A bioavailability study on microbeads and nanoliposomes fabricated by dense carbon dioxide technologies using human-primary monocytes and flow cytometry assay.

Supercritical Emulsion Extraction (SEE) and Supercritical assisted Liposome formation (SuperLip), use dense gases such as carbon dioxide (dCO2) to fabricate advanced micro/nanocarriers. SEE uses dCO2 to extract solvent from the oily phase of an emulsion and obtain biopolymer microbead; For this study, poly-Lactic Acid (PLA) microbeads of 1 ±â€¯0.2 µm in mean size loaded at 1 µg/mgPLA with Rhodamine B (ROD) were prepared by SEE; the beads showed a solvent residue lower than 10 ppm and encapsulated the fluorochrome with an efficiency of 90%. SuperLip uses dCO2 to enhance lipid/ethanol/water mixing and to promote the ethanol extraction from liposome suspension. In this case, phosphatidyl-choline (PC) vesicles with a mean size of 0.2 ±â€¯0.05 µm and loaded with Fluorescein Iso-ThioCyanate (FITC) at 8 µg/mgPC were prepared; small unilamellar structure was observed for all the vesicles with FITC encapsulation efficiency of 80%. Ethanol residue of 50 ppm was measured in all the liposome suspensions. The bioavailability of microbeads and nanoliposomes was assessed through incubation with human monocytes previously isolated from healthy donors' blood. A specifically optimized protocol that allowed their quenching on the cell surface was developed to monitor by flow cytometer assay only the cell population that effectively internalized the carriers. When microbeads were tested, the percentage of alive internalizing monocytes was of about 30%. An internalization of 96.1 ±â€¯21% was, instead, obtained at dosage of 0.1 mg/mL for nanoliposomes. In this last case, monocytes showed a vitality of almost 100% after vesicles internalization at all the concentrations studied; on the other hand, cell apoptosis progressively increased in a dose/response manner, after polymer microbeads phagocytosis. The proposed data suggested that dCO2 technologies can be reliably used to fabricate intracellular carriers.

Motor phenotype is not associated with vascular dysfunction in symptomatic Huntington's disease transgenic R6/2 (160 CAG) mice.

Whereas Huntington's disease (HD) is unequivocally a neurological disorder, a critical mass of emerging studies highlights the occurrence of peripheral pathology like cardiovascular defects in both animal models and humans. The overt impairment in cardiac function is normally expected to be associated with peripheral vascular dysfunction, however whether this assumption is reasonable or not in HD is still unknown. In this study we functionally characterized the vascular system in R6/2 mouse model (line 160 CAG), which recapitulates several features of human pathology including cardiac disease. Vascular reactivity in different arterial districts was determined by wire myography in symptomatic R6/2 mice and age-matched wild type (WT) littermates. Disease stage was assessed by using well-validated behavioural tests like rotarod and horizontal ladder task. Surprisingly, no signs of vascular dysfunction were detectable in symptomatic mice and no link with motor phenotype was found.

Assembly of the CD8alpha/p56(lck) protein complex in stably expressing rat epithelial cells.

We have previously characterized the biogenesis of the human CD8alpha protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8alpha/p56(lck) protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56(lck), or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8alpha, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8alpha established hetero-oligomeric complexes with p56(lck), as revealed by co-immunoprecipitation assays performed with anti-CD8alpha antibody. Moreover, p56(lck) bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56(lck) binds rapidly to CD8alpha and most of the p56(lck) is bound to CD8alpha at steady state; (2) CD8alpha/p56(lck) protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56(lck) reaches the cell surface.

Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2.

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.

Sequence and expression of the monkey homologue of the ER-golgi intermediate compartment lectin, ERGIC-53.

We obtained the cDNA sequence of the monkey homologue of the intermediate compartment protein ERGIC-53 by both cDNA library screening and RT-PCR amplification. The final sequence of 2422 nts of the monkey ERGIC-53 cDNA is 96.2% identical to the human ERGIC-53 cDNA and 87% and 67% identical to the rat and amphibian cDNA, respectively. The translated CV1 ERGIC-53 protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat p58 and 66. 98% to the Xenopus laevis protein. Southern blot analysis of multiple genomic DNAs shows the presence of sequences similar to ERGIC-53 in different species. ERGIC-53 is expressed as a major transcript of about 5.5 kb in either monkey CV1 or in human CaCo2. A shorter transcript of 2.3 kb was detected in both cell lines and in mRNAs derived from human pancreas and placenta.

Regulation of ZiRF1 and basal SP1 transcription factor MRE-binding activity by transition metals.

The metal-dependent activation of metallothionein (MT) genes requires the interaction of positive trans-activators (MRFs) with metal-regulatory (MRE) regions of MT promoters. In this report, we examined the role of transition metals in modulating the MRE-binding activities of two different MRE-binding proteins: the metal-regulated factor ZiRF1 and the basal factor SP1. We showed the ability of both proteins to interact with a similar sequence specificity with the cognate target site (MRE-S) of another known MRE-binding protein, mMTF1. We next evaluated the role of metal ions in modulating the MRE-binding activity of recombinant ZiRF1 and basal SP1 proteins by measuring the effect of different metal chelators on DNA interaction. We observed a dose-dependent inhibition of the GST-ZiRF1/MRE-binding activity using three different metal chelators: EDTA, 1,10 PHE and TPEN. Interestingly, EDTA treatment failed to inhibit the recombinant SP1 MRE-binding activity while the effect of 1,10 PHE was comparable to that obtained analyzing 1,10 PHE-treated GST-ZiRF1. The MRE-binding complexes detected in cell extracts showed a response to metal chelator treatment very similar to that displayed by the recombinant ZiRF1 and SP1 proteins. The hypothesis of mutual interactions of both basal and metal-regulated transcription factors with the same metal-regulatory regions is discussed.

Interactions of the zinc-regulated factor (ZiRF1) with the mouse metallothionein Ia promoter.

A mouse cDNA clone, M96, encoding a metal-regulating-element (MRE)-binding protein, was analysed for its ability to act as a metal-regulated transcription factor. The metal depletion of a glutathione S-transferase (GST)-M96 fusion protein showed that Zn2+ ions modulate the MRE-binding activity, suggesting that the M96-encoded protein is a Zn2+-regulated factor (ZiRF1). The methylation interference assay showed the specific interactions of ZiRF1 with the MRE, MREd/c, present on the mouse metallothionein Ia promoter. Point mutations of the MREd/c nullified the metal-regulatory properties of this region. In mouse L-cell nuclear extracts, mobility-shift assays revealed a Zn2+-dependent MRE-binding complex (MBC) with DNA-recognition properties similar to those of ZiRF1. Antibodies raised against purified GST-ZiRF1 were able to specifically recognize MBC in Western-blot analyses. Competition analysis of MRE-binding proteins from mouse NIH3T3 cells with oligonucleotide matching the binding sites for SP1 and MTF1 confirmed that both the basal SP1 and the metal-regulated MBC/ZiRF1 interact with the MREd/c region. The significance of mutual interactions with the metal-responsive promoter regions of either metal-regulated or basal transcription factors is discussed.

Analysis of metal-regulated metallothionein and heat shock gene expression in HeLa-derived cadmium-resistant cells.

The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd2+ and Zn2+ exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd(2+)-exposed H454 cells at levels comparable to the ones present in Cd(2+)-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes.

Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system.

A new method for isolation of cDNA clones encoding sequence-specific DNA-binding proteins is described. This method, the one-hybrid system, is based on the use of reporter genes whose transcription can be activated through synthetic cis elements recognized by the sought-after DNA-binding protein. These reporter genes are used for in vivo screening of a library of cDNAs fused to a DNA fragment encoding the GAL4 activation domain. cDNA clones expressing the appropriate fusion proteins lead to activation of these reporter genes in transformed yeast cells. We have used this approach to isolate a mammalian cDNA clone encoding a sequence-specific DNA-binding protein that recognizes the metal response elements (MREs) of the metallothionein (MT) genes. The protein encoded by this cDNA, M96, shows similarity to the trithorax proteins. Expression of a functional DNA-binding form of M96 requires Zn2+ ions. The recombinant protein binds to several different MREs but fails to recognize nonfunctional mutant MREs. M96 may be involved in the activation of MT genes in response to heavy-metal ions.

Interactions of nuclear proteins from uninduced, induced and superinduced HeLa cells with metal regulatory elements MRE3 and 4 of the human metallothionein IIa-encoding gene.

Transcriptional activation of metallothionein (MT)-encoding genes(MT) is regulated during heavy metal induction by short non-identical repeats, termed 'metal regulatory elements' (MRE), present in multiple imperfect copies in MT promoter regions of eukaryotes. Using mobility shift assays, we have studied the interaction between the human MRE 3 and 4 regions (hMRE3/4) of the MTIIa promoter and nuclear proteins from uninduced and Cd(2+)-induced HeLa cells, and from Cd(2+)-superinduced H454 cells, a HeLa-derived Cd(2+)-resistant cell isolate which overexpresses hMTIIa after exposure to metal. A specific complex with a similar electrophoretic mobility was formed in all three extracts. Dialysis of the extracts using EDTA inhibited the formation of the complexes, which could be reconstituted only after the addition of Zn2+. UV cross-linking analyses of the specific complexes formed by the three nuclear extracts interacting with the hMRE3/4 region revealed that in all of them polypeptides were present having similar electrophoretic mobilities and different molecular masses. Mobility shift assays showed no major differences in the binding of nuclear proteins from induced or uninduced cells. Proposed models of activation of metal-induced MT transcription are discussed.

Serum beta 2-microglobulin levels and p24 antigen, lymphocyte depletion and disease progression in human immunodeficiency virus infection.

Abnormally elevated serum beta 2-microglobulin levels have been associated with progression of human immunodeficiency virus disease. In this study we have analyzed the relationship between serum beta 2-microglobulin levels of patients at different stages of the disease and serological and immunological parameters commonly used for monitoring the infection. The investigation was performed on 150 patients and 30 controls during the period from March 1989 to March 1990. At that time, 30 patients had the acquired immunodeficiency syndrome or its related complex and 120 had persistent generalized lymphadenopathy or were asymptomatic. Thirty-nine antibody-negative subjects, belonging to a high-risk group for the acquired immunodeficiency syndrome, were used as controls. All patients had normal renal function. There was a significant relationship between increased serum beta 2-microglobulin levels and the presence of p24 antigen, a decrease in the total number of lymphocytes (less than or equal to 1500/mm3) and a decrease in CD4+ T lymphocytes (less than or equal to 200/mm3). No significant relationship between serum beta 2-microglobulin levels and CD3+ T lymphocytes was found.

A nuclear factor binds to the metal regulatory elements of the mouse gene encoding metallothionein-I.

The ability of vertebrate metallothionein (MT) genes to be induced by heavy metals is controlled by metal regulatory elements (MREs) present in the promoter in multiple, non-identical copies. The binding specificity of the mouse L-cell nuclear factor(s) that interact with the element MREd of the mouse MT-I gene was analyzed by in vitro footprinting, protein blotting, and UV cross-linking assays. In vitro footprinting analyses revealed that synthetic oligodeoxynucleotides (oligomers) corresponding to the metal regulatory elements MREa, MREb, MREc, MREd and MREe of the mouse MT-I gene, as well as the MRE4 of the human MT-IIA gene and the MREa of the trout MT-B gene, all competed for the nuclear protein species binding to the MREd region of the mouse MT-I gene, the MREe oligomer being the weakest competitor. In addition, protein blotting experiments revealed that a nuclear protein of 108 kDa, termed metal element protein-1 (MEP-1), which specifically binds with high affinity to mouse MREd, binds with different affinities to the other mouse MRE elements, mimicking their relative transcriptional strength in vivo: MREd greater than or equal to MREa = MREc greater than MREb greater than MREe greater than MREf. Similarly, human MRE4 and trout MREa bind to MEP-1. A protein similar in size to MEP-1 was also detected in HeLa-cell nuclear extracts. In UV cross-linking experiments the major protein species, complexed with mouse MREd oligomers, migrated on a denaturating gel with an apparent Mr of 115,000 and was detected using each of the mouse MRE oligomers tested. These results show that a mouse nuclear factor can bind to multiple MREs in mouse, trout, and human MT genes.

In vitro activation of isophosphamide and trophosphamide to metabolites mutagenic for bacteria.

The ability of S9 liver fractions from uninduced rats to activate isophosphamide (IP) and trophosphamide (TP) to metabolites mutagenic for bacteria was compared to that of S9 fractions prepared from rats pretreated in vivo with three inducers of hepatic monooxygenase. Pretreatment of rats with phenobarbital (PB) and Aroclor 1254 increased IP and TP mutagenic activation by S9 fractions as compared to control and 3-methylcholanthrene (3-MC)-induced rat liver S9. Furthermore, the effect of mixed-function oxidase inhibitors, such as alpha-naphthoflavone, metyrapone and SKF 525-A on S9-mediated mutagenic activation of IP and TP was investigated. The data obtained suggest the involvement of a PB-inducible form of cytochrome P-450 in the activation of IP and TP to mutagenic species.

[Hematologic characterization and analysis of genetic transmission in a case of double heterozygosity Hb Lepore/beta-thalassemia]. / Caratterizzazione ematologica e analisi della trasmissione genetica di un caso di doppia eterozigosi Hb Lepore/beta-talassemia.

Double heterozygosis condition for hemoglobin variants induce clinical syndromes known as intermediate thalassemias. Their diagnosis is often of certain difficulty because of their low frequency and heterogeneity of clinical expressions. We report a case of a 4 year child admitted to our medical center with a story of hepatosplenomegaly. An appropriate hematological study on patient's family permitted a diagnosis of double heterozygosis for Hb Lepore and beta-thalassemia. Results of hematological investigation are reported.

[Toxic genetic effects of flunitrazepam]. / Effetti genotossici del flunitrazepam.

The mutagenic activity of Flunitrazepam, the active ingredient of the drug Rohypnol, has been investigated by using the Salmonella/microsome mutagenicity test. A dose-related mutagenic effect was observed on Salmonella typhimurium strain TA 100 either in the absence or in the presence of a rat liver microsomal fraction (S9) as in vitro metabolic activation system. By adopting a modification of the Salmonella test, the mutagenicity of urines from rats or patients treated with the drug was evaluated. In these cases mutagenic activity was detected toward the Salmonella strains TA 98 and TA 100 both in presence and in absence of the metabolic activation system. The data indicate that Flunitrazepam and/or its urinary metabolites can induce both base-pair substitutions or frame-shift point mutations.