Perspectives of choroidal neovascularization therapy.

Vision loss secondary to Choroidal Neovascularization (CNV) is becoming a major disease condition in developed world. CNV is typically secondary to Age-related Macular Degeneration (AMD) and these conditions are major, and also substantially increasing, causes of blindness among aged people. Several therapeutic options are currently available to treat CNV with variable efficacy on disease progress. Among existing treatments there are laser photocoagulation, photodynamic therapies, local corticosteroids and, more recently, the use of anti-angiogenic factors. Although by these treatments very effective results are obtained and their further improvement is still possible, it is also reasonable and necessary to look for more successful and definitive alternatives. The research in this direction is already very active and it can be expected that applications of the more recent molecular technologies will bring important advances also for CNV. These will likely regard the use of gene therapy and of new target specific factors. Gene therapies methodologies are rapidly becoming closer to current clinical use and, since the eye is a particularly favourable organ for drug delivery, their ocular use is probably going to be among the first successful applications of these techniques. In addition to its specific technology, gene therapy requires the knowledge of specific genes to be modulated to adequately affect pathogenesis and progression of the disease in which has to be applied. This will also be true for the use of novel target specific drugs such as antibodies and other molecules able to affect cellular factors and pathways also related to disease development. For this reason, a major direction of future CNV therapies will be the identification of specific gene, gene products, metabolic pathways and metabolites related to the disease. This information, in addition to be suitable for gene and target specific therapies, will also allow the development of new procedures to improve diagnosis and/or prognostic evaluation of the disease.

Migraine and coronary artery disease: an open study on the genetic polymorphism of the 5, 10 methylenetetrahydrofolate (MTHFR) and angiotensin I-converting enzyme (ACE) genes.

Genetic factors that increase susceptibility to oxidative stress, endothelial disfunction and, possibly, stroke include angiotensin-converting enzyme gene deletion polymorphism (ACE-DD) and the methylentetrahydropholate reductase (MTHFR) C677-TT polymorphism. The relationship of ACE-DD genotype to ischemic stroke and cardiovascular disease is controversial, but it has been independently linked to lacunar infarction, in the absence of carotid atheroma. Lea et al. (2005) reported that the ACE DD genotype acts in combination with the MTHFR T/T genotype to increase migraine susceptibility, with the greatest effect in those with aura. The "TT" polymorphism is also associated with an increased risk of migraine with aura, independent of other cardiovascular risk factors. The aim of our study was to evaluate the incidence of ACE and MTHFR genes polymorphisms in a consecutive series of migrainous patients and of patients affected by myocardial infarction. We studied a series of 103 migrainous patients (1), whose age was between 13 and 75 years (81 suffering from migraine without aura, MwA, 9 from migraine with aura, MWA, 13 from mixed forms MwA-MWA, according to ICHD-II 2004 criteria) and of 336 patients (2) suffering from ischaemic cardiopathy (myocardial infarction, MI). The analysis, based on Polymerase Chain Reaction (PCR) and on reverse-hybridization, showed as follows: MTHFR (C677T): 60 patients (58%) (1) and 186 (56%) (2) were heterozygous; 9 patients (9%) (1) and 54 (16%) (2) were mutated. The result of 1 patient (2) was unknown. MTHFR (A1298C): 54 patients (52%) (1) and 146 (44%) (2) were heterozygous, 7 patients (7%) (1) and 33 (10%) (2) were mutated. The result of 1 patient (2) was unknown. ACE (evaluated on 101 patients (1) and 245 (2)): 45 patients (43%) (1) and 133 (54%) (2) had an ID genotype; 42 (41%) (1) and 87 (36%) (2) had a DD genotype. The results of our study confirm the high incidence in the genetic polymorphisms ACE and MTHFR in migraineuse. These data are confirmed in the sample of patients affected by myocardial infarction. This gives evidence of a strong relationship between migraine and major vascular diseases and let us hypothesize an important role of ACE and MTHFR system in the pathogenetic model of migraine for its capability to interfere with the endothelial regulation tone. Once an effective role in the genesis of migraine and in the increased risk of migrainous patients to evolve into an ischemic pathology has been obviously assigned to this genetic mutation, future researches must aim through wider and more controlled casistics also to clarify the role that drugs acting on these systems may have on the resolution of these diseases.

Transcriptional response of a human colon adenocarcinoma cell line to sodium butyrate.

Taking advantage of the DNA array screening technology, we analysed the effect of sodium butyrate on mRNA transcription in human HT29 colon adenocarcinoma cells. Out of 588 mRNA species analysed, only 119 resulted expressed. Among these, 60 exhibited a variable degree of modulation after butyrate treatment. Genes linked to the cell growth, apoptosis and oxidative metabolism appeared the most significantly affected. Furthermore, many of the differentially expressed genes are transcription factors and this may account for the variability of the biological effects of butyrate. The pattern of butyrate-affected genes may represent a reference in further analyses of gene expression of intestinal cells and tissues.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Differential expression of antiapoptotic genes in human endometrial carcinoma: bcl-XL succeeds bcl-2 function in neoplastic cells.

OBJECTIVES: Previous histochemical observations have suggested a possible involvement of the bcl-2 family genes in the acquisition of neoplastic phenotype of the endometrium. Since knowledge of the type and function of genes controlling the transformed cell may result in new diagnostic, prognostic, and therapeutic approaches, we have investigated at the molecular level the biological role of bcl-2 family genes in endometrial neoplastic cells. METHODS: To investigate the relationship between the sensitivity to apoptosis and the expression of the bcl-2 family genes, we set up a model system consisting of four human endometrial carcinoma cell lines. This system constitutes an array of two cell pairs presenting, respectively, endometrioid and adenosquamous phenotypes. G2 and G3 gradings are represented within each pair; in addition, each set contains one cell line that is apoptosis-sensitive and one that is resistant. Transfection of bcl-2 and bcl-XL into apoptosis-sensitive cells was used to monitor the biological function of protective genes. RESULTS: A differential pattern of expression of bcl-2 family genes was observed in apoptosis-sensitive versus resistant cells, independent from the histological subtype. Resistant lines exhibited high amounts of Bcl-XL and low amounts of Bcl-2. Bax expression clearly correlates with cellular susceptibility to apoptosis. Transfection of bcl-XL resulted in a dose-dependent enhancement in resistance toward apoptosis. In contrast, the main effect of bcl-2 constitutive overexpression was to drastically abate the proliferative potential of transfected cells. CONCLUSIONS: These data demonstrate, at the molecular level, that bcl-XL is selected as an apoptosis-protective gene in place of bcl-2 while bax retains its dominant proapototic role.

PED/PEA-15 gene controls glucose transport and is overexpressed in type 2 diabetes mellitus.

We have used differential display to identify genes whose expression is altered in type 2 diabetes thus contributing to its pathogenesis. One mRNA is overexpressed in fibroblasts from type 2 diabetics compared with non-diabetic individuals, as well as in skeletal muscle and adipose tissues, two major sites of insulin resistance in type 2 diabetes. The levels of the protein encoded by this mRNA are also elevated in type 2 diabetic tissues; thus, we named it PED for phosphoprotein enriched in diabetes. PED cloning shows that it encodes a 15 kDa phosphoprotein identical to the protein kinase C (PKC) substrate PEA-15. The PED gene maps on human chromosome 1q21-22. Transfection of PED/PEA-15 in differentiating L6 skeletal muscle cells increases the content of Glut1 transporters on the plasma membrane and inhibits insulin-stimulated glucose transport and cell-surface recruitment of Glut4, the major insulin-sensitive glucose transporter. These effects of PED overexpression are reversed by blocking PKC activity. Overexpression of the PED/PEA-15 gene may contribute to insulin resistance in glucose uptake in type 2 diabetes.

Identification of differentially expressed mRNAs in normal and neoplastic (adenocarcinoma) human endometrium.

Individual mRNA species from normal and neoplastic endometrium obtained from the same patient were comparatively studied by exploiting the "differential display" methodology. The mRNAs were extracted from tissues, reverse transcribed, and amplified by PCR using appropriate primers. The cDNA electrophoretic bands which were frankly different on the basis of their quantitative expression were excised from the gels and reamplified. Each of these sequences was subsequently screened for the capacity to hybridize to RNA preparations from normal endometrium and endometrial adenocarcinoma samples, first from the original patient and then from a group of selected patients. Of many, only two sequences, thereafter named N5.5 and T16.2, respectively, successfully passed the two sieving tests and were chosen for further analysis. It appears that N5.5 recognizes an mRNA which is expressed more abundantly in normal than in neoplastic (adenocarcinoma) endometrium; in contrast, T16.2 seems to preferentially recognize an mRNA which is expressed in tumoral tissues. These two sequences have been cloned and sequenced; they do not show any identity or significant similarity to any other known sequence.

Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation.

The blue beam of an Argon laser (488 nm) has been focused on the cell membrane in the presence of phenol-red, an usual component of cell culture media, through a 100 x objective. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously within 1-2 minutes. No evident sings of cell injury or hurt have been observed afterward. Plasmid DNA, purposely added to culture fluid, easily slips in the cytoplasm; utilizing such approach, thereafter indicated as "optoporation', we have successfully transfected two genes, namely beta-galactosidase and chloramphenicol-acetyl-transferase in murine NIH3T3 fibroblasts. Therefore optoporation represents an additional procedure for gene transfer with several advantages over already available methods: (1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances; (2) it is a very mild treatment virtually suitable for any cell type and (3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).

Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease.

The gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. pylori pathogenesis and the development of therapeutics and vaccines.

Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.

Immunogenicity of anti-idiotypic antibodies and of their F(ab')2 fragments.

Within the idiotype/anti-idiotype network, immunoglobulins act alternatively as reactive molecules and as antigens. To investigate the antigenic properties of immunoglobulins, we evaluated the immunogenicity in rabbits of three murine monoclonal anti-idiotypic antibodies and of their F(ab')2 fragments. These antibodies, bearing the internal image of a human melanoma antigen, may be useful in view of a human therapeutic application. The effect was evaluated as specific anti-anti-idiotypic response, related to the immunogenicity of the idiotypic epitopes in the combining sites of the immunoglobulins, and as total anti-murine immunoglobulin response, which represents the recognition of all the immunological determinants of the molecule. The results showed that the administration of the F(ab')2 fragments results in either higher or similar degrees of anti-anti-idiotypic immunization, compared to those induced by the whole immunoglobulins. Nevertheless, when anti-anti-idiotypic immunogenicity was increased, the anti-murine response did not increase proportionally. This suggests that the use for in vivo administration of F(ab')2 fragments is more convenient than the use of their original molecules, since this results, at least, in a similar or eventually in an increased specific immunogenicity, while the possibility of aspecific recognition is reduced.

Unique structure of glycopeptide from alpha-fetoprotein produced in human hepatoma cell line, as determined by 1H-nuclear magnetic resonance spectroscopy.

Determination of alpha-fetoprotein is used in diagnosis of tumors and neural tube defects. A good reliable source of alpha-fetoprotein would be an obvious advantage to the preparation of diagnostic reagents and their standardization. We have recently developed a method for the production of alpha-fetoprotein from a human hepatoma cell line. This method, which is suitable for scaling up, allowed us to produce 40 g of alpha-fetoprotein from culture supernatant liquid through a simple purification procedure. We have previously shown this protein to be identical to alpha-fetoprotein produced from other sources. However, because the presence of different glycoforms has been reported in alpha-fetoprotein preparations, both from human sources and from other species, it was important to establish the type and extent of glycosylation of alpha-fetoprotein prepared by our method. By using 1H-NMR spectroscopy we were able to establish that our product contains a single N-linked biantennary, fully sialylated complex-type oligosaccharide, typical of human hepatomas.

Thyroid hormone effect on alpha-fetoprotein and albumin coordinate expression by a human hepatoma cell line.

The action of triiodothyronine on the production of alpha-fetoprotein and albumin in serum-free cultures of Hep G2 human hepatoma cells was examined. Our data showed that a marked inhibition (up to 8-fold) of alpha-fetoprotein secretion and an increase in albumin (up to 4-fold) are produced by 10(-8) M triiodothyronine. These effects were slow in their onset and for completion required 20-25 days of treatment with the hormone. However, an exposure of the cells to triiodothyronine for only the first 4 h was sufficient to affect, in a similar way, the secretion of alpha-fetoprotein and albumin when measured 15 days after treatment. The secretion of the two proteins parallels their intracellular levels. The decrease in alpha-fetoprotein production can be explained by a reduction of the RNA coding for the protein. The same is essentially true also for albumin increased secretion and related mRNA expression.

Synthesis and characterization of a recombinant fragment of human alpha-fetoprotein with antigenic selectivity versus albumin.

A DNA sequence coding for human alpha-fetoprotein amino acid sequence 38-119 was synthesized and cloned in a bacterial expression vector. The alpha-fetoprotein sequence was selected as the least homologous to albumin, since the two proteins have an overall amino acid identity of approximately 38%. A chimeric protein was obtained which was purified by preparative electrophoresis and characterized in its primary structure by fast atom bombardment mass spectometry. About 70% of the alpha-fetoprotein sequence was physically mapped and found to correspond to the amino acids encoded in the synthetic gene. The use of this recombinant protein allowed the selection of monoclonal antibodies recognizing both the recombinant fragment and native alpha-fetoprotein. These antibodies should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.

Monoclonal antibodies recognizing a recombinant portion of human alpha-fetoprotein with antigenic selectivity versus albumin.

Determination of serum alpha-fetoprotein is useful in the clinical management of liver cancer, but it has not been particularly helpful in the early diagnosis of this disease, since also non-neoplastic liver diseases may result in small increases of its serum concentration. To improve the clinical performance of this assay, we have previously developed an in vitro culture system, in which the expression of alpha-fetoprotein and albumin could be coordinately modulated by thyroid hormone. This system allowed large scale production and purification of native alpha-fetoprotein to be used as reference material. In addition, we synthesized and cloned in a bacterial expression vector a DNA sequence coding of human alpha-fetoprotein amino acid sequence 38-119. This alpha-fetoprotein sequence was chosen since it is the least homologous to albumin, being the amino acid sequence of the two proteins extremely similar with an overall identity of about 38%. Now we have obtained three hybridomas recognizing with high affinity and specificity both the recombinant fragment and native alpha-fetoprotein. These antibodies, which therefore recognize the native protein in the amino acid sequence 38-119, should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.

High-yield and high-degree purification of human alpha-fetoprotein produced by adaptation of the human hepatoma cell line Hep G2 in a serum-free medium.

The human hepatoma cell line Hep G2 secretes both albumin and alpha-fetoprotein when grown in the presence of serum. The present report describes how adaptation to growth in serum-free medium results in a progressive switch in the expression of the two proteins; i.e., alpha-fetoprotein becomes the main protein secreted while albumin production is greatly reduced. The culture supernatant obtained, being very enriched in the protein, allows the development of a purification procedure by preparative electrophoresis. By this procedure it is possible to easily obtain large amounts of alpha-fetoprotein from a constant and unlimited source. The availability of these protein preparations should improve the reproducibility and the quality of standardization in clinical immunoassays for alpha-fetoprotein and should permit a more accurate study of the structure and biological functions of the protein.

Transcriptional regulation by thyroid hormone of an mRNA homologous to a protease inhibitor.

We have previously cloned a cDNA of a rat liver mRNA, designated 4-12B, markedly induced by triiodothyronine (T3) at a pretranslational level [Magnuson, M.A., Dozin, B., & Nikodem, V.M. (1985) J. Biol. Chem. 260, 5906-5912]. Here we show that this hormonal effect is due in part to an increase of the rate of transcription of the 4-12B gene. In addition, the nucleotide sequence of 4-12B cDNA has been determined, revealing significant similarity with the sequences of the superfamily of serine protease inhibitors and a very high homology with contrapsin, a mouse serum trypsin inhibitor, at the level of nucleotide and amino acid sequence (77.9 and 66.8%, respectively). The optimized alignment of the putative reactive center region of 4-12B with four related members of this superfamily revealed that lysine-serine residues are located at the reactive site or adjacent to it, thus suggesting that the triiodothyronine-regulated rat 4-12B mRNA might code for a protease inhibitor with trypsin-like specificity. Although not enough data are presently available to assign definitively antitryptic activity to this protein, the high degree of similarity with members of the superfamily of serine protease inhibitors leaves no doubt that 4-12B is a member of this superfamily.

Coding nucleotide sequence of rat liver malic enzyme mRNA.

The nucleotide sequence of the mRNA for malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) from rat liver was determined from three overlapping cDNA clones. Together, these clones contain 2078 nucleotides complementary to rat liver malic enzyme mRNA. The single open reading frame of 1761 nucleotides codes for a 585-amino acid polypeptide with a calculated molecular mass of about 65,460 daltons. The cloned cDNAs contain the complete 3'-noncoding region of 301 nucleotides for the major mRNA species of rat liver and 16 nucleotides of the 5'-noncoding region. Amino acid sequences of seven tryptic peptides (67 amino acids) from the purified protein are distributed through the single open reading frame and show excellent correspondence with the translated nucleotide sequence. The putative NADP-binding site for malic enzyme was identified by amino acid sequence homology with the NADP-binding site of the enoyl reductase domain of fatty acid synthetase.