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Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire ABO coding sequence, splice junctions, proximal promoter, and intron 1 enhancer. Amplification products were sequenced by next-generation or Sanger dideoxy methods, either directly or after cloning into a bacterial plasmid vector. Eight unreported alleles were found in the 15 cases analyzed. Alleles ABO*A(28+1C) and ABO*A(29–5G) harbor variants that alter the consensus sequence at the intron 1 donor and acceptor splice sites, respectively. The other alleles harbor variants that alter the consensus sequence at transcription factor–binding sites in the intron 1 enhancer: specifically, ABO*A(28+5792T), ABO*A(28+5859A), and ABO*A(28+5860G) at GATA-1 sites; ABO*B(28+5877T) and ABO*B(28+5878G) at a RUNX1 site; and ABO*A(28+5843A) at or near a C/EBP site. Molecular and serologic characterization of ABO alleles can help in their future identification and in the resolution of discrepancies.

Unusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entireUnusual and discrepant ABO phenotypes are often due to genetic variants that lead to altered levels or activity of ABO transferases and consequently to altered expression of ABO antigens. This report describes eight genetic alterations found in 15 cases with reduced or undetectable expression of ABO antigens. Forward and reverse ABO grouping was performed by standard gel or tube methods. Adsorption-heat elution and saliva testing for H and A substances followed the AABB technical manual procedures. Genomic DNA extracted from whole blood was PCR-amplified to cover the entire

Vegetable crops of Brassica oleracea contain significant amounts of antioxidant compounds, such as glucosinolates (GLS) which, upon disruption of the tissue, are broken down by the endogenous enzyme myrosinase (MYR), resulting in production of isothiocyanates, thiocyanates, nitriles, goitrin and epithionitriles. In particular, isothiocyanates are known to exert beneficial effects for human health. Several studies describe isothiocyanates (ITCs) as inhibitors of mitosis and stimulators of apoptosis in human tumor cells. The Sicilian black broccoli is a neglected crop of Mt. Etna towns showing a particular GLS profile mainly represented by GRA and NGBS and their derivatives. We investigated the effects of different concentrations (0-25 μM) of various GLS: gluconasturtiin (GNT), glucoraphanin (GRA), progoitrin (PRO), sinigrin (SIN), glucoiberin (GIB), gluconapin (GNA), sinalbin (GSI), glucobrassicin (GBS), glucobrassicanapin (GBN) and glucoerucin (GER) on the growth of HT29 (colon cancer) and A2058 (melanoma cancer) cells, after 24 h of treatment, in presence or not of MYR (0.018 U, in situ method). The same cell lines were also exposed to the Sicilian black broccoli juice extract (from leaves and stems) at different amount (0.05, 0.1, 0.5, 1, and 5%), with and without MYR. Cellular viability was evaluated by MTT assay. GLS, alone and in presence of MYR, elicited different grades of toxicity in both cell lines models. GRA and GNT showed less toxicity than their breakdown products and opposite results were obtained with GBS and GBN. Black broccoli juice showed to be less toxic in presence of MYR; this effect is particular evident at higher juice percentages (1 and 5%), corresponding to 5 and 25 µM, respectively of GRA and NGBS. Certainly, juice extract activity on in vitro cell lines cannot only be explained by the peculiar GLS profile; probably, the interaction of others compounds, such as polyphenols, have to be taken into account. In conclusion, not all breakdown GLS products show more toxicity than their precursors, at least in the in vitro cell cancer models we utilized.

We describe a Sicilian family presenting a recessive form of hypercholesterolemia harboring a mutation of the autosomal recessive hypercholesterolemia (ARH) gene. In two of the three sibs, a 26-year-old male and a 22-year-old female, a severe hypercholesterolemia was diagnosed with very high levels of plasma cholesterol (15.9 and 12.2 mmol/l, respectively); tendon xanthomatas and xanthelasms were present and in the male proband was documented a diffuse coronary atherosclerotic disease with a rapid and fatal progression. Both the parents had normal or slightly increased levels of plasma cholesterol. All causes of secondary hypercholesterolemia were ruled out as well as an involvement of the LDL receptor or apoB genes. β-Sitosterol plasma levels were in the normal range. Cultured fibroblasts from skin biopsy from parents and the two probands displayed a normal ability to bind and degrade 125 I-LDL. Direct sequencing of ARH gene demonstrated the presence of a 432insA mutation in homozygosis in the two probands; parents were heterozygotes for the same mutation. This mutation is the first report of a mutation of the ARH gene responsible for recessive forms of hypercholesterolemia in Sicily.

The expression of vimentin and alpha-smooth muscle (alpha-SM) actin was examined in 10 human temporomandibular joint (TMJ) disc samples, with internal derangement and in two control specimens, in order to evaluate the phenotypic characteristics of TMJ disc cells in relationship to histological findings. This was accomplished by means of monoclonal antibodies specific for vimentin and alpha-SM actin and immunocytochemical technique. The study, revealed that every disc cell constantly expressed vimentin. Scattered alpha-SM actin positive cells could be appreciated in normal TMJ discs and tissues with minor pathological findings. In TMJ discs with severe alterations, i.e. tears and clefts, almost fibroblast-like cells, fibrochondrocytes and chondrocyte-like cells were strongly immunolabelled by anti-alpha-SM actin antibody. According to these findings it can be assumed that vimentin is expressed by all disc cell populations and it appears not to be influenced by any disease condition of the disc; on the other hand the up-regulation alpha-SM actin immunolabelling seems to be correlated to histopathological findings of tears and clefts. Cells, with a contractile phenotype, close to such defects, could be involved in disc tissue contraction and repair. The plasticity of disc cell populations which evolve towards a different phenotype when subjected to action of macro- and micro-environmental factors is also supported.

Bovine seminal ribonuclease is a member of the RISBAses (ribonucleases with special biological actions) family. It exerts specific anti-tumor, embryotoxic, aspermatogenic and immunosuppressive activities. The cytotoxic effect of bovine seminal ribonuclease on tumor cells is accompanied by the induction of apoptosis. We provide ultrastructural and flow cytometry evidence of apoptotic death following bovine seminal ribonuclease treatment, in normal cells and phytohemagglutinin-stimulated lymphocytes. Transmission and scanning electron microscopy, which were fully supported by flow cytometry data, showed typical features of apoptosis, such as decreased cell size, chromatin condensation, fragmentation in micronuclei, and the presence of apoptotic bodies.

The expression pattern of the cell adhesion molecule CD44 standard form (CD44H) in dysfunctional human temporomandibular joint (TMJ) discs was studied immunohistochemically and compared with normal disc pattern in order to evaluate the expression of this adhesion molecule and correlate it to histopathological changes. Immunohistochemistry with anti-CD44H antibodies was performed on paraffin sections of pathological and normal discs. In normal TMJ discs, a moderate immunolabelling with anti-CD44H antibodies was detectable in fibroblastlike cells, in the few fibrochondrocytes and in chondrocytelike cells. In dysfunctional discs, the staining pattern and intensity varied according to the histopathological findings of the specimens. The TMJ discs showing abnormal collagen arrangement or fragmentation of collagen fibres presented overall the same immunolabelling pattern of normal discs. In the discs showing areas of fibrocartilaginous metaplasia, CD44H expression was up-regulated in fibrochondrocytes and fibroblastlike cells, especially around the chondroid tissue. Overall, these results suggest that CD44H mediates the binding of some ECM proteins in TMJ disc cells. The up-regulation of CD44H observed in some dysfunctional TMJ discs seems to indicate a prevention of apoptosis in fibroblastlike cells and an important role in phenotypical change of fibrochondrocytes into chondroblastlike cells, enabling the aggregation of chondroid tissue pericellular matrix components.

The aim of our work was to study the allelic frequencies and distribution within the 15 forensic STR loci in a group of 440 unrelated Sicilian subjects. Afterwards we have evaluated the genetic equilibrium among the most recurrent allele loci and have compared our data to those already published by other authors referring to different populations.

Cells involved in atherogenesis produce growth factors crucial for the progression of the atherosclerotic lesions. One of them is the heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, synthesized as a transmembrane precursor (proHB-EGF). This anchored insoluble juxtacrine growth factor can be converted into a soluble molecule with paracrine activity and mature HB-EGF is released in the extracellular matrix from the cell surface. HB-EGF is a potent stimulator of cell proliferation, migration and cell motility and several studies show that HB-EGF is associated with pathologies of hyperplasia of smooth muscle cells including atherosclerosis.We localized HB-EGF by immunohistochemistry within the atherosclerotic lesions collected from right or left internal carotid artery of 20 patients with evident clinical symptoms.In the 20 samples we tested, the proportion of positive samples was significant. Considering the only positive samples the proportion difference related to the gender of patients was highly significant.The aim of our investigation was to better understand if this growth factor exerts its role through a juxtacrine or paracrine mechanism, or both in the process of atherogenesis. According to the results, the paracrine role of HB-EGF was clear.

Vasoactive intestinal peptide (VIP) is an important member of the group of neuropeptides that appears to be involved in the regulation of prostatic growth and function. Here we studied VIP receptors in membranes from human benign hyperplastic prostate. Accordingly to observations in rat prostatic membranes, [125I]VIP binding to human prostatic membranes suggested two classes of binding sites with high Kd = 0.22 nM) and low (Kd = 37.7 nM) affinities. VIP bound in human and rat membrane preparations to a common VIP/pituitary adenylate cyclase-activating peptide (PACAP) receptor, as VIP, PACAP-27, and PACAP-38 were equipotent for competition of [125I]VIP binding. A PACAP-preferring receptor appears to be expressed in human prostate, since [125I]PACAP binding was displaced with more potency by PACAP than by VIP, and a messenger RNA corresponding to type I PACAP receptor was found. Cross-linking experiments suggested a VIP receptor of about 71 kDa in human and 52 kDa in rat prostates. The binding of [125I]VIP to membranes and the labeling of the bands observed after electrophoresis were competitively inhibited by GTP, suggesting the coupling of VIP receptors to a G protein. Moreover, after solubilization and cross-linking, we observed a 120-kDa band that corresponded to the VIP receptor-alpha s association. VIP stimulated adenylyl cyclase activity in a dose-dependent manner, but the potency and/or the efficacy of VIP were lower in all human preparations studied than in rat prostatic membranes. In conclusion, this study clearly demonstrates the expression of VIP/PACAP common receptors associated with alpha s protein in human prostate and suggests that these neuropeptides could play an important and complex role in the physiology and pathophysiology of this human gland.

The aim of this study is to analyze the possibility of DNA typing, on the specimen recovered at the crime scene. The optimization of extraction was carried out via an experimental system to reproduce the contact with the victim. The results obtained will be described in detail.

The aim of this study is to clarify the prevalence of gallbladder benign neoplasms, their ultrasonographic appearance and their relationship with gallbladder lithiasis and cancer.This study was carried out on 9000 consecutive patients having ultrasound of upper abdomen. Only adenomas and papillomas are considered as true benign neoplasms of the gallbladder. Adenomiomatosis and cholesterol polyps, often erroneously labelled as benign neoplasms, were excluded. Patients were followed-up by ultrasound every three months up to two years.The prevalence of benign neoplasms was 1.19%. Papillomas were found more frequently than adenomas both in males (68.51%) and in females (94.33%). Gallstones were not concomitant with benign neoplasms in any case. Neither stones nor growth of gallbladder benign neoplasms were recorded within the two-year follow-up period.Papillomas were more frequent than adenomas. No gallstone was concurrent with gallbladder benign neoplasms in our series. However, when gallstones are evidenced at ultrasound, further attention is recommended to discover probable concomitant neoplasms. Papillomas and adenomas more than 1 cm in diameter should be quarterly followed-up, while smaller masses could be six-monthly controlled. Surgery should be indicated for large-sized or rapidly growing masses because of the risk for cancer development.

Hepatitis C virus (HCV) is one of the most common blood-borne pathogens transmitted from patients to health care workers (HCWs). The Centers for Disease Control and Prevention (CDC) have developed a set of universal precautions to help prevent transmission of blood-borne pathogens between patients and HCWs in health care settings. HCV infection status among HCWs and proportion of HCWs experiencing occupational blood exposure accidents were monitored to assess the risk of HCV infection among HCWs at a hospital in Catania, Italy.The number of HCWs reporting occupational blood exposure accidents during 1999 and 2004 were compared to examine whether there was any change in the incidence of these accidents among 900 HCWs. HCV infection status of these HCWs was also analyzed in 1999 and 2004 to determine how many were infected with HCV during this time period.HCV infection was detected in 21 out of 900 subjects in 1999. The remaining 879 HCWs remained HCV-negative until they were last tested in 2004. There was a statistically significant decrease in the number of HCWs that experienced occupational blood exposure accidents from 306 in 1999 to 240 in 2004 (P = 0.001).The finding that all 871 HCV-negative HCWs remained HCV-negative from 1999 until 2004 supports the view that the set of universal precautions recommended by the CDC are helpful for preventing HCV transmission from patients to HCWs. HCWs must continue following these precautions to prevent transmission of HCV and other blood-borne pathogens between patients and HCWs in the future.

Many Y chromosome short tandem repeats (STRs) have been studied and characterized over the past years. A few Y-STRs multiplex kits have been placed on the market for forensic and population study purposes. The aim of our work was to analyse a sample of Sicilian male individuals to evaluate the allelic frequency and, thus, the possibility to implement a genetic database. An 11 loci Y-STR Typing kit was used to yield haplotype profiles from male DNA; amplification products were detected on ABI PRISM 310 Genetic Analyzer and examined by Genemapper v 3.2 (Applied Biosystems). The population sample subjected to the screening resulted to be very different with regards to certain loci whereas for other loci the allelic profile was less variable. Despite a minor discrimination power within the entire population, Y-STRs represent a valid tool to simplify male/female DNA mixture interpretation which is a major challenge when biological traces are found in case of sexual assault.

Background and Aims: Type III hyperlipoproteinemia, or dysbetalipoproteinemia, is commonly associated with apolipoprotein E2 homozygosity (Cy Background and Aims: 12, Cy Background and Aims: 58). Apo E2-Christchurch (Arg136→Ser), a rare mutation of the Apo E gene, located in the receptor-binding domain of the protein, has been found to be associated in the vast majority of cases of dysbetalipoproteinemia. Methods and Results: This is the first report of two Italian kindreds carrying the Arg136→Ser mutation. One family is a four-generation kindred from Genoa (Liguria, Italy) with a high rate of mortality due to coronary artery disease: the proband was a 51-year-old woman with previous myocardial infarction and residual angina, severe carotid atherosclerosis, peripheral arterial vascular disease and arterial hypertension. The other family was identified in Palermo (Sicily, Italy): the proband was an overweight 62-year-old man with a mixed form of hyperlipidemia. The mutation, which was identified by means of Apo E genotyping followed by direct sequencing, co-segregated with the same haplotype in the two families. Conclusions: The family histories and clinical examinations of these subjects clearly show that the Apo E Arg136→Ser variant fully expresses a type III phenotype in association with a second allele coding for Apo E2, and only partially in association with a second allele coding for Apo E4.

CD surface molecules mediates cell activation and signaling. In particular, CD14 on blood monocytes mediate monocyte/macrophage activation by lipopolysaccharide. Lipopolysaccharide and its receptor, CD14, have been implicated in atherogenesis. It has been recently shown that a C(-260)T polymorphism in the promoter of the CD14 receptor may be a risk factor for coronary artery disease. Recently this association has been questioned because no increased risk was found with the T allele, even in the homozygous state. In the present study we investigated a possible association between the C(-260)T polymorphism in the CD14 promoter and acute myocardial infarction. Two hundred and thrteen patients with and acute myocardial infarction 213 healthy controls were included in the study. Genotype frequencies of the C(-260)T polymorphism in the CD14 promoter were determined by polimerase chain reaction and the amplified product was cleaved with HaeIII. The frequency of the T allele was not significantly different in patients compared with controls. In this study we were not able to detect differences of frequency of the allele T (-260) in the promoter of the CD 14 receptor gene in survivors of myocardial infarction and controls.

Familial defective apolipoprotein (apo) B-100 together with familial hypercholesterolemia are the two common genetic conditions that cause hypercholesterolemia. Familial defective apolipoprotein B-100 is due to mutations around codon 3500 of the apo B gene. The most-characterized mutation is a G>A transition at nucleotide 10,708 that results in the substitution of arginine by glutamine at codon 3500 (Apo B Arg3500Gln). Two other mutations are caused by a C>T transition, one at nucleotide 10,800 (Apo B Arg3531Cys) and the other at nucleotide 10,707 (apo B Arg3500Trp). In the present study we describe three new Italian cases of familial defective apolipoprotein B-100 (Apo B Arg3500Gln), one from the Liguria region and two from Sicily, and the haplotype of the apo B gene co-segregating with the mutation. By screening two groups of probands, clinically diagnosed as having Familial Hypercholesterolemia (700 from mainland Italy and 305 from Sicily), the prevalence of familial defective apolipoprotein B-100 due to Arg3500Gln was found to be very low (0.28% and 0.65%, respectively). The Arg3531Cys mutation was not detected in any proband. In the three new families with Arg3500Gln mutation in the present study and in one previously described in Italy, the mutation was associated with a unique apo B haplotype, which is consistent with data previously reported for Caucasian patients [XbaI-, MspI+, EcoRI-, presence of the 5' signal peptide insertion (Ins) allele, and the 49-repeat allele of the 3'-VNTR].

Heat shock protein (HSP) 27 is a member of the small HSP family that plays a part in the regulation of epithelial cell growth and differentiation, wound healing, apoptosis and cell protection against inflammatory cytotoxicity mediators. Thus the expression of HSP27 was investigated immunohistochemically in periapical granulomas with epithelial rests of Malassez and in radicular cysts. Anti-HSP27 mouse monoclonal antibody and peroxidase-labeled streptavidin-biotin standard technique were used to study the expression of HSP27. Proliferating epithelial cell rests, and islands of epithelium and epithelial lining of microcysts strongly reacted throughout all layers, whereas radicular cysts epithelial lining presented mainly a moderate suprabasal staining pattern. However both the proliferating epithelial cell rests and the radicular cysts shared an over-expression of HSP27 immunostaining intensity in coincidence with the presence of local infiltration of immune cells. HSP27 may play several roles in periapical lesions that include contributing to the migration of epithelial cell rests and an increased resistance both to necrotic and apoptotic cell deaths.

The expression pattern of the cell adhesion molecule CD44 standard form (CD44H) in dysfunctional human temporomandibular joint (TMJ) discs was studied immunohistochemically and compared with normal disc pattern in order to evaluate the expression of this adhesion molecule and correlate it to histopathological changes. Immunohistochemistry with anti-CD44H antibodies was performed on paraffin sections of pathological and normal discs. In normal TMJ discs, a moderate immunolabelling with anti-CD44H antibodies was detectable in fibroblastlike cells, in the few fibrochondrocytes and in chondrocytelike cells. In dysfunctional discs, the staining pattern and intensity varied according to the histopathological findings of the specimens. The TMJ discs showing abnormal collagen arrangement or fragmentation of collagen fibres presented overall the same immunolabelling pattern of normal discs. In the discs showing areas of fibrocartilaginous metaplasia, CD44H expression was upregulated in fibrochondrocytes and fibroblastlike cells, especially around the chondroid tissue. Overall, these results suggest that CD44H mediates the binding of some ECM proteins in TMJ disc cells. The up-regulation of CD44H observed in some dysfunctional TMJ discs seems to indicate a prevention of apoptosis in fibroblastlike cells and an important role in phenotypical change of fibrochondrocytes into chondroblastlike cells, enabling the aggregation of chondroid tissue pericellular matrix components.

Clinical epidemiologic studies carried out in smokers versus non smokers support the concept that tobacco-related factor may affect the periodontal tissues and wound healing.In this study, the role of nicotine on the integrin alpha 2 chain immunocytochemical expression, in human gingival fibroblasts (HGF) was investigated in vitro. The kinetic induction of this type of integrin on HGF in response to increasing percentage of nicotine was been characterized. A human gingival fibroblast strain, derived from a healthy individual with non-inflamed gingiva, was used in this study. The cells were then grown on acetylated microscope slides and fixed with cold ethanol, samples were then incubated for 16 to 19 hrs at 4 degrees C to anti-human alpha 2 integrin chain monoclonal antibody.In control cultures and in HGF treated with 0.025 microM nicotine the initial higher expression of alpha 2 chain decreased (grade 1 in both culture) while in HGF treated with 0.075 microM increased (grade 3). After 48 hours HGF exposed to 0.075 microM nicotine, increased further their expression of alpha 2 chain.The results obtained demonstrate a dose-time dependent nicotine influence on immunocytochemical expression of alpha 2 integrin chain. These data suggest that nicotine may promote a collagene breakdown via an increase of alpha 2 integrin chain expression.

S-100 protein was detected immunohistochemically in diseased human temporomandibular joint discs with different degrees of pathology, and the findings compared with those of normal discs. In normal discs, large nerve trunks in the posterior ligament were strongly stained by anti-S-100 antiserum; the very few chondrocyte-like cells sometimes showed faint staining, while no staining was observed in any fibrochondrocyte-like or fibroblast-like cell. In dysfunctional discs, S-100 protein immunostaining seemed to correlate with structural pathological findings. The discs showing an abnormal collagen arrangement or fragmentation of collagen fibres presented overall the same immunolabelling pattern as normal discs. In discs with fibrocartilaginous metaplasia and dystrophic cartilage formation, fibrochondrocyte cells showed a very strong immunoreaction for S-100 protein and fibroblast-like cells in some instances were also positive. These findings suggest that S-100 upregulation in disc cells can be considered an attempt at tissue repair by chondroid metaplasia following an injury in that it enables fibroblast-like cells and fibrochondrocytes to acquire a chondrogenic phenotype.

The LDL-receptor gene point mutation FH-Genoa/Palermo is the most frequent mutation responsible for Familial Hypercholesterolemia in Sicily. The mutation does not introduce or abolish any useful restriction site. We establish a GeneComb-based strategy to identify this mutation in a population of Sicilian unrelated clinically diagnosed FH probands. The method was very sensitive and specific; 12 out of 90 (13.3%) unrelated FH probands were found to carry the FH-Genoa/Palermo mutation. According to these results, the FH-Genoa/Palermo is the more frequent LDL-receptor gene mutation among the Sicilian FH patients. Moreover FH-Genoa/Palermo is the mutation cluster to date more represented in Southern Italy.

Palmar fibromatosis is characterized by changes in the expression of extracellular matrix (ECM) proteins and re-organization of the interactions between cellular and extracellular compartments. We compared the expression and distribution of alpha2beta1, alpha6beta1, alpha5beta1 integrins and their corresponding extracellular ligands, type IV collagen, laminin and fibronectin in palmar fibromatosis nodules from 24 patients. Depending on the degree of cellularity and fibrosis, three different histologic phases were identified: proliferative, involutional, and residual. Immunohistochemistry revealed that only stromal cells of the highly cellular areas of both proliferative and involutional phases were myofibroblasts, as demonstrated by their cytoplasmic positivity for alpha-smooth-muscle actin. The ECM, surrounding these cells, was strongly and diffusely positive to type IV collagen, laminin and fibronectin, whereas no immunoreactivity was found in the ECM of the fibrotic and hypocellular areas of both involutional and residual phases. Immunostaining for alpha2beta1, alpha6beta1 and alpha5beta1 integrins revealed that alpha5beta1 integrin was expressed and restricted to the myofibroblast-rich cellular areas, whereas no expression was detected for alpha2beta1 and alpha6beta1 integrins. By examining serial sections, a co-localization of alpha5beta1 integrin and fibronectin was observed in the myofibroblast-rich cellular areas, indicating that, in palmar fibromatosis, a co-ordinate expression between cellular and extracellular ligand is detected only for the alpha5beta1 integrin/fibronectin complex. These findings suggest that the alpha5beta1 integrin/fibronectin complex may be involved in regulating the interactions between myofibroblasts and ECM in palmar fibromatosis.

Introduction Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen. The objective of this study was to verify the proregenerative effects of VEGF in an experimental model of acute liver failure. Materials and methods Sixty four rats that underwent intraperitoneal injection of carbon tetrachloride (CCl 4 ) were randomly divided into two groups: group B animals received intravenous injection of VEGF 164 1 hour following CCl 4 poisoning. Group A hosts were untreated. To obtain daily liver function tests (LFTs) and histological samples, on each day up to 8 days we sacrificed four rats in each group. Results The laboratory examinations showed notable alteration of LFTs in group A, while group B revealed only slight changes. The histological examination showed greater liver damage in group A compared with group B. Conclusion Our results suggest that administration of exogenous VEGF protects the liver from CCl 4 -induced acute hepatic failure. Further studies are underway to assess whether exogenous VEGF is effective in other liver injuries.

Apolipoprotein H is a protein of about 50 kilodaltons, structurally related to the regulators of the complement activation family. Its physiological function is poorly understood but it has been implicated in lipid metabolism and coagulative pathways. The major site of synthesis is thought to be the liver. Several reports indicate that apolipoprotein H is the antigen of the antiphospholipid antibodies and also behaves as an acute-phase reactant. Moreover, 40% of plasma apolipoprotein H is associated with very low-density lipoprotein, high-density lipoprotein, and postprandial chylomicrons. In this study we investigated other sites of synthesis by reverse transcription/polymerase chain reaction and we found apolipoprotein H mRNA expression in intestinal cell lines and tissues. Immunohistochemistry was performed on various fresh and paraffin-embedded tissues and apolipoprotein H was immunolocalized in the cytoplasm of hepatocytes and epithelial cells from colon and jejunum. This study indicates that apolipoprotein H is expressed at both mRNA and protein levels in enterocytes.

The two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP27 and PACAP38, are neuropeptide hormones related to the vasoactive intestinal peptide/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylyl cyclase and phospholipase C have been recently identified. We have investigated the expression of PACAP-Rs in undifferentiated and differentiated PC-12 cells. PACAP27 and PACAP38 failed to significantly increase cAMP or [3H]inositol monophosphate levels in undifferentiated PC-12 cells treated with vehicle, insulin-like growth factor I, or epidermal growth factor but greatly elevated levels after differentiation with nerve growth factor (NGF) or basic fibroblast growth factor. PACAP responsiveness increased significantly after 24 hr of NGF treatment, reaching a maximum within 4 days. At this time of differentiation, the effect of PACAP was dose dependent between 1 nM and 0.1 microM, whereas vasoactive intestinal peptide, at the maximal dose of 10 microM, slightly increased cAMP formation and failed to affect [3H]inositol monophosphate content. Radioreceptor assays, performed with 125I-PACAP27, revealed the induction of high affinity type I PACAP receptors in differentiated PC-12 cells. Using reverse transcription-polymerase chain reaction methodology, we showed the absence of type I PACAP receptor mRNAs in undifferentiated PC-12 cells and the expression of PACAP-R-hop mRNA after NGF or basic fibroblast growth factor treatment. The increased PACAP responsiveness induced by these growth factors in PC-12 cells may therefore result from the expression of the PACAP-R-hop isoform, positively coupled to both adenylyl cyclase and phospholipase C.

The p53 tumor suppressor gene plays a role in controlling a G1 phase checkpoint. The WAF1/CIP1 gene with encodes p21WAF1/CIP1 protein, an inhibitor of cyclin-dependent kinases, is a downstream mediator of p53 function. We examined expression of the WAF1/CIP1 gene and its relationship to growth arrest and differentiation in p53-null human leukemic cell lines. We show that p53-independent induction of WAF1/CIP1 occurs in human leukemia cells treated with 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, or IFN-gamma but not with retinoic acid, vitamin D3, or DMSO. Furthermore, WAF1/CIP1 induction correlates with growth arrest associated with monocyte-macrophage differentiation. The present studies support the idea that WAF1/CIP1 gene expression can be regulated through multiple mechanisms, suggesting that strategies may be designed to restore the G1 checkpoint controls in p53-null cells by targeting these p53-independent mechanisms of WAF1/CIP1 induction.

Overexpression of wild-type p53 protein has been shown to induce arrest in the G1 stage of the cell cycle and to transactivate expression of the gene that encodes the 21-kDa Waf1/Cip1 protein, a potent inhibitor of cyclin-dependent kinase activity. p53-dependent G1 arrest is accompanied by decreased expression of the B-myb gene, a relative of the c-myb cellular oncogene. In this study we show that B-myb expression is required for cells to progress from G1 into S phase and that high levels of ectopic B-myb expression uncoupled from cell cycle regulation rescues cells from p53-induced G1 arrest even in the presence of Waf1/Cip1 transactivation and inhibition of cyclin E/Cdk2 kinase activity. Cotransfection experiments with p53 expression plasmids and expression plasmids encoding in-frame deletion mutations in B-myb coding sequences indicate that the DNA-binding domain of the B-Myb protein is required for this activity. These results provide evidence of a bypass of p53-induced Waf1/Cip1-mediated cell cycle regulatory pathways by a member of the myb oncogene family.

The proliferative capacity of the immune system is impaired in elderly subjects and the expression of various genes involved in cell cycle progression is reduced in PHA stimulated lymphocytes during the aging process. Macrophages play a fundamental role in the immune system response. It has recently been demonstrated that the process of macrophage activation is accompanied by a rapid, transient rise of ornithine decarboxylase (ODC) mRNA levels. In fact, the ODC gene seems to be involved in macrophage activation and differentiation. The authors demonstrated that the steady-state levels of ODC mRNA and the correlated superoxide anion production are lower in the monocytes of elderly subjects with respect to those in young subjects used as control. These results confirmed the impaired immune function of the elderly.

The c-myb protooncogene, which is preferentially expressed in hematopoietic cells at the G1/S boundary of the cell cycle, encodes a transcriptional activator that functions via DNA binding. The regulatory mechanisms governing this specific pattern of expression are not fully understood, although human c-myb expression appears to be positively autoregulated via myb-binding sites in the 5'-flanking region of the c-myb gene (Nicolaides, N. C., Gualdi, R., Casadevall, C., Manzella, L., and Calabretta, B. (1991) Mol. Cell. Biol. 11, 6166-6176). To determine the contribution of other transcription regulators such as JUN family members in the control of c-myb expression, transient expression assays were carried out which revealed a 6- to a 15-fold enhancement by c-Jun and JunD, but not JunB, in chloramphenicol acetyltransferase reporter gene expression driven by different segments of the human c-myb 5'-flanking region. An Ap1-like element located at nucleotide -149 from the c-myb initiation site appears to be required for this transactivation upon binding to a nuclear protein complex containing c-Jun and JunD, since site-directed mutations of this Ap1-like element abolished c-Jun and JunD binding and transactivation. Exposure of phytohemagglutinin-stimulated peripheral blood mononuclear cells to c-jun and junD antisense oligodeoxynucleotides resulted in a 46 and 43% inhibition of T-lymphocyte proliferation that was accompanied by a decrease in c-myb mRNA levels as compared with sense-treated cultures. Because T-lymphocytes induced to proliferate express c-jun and junD before c-myb, these data suggest a mechanism whereby c-Jun and JunD contribute to the transcriptional activation of c-myb that, in turn, is maintained at the G1/S transition and during S phase by positive autoregulation.

The protooncogene c-myb is the cellular equivalent of the viral transforming oncogene v-myb. When human c-myb is constitutively expressed in Balb/c3T3 cells it abrogates their absolute requirement for insulin-like growth factor 1 (IGF-1). We show now, in two different cell lines, that the constitutive expression of the protooncogene c-myb causes an increase in both IGF-1 and IGF = 1 receptor mRNA levels. This increase in mRNA levels is due, at least in part, to an increase in the rate of transcription since, by run-on assay, cells carrying the human c-myb cDNA show a 3-fold increase in transcriptional rates in comparison to the control parent cell lines. The increased expression of IGF-1 receptor mRNA also results in an increased number of IGF-1 binding sites per cell. Although some oncogenes have been described that are homologous to growth factors, or growth factor receptors, c-myb seems to represent a novel way of oncogene action inasmuch as it increases the expression of both a growth factor receptor and its ligand, thus establishing a quasi-autocrine mechanism which modifies the growth factor requirements of the cell and its growth regulation.

Tk-ts13 cells are BHK-derived fibroblasts that have a G1-specific temperature sensitive (ts) mutation so that the cells arrest in the G1 phase of the cell cycle at the restrictive temperature of 39.6 degrees. We have introduced into these cells a plasmid carrying the human c-myb cDNA under the control of the early SV40 promoter. The resulting cell line, ts13 myb cells, express the c-myb RNA and protein and, when serum-stimulated, they undergo one round of DNA replication even at the restrictive temperature. Under the same conditions, the parent cell line (tk-ts13 cells) are blocked in G1 and fail to replicate DNA. We have investigated the expression of two late growth-regulated genes, PCNA and histone H3, in tk-ts13 and in ts 13 myb cells, both at permissive and restrictive temperature. At the permissive temperature of 34 degrees, the mRNA levels for PCNA and histone H3 increase markedly after serum stimulation in both cell lines, reaching a peak at the time of DNA synthesis. At the restrictive temperature of 39.6 degrees, the mRNA's for PCNA, and histone H3 are detectable in serum-stimulated ts13 myb cells while they are not detectable in the parent tk-ts13 cell line. Run-on transcription assays indicate that these 2 genes are transcribed in tk-ts13 cells equally well at permissive and nonpermissive temperatures, and that the presence of the myb product does not significantly increase their rates of transcription. The stability of the respective mRNA's is roughly the same at either temperature and in both types of cells. These results indicate: (1) a constitutively expressed c-myb gene product confers to fibroblasts the ability of temporarily by-passing a ts block in the G1 phase of the cell cycle and (2) the myb product, under these conditions, regulates the mRNA levels of PCNA and histone H3 either directly or indirectly by a post-transcriptional mechanism.

The LDL-receptor gene point mutation FH-Genoa/Palermo is the most frequent mutation responsible for Familial Hypercholesterolemia in Sicily. The mutation does not introduce or abolish any useful restriction site. We establish a GeneComb-based strategy to identify this mutation in a population of Sicilian unrelated clinically diagnosed FH probands. The method was very sensitive and specific; 12 out of 90 (13.3%) unrelated FH probands were found to carry the FH-Genoa/Palermo mutation. According to these results, the FH-Genoa/Palermo is the more frequent LDL-receptor gene mutation among the Sicilian FH patients. Moreover FH-Genoa/Palermo is the mutation cluster to date more represented in Southern Italy.

Oncogenes play a major role in the control of proliferation in animal cells. Because the growth of cell populations is regulated by the stimulatory and inhibitory growth factors in the environment, we have attempted in this review to relate the function of oncogenes to the mechanism of action of growth factors. This correlation allows a description of the cell cycle that rests on a molecular basis and in which protooncogenes figure prominently as the cellular messengers of the environmental growth stimuli.

The LDL-receptor gene point mutation FH-Genoa/Palermo is the most frequent mutation responsible for Familial Hypercholesterolemia in Sicily. The mutation does not introduce or abolish any useful restriction site. We establish a GeneComb™-based strategy to identify this mutation in a population of Sicilian unrelated clinically diagnosed FH probands. The method was very sensitive and specific; 12 out of 90 (13.3%) unrelated FH probands were found to carry the FH-Genoa/Palermo mutation. According to these results, the FH-Genoa/Palermo is the more frequent LDL-receptor gene mutation among the Sicilian FH patients. Moreover FH-Genoa/Palermo is the mutation cluster to date more represented in Southern Italy. Hum Mutat 13:412–412, 1999. © 1999 Wiley-Liss, Inc.

We have studied modifications of LDH isoenzymes pattern in normal human gastric mucosa as well as in adenocarcinoma and precancerous lesions of the stomac (gastritis and ulcer); samples from the injured and the surrounding non-injured area were examined, drawing up the isoenzymes, using Tris-buffer pH 7,4 at 4 degrees C and performing the determination within 1 h - because of the high chronolability of the fractions LDH and LDH by cellogol electrophoresis separation. We have always noticed - especially in samples from adenocarcinoma- a shifting toward the M chains, with a clear increase of the fractions LDH4 and LDH5; this has been noticed even in the surrounding non-injured area.