Nickel-induced proteins in human HaCaT keratinocytes: annexin II and phosphoglycerate kinase.

It has been established in previous in vitro experiments with human HaCaT keratinocytes that nickel becomes cytotoxic at concentrations higher than 100 microM and that it is accumulated mainly in the cytosolic fraction (Ermolli et al., 2000). The aim of this work was to search possible biomarkers of metal insult, i.e. nickel-binding proteins or proteins differentially expressed in the cytosolic fraction of nickel-exposed cells (up to 1 mM nickel) as compared to controls. Cytosolic proteins were studied by isoelectric focusing (IEF) and two-dimensional gel electrophoresis (2-DE). Separation by IEF revealed nickel-induced changes in the abundance of cytosolic proteins as visualised with nickel-nitrilo-triacetic-alkaline phosphatase (Ni-NTA-AP) in blots. The cytosolic fraction of cells incubated with nickel, at concentrations over 100 microM, showed nickel binding components which were absent or present in significantly lower amounts in control cells. These proteins had isoelectric points (pIs) 6.9, 7.7 and 8.5. After 2-DE silver- and protein staining significantly increased abundance of four proteins was observed. Their pI values corresponded to those of the nickel binding ones seen after IEF. A protein with pI 6.9 had a molecular weight estimated to 38 kDa, two proteins with pI around 7.7 showed molecular weights of 57 and 22 kDa, respectively and another protein with pI of 8.5 had a molecular weight of 33 kDa. The increased abundance of these components, both in IEF experiments and in 2-DE, correlated with the nickel concentration in the culture media. N-terminal amino acid sequencing and database search allowed identification of one a protein as phosphoglycerate kinase and another one as annexin II. The involvement of these proteins in cellular functions and their possible implications in the mechanism of nickel toxicity in keratinocytes are discussed. Some of these proteins may be biomarker candidates for effects of nickel exposure in human keratinocytes.

Nickel, cobalt and chromium-induced cytotoxicity and intracellular accumulation in human hacat keratinocytes.

Nickel, cobalt and chromium can induce allergic contact dermatitis (ACD) and may provoke irritant reactions in the skin. This study aimed at investigating cytotoxicity and cell viability along with intracellular metal accumulation in HaCaT human keratinocytes exposed to soluble forms of nickel, cobalt or chromium. The EC50 (24 h) values as detected by MTT test were 30 microM for sodium chromate (Na2CrO4), 475 microM for cobalt chloride (CoCl2) and 600 microM for nickel chloride (NiCl2). Chromium chloride (CrCl3) was not toxic up to 1 mM. No clear effects were observed after 4 h, but 24-h treatments with 1 mM CoCl2 or 10 microM Na2CrO(4) were found to almost completely abolish the ability of the cells to form colonies, whilst 1 mM NiCl2 reduced cellular survival to only 70% of control cultures. Intracellular accumulation of metals was evaluated by the use of radioisotopes at the EC50 value and at 1/10-1/5 of this concentration. Accumulation of Na2(51)CrO4 was linear with increasing dose. This was not the case for 63NiCl2 and 58CoCl2. All the metals were accumulated preferentially in the cytosols; 96% or more for 63NiCl2, approximately 90% for 58CoCl2 and 60-70% for Na2(51)CrO4. Finally, it was observed that HaCaT human keratinocytes can concentrate the metals present in the media up to 3.9 and 12.5 times for NiCl2 and CoCl2, respectively, and up to 167 for Na2CrO4. These striking metal intracellular accumulation patterns, which have not been earlier described in keratinocytes, highlight the relevance of searching for specific biomarkers of early cellular toxic effects, such as cytosolic proteins that bind the metals.

[Portal cavernoma. Observation of a case and clinico-nosographic considerations (author's transl)]. / Cavernoma portale. Osservazione di un caso e considerazioni clinico-nosografiche.

The clinical case of a sixty-two year old woman suffering for about six years from rare episodes of cramplike pains in the right hypochondriac region, radiated to the homolateral scapula, is described. After diagnosis of biliary calculosis, the patient was admitted to hospital and her gallbladder removed. During the operation a number of venous ectasias of the portal system were evidenced at the hepatic hilus, and their biopsy led to a diagnosis of portal cavernoma. The possible part played by the cavernoma in producing the patient's clinical picture is discussed, with reference also to other cases of cavernoma of varying location. This is followed by a review of the literature, in which stress is placed on the different aetiopathogenetic interpretations of angiomas and in particular of portal cavernomas. The possible evolution of angiomas and present treatment trends are also mentioned.

[Indications for cerebral revascularization surgery (author's transl)]. / Indicazioni agli interventi di rivascolarizzazione cerebrale.

Cerebrovascular diseases are among the most widespread. In the USA alone there is at any time a population of 1.6 million people with current stroke syndrome or the sequelae thereof. The most effective weapon to combat this disease is prevention, and here reference is made in particular to surgical prevention, especially through endoarterectomy of the extracranial internal carotid, and to vertebrobasilar circulatory failure.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.