redshifted intense spectra of the complexes of (R)- or (S)-1-phenylethylamine hydrogenperchlorate salt (PEA) with the achiral diester-pyridino-18-crown-6 host 4 are indicative of merging the pi electron systems into one joint charge transfer chromophore. The appearance of weak bands with alternating sign in the spectrum of PEA complexes of the achiral "parent" pyridino-18-crown-6 host (1) indicates the presence of two or more conformers. The CD spectra of the complexes of achiral phenazino-18-crown-6 host 6 with PEA are also determined by pi-pi interaction. In addition to charge transfer bands, CD bands are also induced in the long-wavelength spectral region of the achiral host. The weak pi-pi interaction between the achiral phenazino-18-crown-6 host and methyl phenylglycinate hydrogenperchlorate (PGMA) or methyl phenylalaninate hydrogenperchlorate (PAMA) does not result in a definite spectral effect in the (1)L(a) region of the spectrum of the chiral guest, but its existence is proven by the weak CD bands induced in the long-wavelength spectral region of the achiral host.

Synthetic porcine secretin is highly accurate in pancreatic function testing in individuals with chronic pancreatitis.

The secretin stimulation test is the most sensitive and specific test for pancreatic function. It is usually performed using biologically derived porcine secretin. Several shortages of biologic porcine secretin have occurred in the past few years. The aim of this study was to compare synthetic porcine secretin to biologic porcine secretin in pancreatic function testing in subjects with chronic pancreatitis. Twelve patients with a previously abnormal secretin stimulation test were enrolled. After an overnight fast, each patient underwent a secretin stimulation test on 2 consecutive days using 1 CU/kg biologic porcine secretin or 0.2 [microg/kg synthetic porcine secretin in a randomized fashion. The peak bicarbonate concentration in duodenal juice was used as a measure of pancreatic function. The peak bicarbonate concentration (mean +/- SD) obtained by using biologic porcine secretin and synthetic porcine secretin were 70 +/- 25 mEq/L and 68 +/- 31 mEq/L, respectively (p = 0.58, paired t test; R = 0.964). The accuracy of synthetic porcine secretin in diagnosing pancreatic insufficiency was 100% when compared with biologic porcine secretin. We conclude that synthetic porcine secretin is highly accurate and safe in pancreatic function testing. The 100% purity, excellent diagnostic accuracy, and ready availability make synthetic porcine secretin an attractive choice for secretin stimulation testing.

Diagnosis and staging of islet cell tumors of the pancreas.

Pancreatic endocrine tumors arise from the amine precursor uptake and decarboxylation (APUD) cells of the pancreas and behave in a different fashion both biologically and clinically from pancreatic adenocarcinoma. Gastrinomas and insulinomas are the two most common pancreatic endocrine tumors. Unlike pancreatic adenocarcinoma, in which tumor stage, resectability, and prognosis are determined by the tumor, nodes, and metastasis (TNM) classification, the prognosis of pancreatic endocrine tumors is determined by the presence of liver but not regional lymph node metastasis. This review focuses predominantly on the different diagnostic tools available to the clinician and the relative merits of each modality. The sensitivities of computed tomography, magnetic resonance imaging, somatostatin receptor scintigraphy, endoscopic ultrasound, and angiography with venous sampling for diagnosing islet cell tumors are compared. A diagnostic algorithm for the management of these tumors is provided at the end of the discussion.

Enteral nutrition in clinical practice.

Enteral nutrition has, at long last, found its place in the modulation of disease. Because of its importance in terms of both anabolic and catabolic processes, today's clinician must have a working knowledge of the types of enteral formulations, their delivery and the therapeutic considerations (particularly concomitant medications) that impact on the safety and efficacy of enteral nutrition. The advantages and disadvantages of this therapeutic intervention must be carefully weighed by the clinician, in concert with sound medical principles. Despite the widespread belief that enteral nutrition is superior to parenteral nutrition in humans, data does suggest that there is little difference between the two. Also, associated costs of enteral nutrition in contrast to parenteral nutrition need to be reappraised based on more invasive enteral access and falling parenteral nutrition prices. Although the enteral route is presumed to be the best feeding modality, the clinician must be ever vigilant about the shortcomings of using the gut, especially in the setting of severe inflammation, stenosis or sepsis. The best feeding modality, then, must blend a knowledge of the patients' anatomy, physiology, and disease with considerations of enteral access, timing of delivery, complications, and a myriad of other therapeutic variables (to include concurrent medication administration) that impact on the enteral feeding regimen. This article reviews the basic principles of enteral nutrition in clinical practice. It describes nutritional assessment, routes of administration, selection of feeding formulas based on nutritional needs, interactions with medications, as well as possible complications of enteral feeding.

Pancreatic duct stenting in benign pancreatic disease.

The ability to place endoscopic stents into the pancreatic duct has led to a dramatic increase in stent therapy for benign pancreatic diseases, particularly chronic pancreatitis and pancreas divisum. The overall effectiveness of this therapy remains unknown. This article critically reviews the available literature with a focus on patient selection, efficacy, and risk. The risk of pancreatic duct stenting is only now beginning to be appreciated, and clinicians must understand not only the potential effectiveness of pancreatic duct stenting but also the magnitude of the potential risk when considering this therapy.

[Changes in the cytokine concentration during immunotherapy of superficial bladder tumors]. / Vizelet cytokinek koncentrációjának változása felületes hólyagdaganatok immunterápiája során.

A growing number of data support the importance of urinary cytokines in the BCG immunotherapy of superficial bladder tumours. To investigate kinetics and stability, urinary levels of IL-8, IL-2 and IL-6 cytokines after BCG treatment, were determined. Significant elevation in the level of IL-8 was established immediately following the first BCG instillation and it reached its highest value 4-6 hours after the treatment. During the first three weeks of the treatment and follow-up period IL-8 peaked significantly earlier than IL-2 or IL-6. Its early appearance associated with high stability makes IL-8 a good candidate for being considered as an effective agent with high predictive value in BCG immunotherapy.

Role of interleukin-8 in onset of the immune response in intravesical BCG therapy for superficial bladder cancer.

In intravesical therapy for superficial bladder carcinoma urothelial cells may, through the production of cytokines, contribute to the bacillus Calmette-Guerin (BCG)-induced local immunological reaction and associated antitumor efficacy. The aim of this study was to investigate such a role for the neutrophil-attracting cytokine interleukin-8 (IL-8). The appearance of IL-8 in patients urine after BCG therapy was compared with BCG-induced IL-6 and IL-2 and the stability of IL-8 in urine was tested. Compared to IL-6 and IL-2, a rapid induction of IL-8 was observed, occurring after the first BCG instillation. Urinary IL-8 was highly stable, even after 24 h incubation at 37 degrees C. The IL-8 concentration after the first instillation seemed to be associated with subsequent development of an immune response. Consequently, IL-8 seems an attractive candidate for investigation of its prognostic value for a clinical response to BCG therapy.

Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C.

Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and DAG concentration, suggesting that the binding site of collagen type IV responsible for phospholipase C activation contains the RGD sequence. Together the present results suggest that, in pancreatic acinar cells, RGD sequence(s) within collagen type IV molecules cause activation of tyrosine kinase, probably through one of the integrin receptors, which then stimulates phospholipase C and increases [Ca2+]i.

Gonadectomy abrogates sex differences in the effectiveness of chemical carcinogenesis in mice.

Sham-gonadectomized and gonadectomized male and female mice were given methylcholanthrene s.c. to assess the influence of sex hormones on carcinogenesis. Gonadectomy decreased the concentration of the respective sex hormone and increased the concentration of the opposite sex hormone. The results showed that androgens enhanced the effectiveness of carcinogenesis, while estrogens had the opposite effect. Gonadectomy effectively abrogated the sex differences in tumor induction.

[Adjuvant BCG immunotherapy in the management of superficial bladder tumors]. / BCG immunterápia a felületes hólyagtumorok adjuváns kezelésében.

The net benefit of BCG immunotherapeutic prophylactic effect on recurrence of superficial bladder tumours was investigated. The BCG treatment group consisted of 121 stage Ta, T1 patients, while the control group, 49 patients, was treated only with transurethral resection. During 3-year follow-up recurrence rate in the control group was 55.1%, while in the BCG group in the two-and-a-half-year follow-up it was 23.9%. The yearly repeated 6-week cycles resulted in decrease of recurrence rate to 11.8%. The recurrence indexes were 2.2 and 0.6 in the control group and in the repeated treatment group respectively. A progression rate of 18.8% was recorded in the control group and 4.1% in the treatment group. Finally both clinical and investigative results were summarised and the necessity of repeated immunotherapeutic BCG courses were emphasized.

Evaluation of criteria contributions for the classification of systemic lupus erythematosus.

The American Rheumatism Association (ACR) preliminary and revised criteria for classification of systemic lupus erythematosus (SLE) were evaluated for sensitivity and specificity in a population of 100 patients with SLE and 100 patients with other rheumatic diseases. Bayes' theorem was applied for evaluation of ARA criteria for the classification of SLE and a scoring system was developed which allows simple determination of the probability of SLE. The evaluation revealed considerable differences in values of the ARA criteria. The serologic tests and discoid lupus appeared to be the most distinctive criteria, while Raynaud's phenomenon, oral and nasal ulcers and arthritis were of little value. Comparison of SLE and control patients presenting the same number of criteria revealed that patients with SLE exhibit more distinctive criteria. This finding emphasizes the need for a quantitative evaluation of classification criteria.