Route of jejunal mucosa absorption of trypsin demonstrated by immunofluorescence

Previous studies have demonstrated the absorption of porcine trypsin in isolated jejunal loops from male Wistar rats by open-loop perfusion. The possible routes of absorption were examined in the study reported here. Trypsin (0.5 mg/ml) was dissolved in tyrode solution and perfused at a rate of 0.5 ml/min, at 37 degrees C, for 40 min. Using immunoperoxidase and immunofluorescence techniques, strong reactivity towards anti-TLCK-trypsin antibody was demonstrated through out the enterocyte cytosol. The present data indicate that trypsin was absorbed by enterocytes, probably through a transcellular route.     

Characterization of Cah,a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.     

Protein origami: therapeutic rescue of misfolded gene products

Receptor mutations that elicit loss of function are sometimes equated with defects that ablate receptor-ligand binding or receptor-effector interactions. Similarly, mutationally defective enzymes and ion channels are often viewed as compromised in substrate or ion recognition, respectively. Recent observations, however, suggest that an alternate mechanism may be surprisingly common, namely, that mutations in structural genes may not interfere with the inherent functionality of the affected protein, but nevertheless cause disease by preventing the cell's trafficking machinery from placing the affected protein at the appropriate subcellular compartment (e.g., at the cell membrane). Accordingly, therapies may be devised to ensure the placement of receptors (or other proteins) at locations where they can support cell function.     

Understanding p27(kip1) deregulation in cancer: down-regulation or mislocalization

There is considerable evidence that the inactivation of the cyclin-dependent kinase inhibitor p27(kip1) is a fundamental step for the development of human malignancies. In particular, reduced expression of p27(kip1), due to increased protein degradation, correlates with poor prognosis of patients affected by various types of cancer. The purpose of this mini-review is to present an overview of the current understanding of the alteration of p27(kip1) function in human cancer and to describe the different mechanisms that contributes to it. Particular emphasis is placed on the novel finding of p27(kip1) mislocalization in tumor cells and on the biochemical pathways responsible for p27(kip1) cytosolic accumulation. Finally, we review the possible clinical implications of these observations with respect to prognosis and novel anticancer therapies.     

Image processing,diagnostic information extraction and quantitative assessment in pathology

As we enter the information age we hold strong beliefs in the benefits of digital technology applied to pathology: numerical representation offers objectivity . Digital knowledge may indeed lead to significant information discovery, and, processing systems might be designed to allow a true evolution of capabilities. Questions arise whether the methodology underlying quantitative analysis provides the information that we need and whether it is appropriate for some of the problems encountered in diagnostic and prognostic histopathology. While one certainly would not dispute the value of statistical procedures, the clinical needs call for individual patient targeted prognosis.     

Response to oxidative stress as a welfare parameter in swine

In pigs, the genetic selection for lean, large muscle blocks and fast growth has been linked to an increased prevalence of metabolic diseases such as porcine stress syndrome and mulberry heart disease. These diseases are associated with cardiovascular inadequacy, which may lead to oxidative stress. In the present study, reactive oxygen metabolites (ROMs) and the anti-oxidant power (OXY) in sera of different swine groups were investigated. The following groups were selected (each around 80 kg body weight): wild boars (WB), Cinta Senese (CS), and Landrace x Large White (LxLW), the latter as both specific pathogen-free (SPF) and intensively farmed animals. In addition, a group of LxLW agonic sows (AS) was also investigated; this group is known to be under oxidative stress. Two colorimetric micro-methods were used to measure ROMs and OXY; ROMs were expressed as mM H(2)O(2) and OXY as microM HOCl neutralised. Between groups, average ROM and OXY values were found to be significantly different by one-way ANOVA (P < 0.001). ROM levels were lower in WB (13.41 +/- 1.85) and CS (19.27 +/- 1.68), and highest in LxLW (42.00 +/- 1.36). OXY values ranged from 260.10 +/- 22.13 (WB) to 396.90 +/- 9.83 (LxLW). Only one swine group (the CS group) showed a significant, positive correlation between ROM and OXY values. The AS group even showed a negative correlation between ROM and OXY values. These results imply satisfactory environmental coping occurred only within the CS group. Results are discussed in the light of animal welfare legislation, food safety and consumers' protection.     

Status and metabolism of iron in elite sportsmen during a period of professional competition

The aim of this study was to determine the effect of both acute exercise and maintained training during a period of competition (3 mo, at the start of the season) on iron metabolism in sportsmen on a professional volleyball team. Twelve sportsmen volunteered for this study. The exercise test was performed on a mechanically braked Monark cycle ergometer and consisted of a triangular progressive test. Three blood samples were obtained in each test: at rest, just after exercise, and after recovery. The following hematological parameters were determined: red blood count (RBC), hemoglobin (Hb) and hematocrit (Hto), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total proteins (TP), serum iron (Fe) and total iron-binding capacity (TIBC), ferritin (FER), transferrin (TRF), haptoglobin (HPT), and serum cortisol (COR) concentrations. We have found changes in hematological and biochemical variables related to Fe metabolism during the study. The changes observed could be the result of hemoconcentration processes after exercise and, at least in part, to physical stress and muscular damage. We conclude that athletes, after a period of adaptation, with a good plan of work/recovery series, undergo a biological redistribution on hematological and biochemical parameters concerning Fe metabolism during the training and competition period. Also, daily Fe supplementation could restore and mask the true repercussions of maintained training observed in other sports.     

Apoptotic rate: a new indicator for the quantification of the incidence of apoptosis in cell cultures

BACKGROUND: Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). METHODS: This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. RESULTS: In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. CONCLUSIONS: This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.     

Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies

Selective degeneration of the smallest fibers (papillo-macular bundle) of the human optic nerve occurs in a large number of optic neuropathies characterized primarily by loss of central vision. The pathophysiology that underlies this peculiar pattern of cell involvement probably reflects different forms of genetic and acquired mitochondrial dysfunction.Maternally inherited Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (Kjer disease), the optic atrophy of Leigh's syndrome, Friedreich ataxia and a variety of other conditions are examples of inherited mitochondrial disorders with different etiologies. Tobacco-alcohol amblyopia (TAA), the Cuban epidemic of optic neuropathy (CEON) and other dietary (Vitamins B, folate deficiencies) optic neuropathies, as well as toxic optic neuropathies such as due to chloramphenicol, ethambutol, or more rarely to carbon monoxide, methanol and cyanide are probably all related forms of acquired mitochondrial dysfunction.Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress. Histopathological studies in LHON cases and a rat model mimicking CEON revealed a selective loss of retinal ganglion cells (RGCs) and the corresponding axons, particularly in the temporal-central part of the optic nerve. Anatomical peculiarities of optic nerve axons, such as the asymmetric pattern of myelination, may have functional implications on energy dependence and distribution of mitochondrial populations in the different sections of the nerve. Histological evidence suggests impaired axonal transport of mitochondria in LHON and in the CEON-like rat model, indicating a possible common pathophysiology for this category of optic neuropathies. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting the oligodendrocytes of the optic nerves.     

Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism,antagonism, or inverse agonism?

1. The 5-HT₂ receptors subdivision into the 5-HT_2A/2B/2C subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT₂ receptors role on memory consolidation.2. The SB-200646 (a selective 5-HT_2B/2C receptor antagonist) and LY215840 (a nonselective 5-HT_2/7 receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP).3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT_2A receptor antagonist) and to a LY215840 high dose.4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine; while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs.5. It is suggested that 5-HT_2B/2C receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT₂ receptors remains unclear at this time.6. Notably, the 5-HT₂ receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreased cholinergic, glutamatergic, and/or serotonergic neurotransmission.