Amphiphysin is necessary for organization of the excitation–contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.     

Amphiphysin IIm, a novel amphiphysin II isoform, is required for macrophage phagocytosis

Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive immune response. Amphiphysin II participates in receptor-mediated endocytosis, in part, by recruiting the GTPase dynamin to the nascent endosome. We demonstrate here that a novel isoform of amphiphysin II associates with early phagosomes in macrophages. We have ablated the dynamin-binding site of this protein and shown that this mutant form of amphiphysin II inhibits phagocytosis at the stage of membrane extension around the bound particles. We define a signaling cascade in which PI3K is required to recruit amphiphysin II to the phagosome, and amphiphysin II in turn recruits dynamin. Thus, amphiphysin II facilitates a critical initial step in host response to infection.     

Amphiphysin I phosphorylation on residue threonine 260 in a pentylenetetrazole-induced seizure model

A method to evaluate kinase inhibitor action was reported [L. Morgan, S.J. Neame, H. Child, R. Chung, B. Shah, L. Barden, J.M. Staddon, T.R. Patel, Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system, Neurosci. Lett. 395 (2006) 143–148]. In this, acute administration of the GABA antagonist pentylenetetrazole triggers seizures through glutamate-dependent pathways. Under such conditions, activation of the c-Jun N-terminal kinase (JNK) pathway was detected in hippocampal extracts. Phosphorylation of the upstream JNK kinase MKK4 was also revealed through use of a phospho-MKK4-specific antibody. Here, this antibody is shown to also react with a protein of ∼125 kDa which underwent increased phosphorylation in response to pentylenetetrazole treatment. The present study aimed to identify the ∼125 kDa protein as it may provide novel insight into signalling, neuronal activity and seizures. Using chromatographic methods and mass spectrometry, the protein was identified as amphiphysin I. This was confirmed by 2D gel analysis and immunoblot with amphiphysin I-specific antibodies. Although the phospho-MKK4 antibody was raised against an MKK4-specific peptide, partial sequence homology between this sequence and a region of amphiphysin was discerned. New antibodies raised against the phospho-threonine 260-amphiphysin-specific sequence detected increased phosphorylation in response to pentylenetetrazole treatment. This particular phosphorylation site does not seem to have been described before, possibly reflecting a novel regulatory aspect of amphiphysin biology. As amphiphysin is involved in the regulation of endocytosis, phosphorylation at this site may play a role in the regulated re-uptake of synaptic vesicles after neurotransmitter release.     

Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis

Dynamin I is dephosphorylated at Ser-774 and Ser-778 during synaptic vesicle endocytosis (SVE) in nerve terminals. Phosphorylation was proposed to regulate the assembly of an endocytic protein complex with amphiphysin or endophilin. Instead, we found it recruits syndapin I for SVE and does not control amphiphysin or endophilin binding in rat synaptosomes. After depolarization, syndapin showed a calcineurin-mediated interaction with dynamin. A peptide mimicking the phosphorylation sites disrupted the dynamin-syndapin complex, not the dynamin-endophilin complex, arrested SVE and produced glutamate release fatigue after repetitive stimulation. Pseudophosphorylation of Ser-774 or Ser-778 inhibited syndapin binding without affecting amphiphysin recruitment. Site mutagenesis to alanine arrested SVE in cultured neurons. The effects of the sites were additive for syndapin I binding and SVE. Thus syndapin I is a central component of the endocytic protein complex for SVE via stimulus-dependent recruitment to dynamin I and has a key role in synaptic transmission.     

Reduction in levels of amphiphysin 1 mRNA in the hippocampus of aged rats subjected to repeated variable stress

Various neurobiological studies of aging indicate that elevated levels of circulating glucocorticoids lead to hippocampal vulnerability to stress, though little is known about the molecular mechanism underlying stress vulnerability in the elderly. We have compared the gene expression profiles in the hippocampus of aged (20 months) and adult (3 months) rats in response to repeated variable stress (RVS) for 4 days, using a cDNA array technique and real-time quantitative PCR, to identify putative genes involved in the mechanism of stress vulnerability in the elderly. We found a significant decrease in the levels of amphiphysin 1 mRNA in aged rats subjected to RVS compared with treated and untreated adult rats or to untreated aged rats. Similarly, we found a significant decrease in hippocampal levels of amphiphysin 1 mRNA in aged rats subjected to RVS for 8 days, but not in those subjected to a single VS. These findings suggest that the decrease in the hippocampal levels of amphiphysin 1 mRNA in response to repeated stress may be involved in the stress vulnerability in the elderly, and may lead to the disturbance of learning and memory under stressful conditions in the elderly.     

Gsα, protein kinase C, cathepsin D, growth factors, estrogen receptor-related protein, and p53 in prolactin cell adenomas and null cell adenomas of the pituitary

The aim of the present study was to assess the prevalence of the expression pattern of different growth factors and growth-related signaling and transport proteins, including the α-subunit of stimulatory G-protein, protein kinase C (PKC), cathepsin D, epidermal growth factor (EGF), transforming growth factor-α (TGF-α), insulin-like growth factor-1 (IGF-1), estrogen receptor-related protein (p29), and p53 in human pituitary tumors. Immunohistochemistry (IHC) was performed by screening tissue sections from 32 patients, including 18 prolactin cell adenomas and 14 null cell adenomas. IHC demonstrated an increased reactivity for Gsα in 6 prolactin cell and 12 null cell adenomas. PKC had a weak immunoreactivity in all pituitary adenomas. Cathepsin D was absent in the majority of pituitary adenomas. The remaining adenomas showed weak positivity in some scattered tumor cells. Low levels of EGF expression were found in 13 prolactin cell and 3 null cell adenomas. Immunoreactivity for TGF-α and IGF-1 was observed in all cases. The prolactin cell adenomas exhibited stronger TGF-α- and IGF-1 labeling compared with the null cell adenomas. The estrogen receptor-related protein (p29) was identified in only one prolactin cell adenoma. p53 protein expression was not present in any pituitary sample. It is suggested that Gsα overexpression, PKC, growth factor, and absent or decreased p29 expression may be involved in pituitary tumorigenesis. p53 and cathepsin D appear to play no consistent role in the pathogenesis of pituitary adenomas.     

Prognostic Significance of p27, Ki-67, and Topoisomerase lla Expression in Clinically Nonfunctioning Pancreatic Endocrine Tumors

Nonfunctioning islet cell tumors or pancreatic endocrine tumors are the most common type of malignant islet cell tumor. Although previously detected usually at an advanced stage because of mass effect, the early detection rate of small localized disease has been increasing. To date it has been difficult to predict the clinical behavior in localized regional nonfunctioning tumors. To investigate potential markers predicting malignancy and poor prognosis in nonfunctioning pancreatic endocrine tumors, we analyzed the expression of Ki-67, topoisomerase IIα (Topollα), and p27, as well as a variety of clinicopathologic parameters in 76 cases of nonfunctioning islet cell tumors (23 benign cases and 53 malignant cases). Ki-67, Topollα, and p27 labeling indices were significantly different between benign and malignant tumors. Expression of Ki-67, Topollα, and p27 were associated with survival in patients with a malignant tumor in a univariate setting. However, only p27 and Topollα were jointly associated with survival in multivariate analysis. Immunohistochemical staining for p27, Topollα, and Ki-67 can be helpful in the diagnosis of nonfunctioning pancreatic endocrine tumor. Analysis of p27 and Topollα may also have potential utility as prognostic factors for malignant tumors.     

TGFB,TGFB Receptors,Ki-67, and p27(Kip)l Expression in Papillary Thyroid Carcinomas

Although most papillary thyroid carcinomas behave as low-grade neoplasms and are generally associated with a good prognosis, some subgroups of these neoplasms represent more aggressive variants. In order to determine if differences in the behavior of these papillary carcinomas were related to expression of growth factors or cell-cycle proteins, we analyzed a series of papillary carcinomas including the conventional or usual type (n=27), tall cell (n=27), diffuse sclerosing (n=5), and columnar cell (n=2) variants for expression of transforming growth factor beta (TGFβ), TGFβ receptors (TGFβ-RI and II, the proliferation marker Ki-67, and for the cell-cycle inhibitory protein p27^Kip1 (p27). All groups of thyroid tumors expressed TGFβ and TGFβ-RI and RII by immunohistochemical staining. These was a marked increase in the Ki-67 labeling index after staining with antibody MIB-1 in the columnar cell tumors compared to the other groups, but this difference was not significant because of the small number of tumors in this group. The cell-cycle inhibitory protein p27 was expressed in all groups and was not significantly different between groups. Normal thyroid cells had a higher labeling index for p27 compared to papillary carcinomas. These results indicate that TGFβ and TGFβ receptors I and II are commonly expressed in the usual and in variant forms of papillary thyroid carcinomas, and that there is decreased expression of p27 protein in all of these neoplasms compared to normal thyroid. The biological basis for the more aggressive behavior of these variants of papillary thyroid carcinoma remains uncertain.     

Lipids in the Processes of Exo- and Endocytosis of Synaptic Vesicles

The phenomenon of synaptic transmission is underlain by the processes of exo- and endocytosis of synaptic vesicles, which involve complex protein mechanisms. The proteins forming the SNARE complex (synaptobrevin, syntaxin, SNAP-25), synaptotagmin, Munc-13, Munc-18, NSF, and α-SNAP, are involved in exocytosis, while other proteins (clathrin, AP-2, epsin, endophilin, amphiphysin, dynamin, synaptojanin, Hsc70, and others) mediate the endocytosis of synaptic vesicles. Recent years have seen the accumulation of data on the critical roles of various lipids in exo- and endocytosis. Particularly interesting results were obtained in relation to the significance of cholesterol, sphingomyelins, phosphoinositides, and phosphatidic and polyunsaturated fatty acids for the exo-endocytic cycle. The involvement of lipid rafts in the recycling of synaptic vesicles is discussed. We present contemporary data, including our own results, on the role of lipids and lipid-modifying enzymes in the processes of exo- and endocytosis.     

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus

Summary.  The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5′-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5′-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3′-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF. JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5′ end of the viral RNA is uncapped. The 3′ end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae. Accepted September 23, 1999     

DNA Aneuploidy, S-phase fraction, nuclear p53 positivity, and survival in non-small-cell lung carcinoma

 Inactivation of the p53 gene plays a key role in tumour biology, probably through a disturbed cell cycle control and an increased genetic instability in p53-inactivated tumours. To learn more about the relationship between p53 alterations, proliferation and genetic instability (DNA aneuploidy) in lung cancer patients, specimens of 220 surgically resected lung carcinomas with clinical follow-up information were examined by immunohistochemistry (p53; CM1) and flow cytometry. Nuclear p53 positivity – found in 49.5% of the tumours – was associated with both high S-phase fraction (SPF) and DNA ploidy aberrations. SPF was higher in p53-positive tumours (15.9 ± 10.2) than in p53-negative tumours (10.3 ± 8.7; P = 0.03). The rate of p53 positivity was higher in 101 DNA-aneuploid and DNA-multiploid tumours (55%) than in 27 diploid and peridiploid carcinomas (33%; P = 0.0512). These results are consistent with an in vivo role of p53 inactivation for increased proliferative activity and development of genomic instability in lung cancer. There was no association between SPF and prognosis. Although prognosis was worse in DNA-aneuploid and multiploid tumours than in diploid, peridiploid and tetraploid carcinomas (P = 0.029), DNA ploidy was not an independent predictor of poor prognosis in multivariate analysis. These data show that DNA-flow cytometry has little prognostic value for patients with resected non-small-cell lung carcinoma. Received: 18 February 1997 / Accepted: 25 March 1997     

Dielectric analysis of blood by means of a raster-electrode technique

Dielectric measurements were made on blood samples containing erythrocytes of varying diameter D and percentage p. For effective measurements of the conductivity γ and the dielectric constant ε, in the frequency range f=10–100 kHz electrode effects were corrected by means of a raster-electrode technique, which is based on the automatic variation of the effective electrode area. The results, which proved to be independent of f, indicate that an increase of haematocrit p is linked with a strong decrease of γ, being essentially independent of D. For low and medium p an increase of ε, resulted from increasing p. For physiological values of p close to 40 per cent, a strong increase of ε, was found with increasing D, indicating possibilities of using the method for rapid determination of D in addition to p. For very high values of p (>60 per cent) ε, showed a distinct decrease. This finding is discussed using a cube model for the particle suspension.     

Genetic heterogeneity in a prostatic carcinoma and associated prostatic intraepithelial neoplasia as demonstrated by combined use of laser-microdissection, degenerate oligonucleotide primed PCR and comparative genomic hybridization

 We combined laser-assisted microdissection from H&E-stained paraffin sections, degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR), and comparative genomic hybridization (CGH) to analyse chromosomal imbalances in small tumour areas consisting of 50–100 cells. This approach was used to investigate intratumour genetic heterogeneity in a case of metastatic prostatic adenocarcinoma and chromosomal changes in areas of prostatic intraepithelial neoplasia (PIN) adjacent to the invasive tumour. In four microdissected invasive tumour areas with different histological patterns (acinar, cribriform, papillary and solid) marked intratumour heterogeneity was found by CGH. Recurrent chromosomal imbalances detected in at least two microdissected tumour areas were gains on 1p32→p36, 2p22, 3q21, 7, 8q21→q24, 11q12→q13, 16p12→p13, 17, 19 and loss on 16q23. Additional chromosomal changes were found in only one of the microdissected areas (gains on 16q21→q23, 20q22 and losses on 8p21→p23, 12p11→q12, 12q21→q26, 13q21→q34, 16q12, and 18q22). In PIN, gains on chromosomes 8q21→q24 and 17 were found in both samples investigated (low and high grade PIN), while gains on chromosomes 7, 11q, 12q, 16p, and 20q and losses on 2p, 8p21→p23, 12q were found only in one PIN area. Controls to ensure reliable CGH results consisted in CGH analyses of (i) approximately 80 microdissected normal epithelial cells, which showed no aberrations after DOP-PCR and (ii) larger cell numbers (approximately 10⁵ or 10⁷ cells) of the primary tumour investigated without DOP-PCR and partially displaying the chromosomal imbalances (gain on 16p12→p13, losses on 2p25, 8p21→p23, 12p11→p12, 12q21→q26, 18q22) found in the small microdissected areas. Microsatellite and FISH analyses further confirmed our CGH results from microdissected cells. The combined approach of laser-assisted microdissection, DOP-PCR and CGH is suitable to identify early genetic changes in PIN and chromosomal imbalances associated with the particular histological patterns of invasive prostatic adenocarcinoma. Received: 30 January 1998 / Accepted: 25 May 1998     

Identification of chromosomal regions linked to premature myocardial infarction: a meta-analysis of whole-genome searches

Myocardial infarction (MI) is a complication of coronary artery disease and the leading cause of death in the Western world. MI is considered a distinct phenotype with an increased genetic component for its premature type. MI’s exact inheritance pattern is still unknown. Genome searches for identifying susceptibility loci for premature MI produced inconclusive or inconsistent results. Thus, a genome search meta-analysis (GSMA) was applied to available genome search data on premature MI. GSMA is a non-parametric method to identify genetic regions that rank high, on average in terms of linkage statistics across genome searches unweighted or weighted by study size. The significance of each region’s average and heterogeneity, unadjusted or adjusted by neighbouring average simulated ranks, was calculated using a Monte Carlo test. The meta-analysis involved five genome searches in Caucasians. Eight regions (6p22.3–6p21.1, 14p13–14q13.1, 13q33.1–13q34, 5p15.33–5p15.1, 8q13.2–8q22.2, 1p36.21–1p35.2, 12q24.31–12q24.33, 8q24.21–8q24.3) were found to have significant average rank by either unweighted or weighted analyses. In addition, region 8q24.21–8q24.3 produced significant low heterogeneity (P _unadjusted=0.03 and P _adjusted=0.05). Four regions (6p22.3–6p21.1, 14p13–14q13.1, 8q13.2–8q22.2, 8q24.21–8q24.3) were not identified by the individual studies. The meta-analysis suggests that these four regions should be further investigated for genes that confer susceptibility to MI.     

Classification of parietal pleural invasion at adhesion sites with surgical specimens of lung cancer and implications for prognosis

Parietal pleural invasion of non-small cell lung cancer (NSCLC) is a factor for poor prognosis, and a tumor of any size that invades the parietal pleura is classified as T3. However, with microscopic invasion beyond elastic fibers of the visceral pleura but no penetration to the parietal pleura at tight adhesion sites (we term this p1–3), classification as to the T factor is unclear. Among 1,179 consecutive patients with NSCLCs who underwent curative surgery between 1980 and 2002, 20 were in this category. Here, a comparison was made with subgroups of p stages IB, II, and IIIA with regard to histology, pleural invasion, and survival rates. To maximize the power of assessing prognostic potential, we set the significance level at 0.10, one-sided. The p1–3 condition sites of the 20 cases were the parietal pleura for 17 cases and the pericardium, diaphragm, and chest wall for one each of the remainder. The 5-year survival rate for these p1–3 patients was 71.6%. Significant differences were observed between p1–3 and IIIA groups. Although the 5-year survival rates did not significantly differ between p1–3 and T3N0 or unequivocal T3 subgroups, the prognosis of p1–3 patients was rather better than that of T3 and identical to T2. It was demonstrated that p1–3 status is not a factor warranting T3 classification for NSCLCs. Considering the prognosis, pathologic p1–3 tumors should be managed as a T2 disease for the present.     

Dedifferentiated peripheral chondrosarcoma: a clinicopathologic,immunohistochemical, and molecular analysis of four cases

Peripheral dedifferentiated chondrosarcoma (DCS) is an exceedingly rare aggressive surface bone neoplasm in which a high-grade sarcoma arises within an osteochondroma. Four such examples were identified in our files, representing 11.1% of all DCS treated at our hospital in the years 1995–2010, and were the object of the present study. The patients were two men and two women ranging in age between 30 and 64 years, with tumors located in the pelvis (n = 2), in the scapula (n = 1), and the tibia (n = 1). Radiologically, there was evidence of a preexisting osteochondroma associated with aggressive osteolytic areas at the base and periphery of the exostosis, extending to the bone segment of origin and to the soft tissues. Immunohistochemical analysis of cell cycle regulators showed consistent loss in the expression of p16 and overexpression of cyclin D1, and to a lesser extent of RB and p53, in the anaplastic compartments of peripheral DCS in comparison with the well-differentiated cartilaginous components, while no significant expression of MDM2 was observed in any of the cases studied. Moreover, PDGFRα was absent in both tumor components, and PDGF-Rβ was strongly and diffusely positive in all the cases. Finally, no mutations were identified in exons 4–9 of the TP53 gene in both cases, showing positivity for p53 in the anaplastic component. In conclusion, our study shows that alterations of genes implicated in the regulation of the G1 to the S phase cell cycle checkpoint contribute to the process of dedifferentiation in peripheral secondary chondrosarcoma (CS), although the molecular mechanisms seem at least in part to differ from those involved in the process of dedifferentiation of central CS. PDGFRβ could represent a potential target for treatments with receptor tyrosine kinase inhibitors in peripheral DCS.     

Immunohistochemical expression of p63, p53 and MIB-1 in urinary bladder carcinoma. A tissue microarray study of 158 cases

P63 is a member of the p53 family, which plays a role in the differentiation of urothelium and is supposed to play a role in urothelial carcinogenesis. P53 and MIB-1 are recognised in many studies as predictive markers of progression, but few studies in the literature have examined p63. The aims of our study were to explore the expression of p63 in bladder carcinomas and to compare this expression to p53 and MIB-1, as well as to stage and grade. Tissue microarrays were performed on 158 urothelial carcinomas (56 pTa, 45 pT1 and 57≥pT2). Immunohistochemical studies were performed with p63, p53 and MIB-1 antibodies. In our study we observed that p63 immunostaining is present in all cell layers in papillary urothelial neoplasm of low malignant potential (PUNLMP), but partially lost in non-invasive papillary urothelial carcinoma low grade (NILGC) and in pT1/≥pT2 bladder cancers. P53 and MIB-1 displayed lower expression in PUNLMP/NILGC vs non-invasive papillary urothelial carcinoma high grade (NIHGC)/pT1, but there was no correlation between the expression of p63, p53 and MIB-1. Our study demonstrates that p63 expression distinguishes between PUNLMP/NILGC and NIHGC/pT1 (p=4.10⁵). A statistical difference disserving pTa and pT1/≥pT2 with a statistical significance (p<10⁻⁶) could also be observed. P63 should be considered as an additional biomarker that might help pathologists to classify their patients.     

Complex translocation involving chromosomes #1, #9, and #22 in a patient with chronic myelogenous leukemia

A case of Philadelphia chromosome positive chronic myelogenous leukemia with a complex translocation involving chromosomes #1, #9, and #22 is described. All cells in the bone marrow showed this rearrangement, and Q-banding analysis showed the predominant karyotype to be 46,XY, t(1;9;22)(p22;q34;q11).     

Evidences of the Involvement of Bak,a member of the Bcl-2 Family of Proteins,in Active Coeliac Disease

Apoptosis of enterocytes is a feature that characterises the development of lesions in coeliac disease (CD). However, the intracellular pathways that lead to apoptosis of enterocytes have not been completely clarified. Bak is a member of the Bcl-2 family of proteins that acts as an endogenous promoter of apoptosis in normal enterocytes. However, its role in coeliac lesions has not been explored. We used small intestinal mucosa from patients with CD to evaluate the differential expression of members of the Bcl-2 family of proteins. Gene expression of Bak was analysed by RT-PCR of biopsies from 14 patients with untreated CD and from 19 controls without CD. In these samples, we also investigated the localisation of the Bak protein by immunohistochemistry and its apoptotic activity. In patients with untreated CD there was a 2.3-fold higher expression of Bak mRNA ( p =0.026), without significant differences in the expression of related genes bax or bcl-2. The higher expression of interferon gamma (IFN &#110 ) ( p =0.036) and the higher number of apoptotic cells identified by the TUNEL method ( p =0.032) confirmed the proapoptotic status in the intestinal mucosa of CD patients. We found a significant positive correlation ( p <0.0001) between the expression of IFN &#110 and Bak mRNA in patients with untreated CD. The expression of Bak protein was higher in patients with CD, and the immunoreactivity was almost restricted to the epithelium. We found that Bak mRNA and its protein were overexpressed in the intestinal lesions of CD patients and that IFN &#110 confers increased susceptibility for enterocytes to undergo apoptosis via upregulation of Bak.     

p66Shc protein, oxidative stress, and cardiovascular complications of diabetes: the missing link

Diabetes affects more than 150 million people worldwide, and it is estimated that this would increase to 299 million by the year 2025. The incidence of and mortality from cardiovascular disease are two- to eightfold higher in subjects with diabetes than in those without, coronary artery disease accounting for the large majority of deaths. Among the full spectrum of biochemical effects of high glucose, generation of oxygen-derived free radicals is one of the main pathophysiological mechanisms linking hyperglycemia to atherosclerosis, nephropathy, and cardiomyopathy. The adaptor protein p66^Shc is implicated in mitochondrial reactive oxygen species (ROS) generation and translation of oxidative signals into apoptosis. Indeed, p66^Shc−/− mice display prolonged lifespan, reduced production of intracellular oxidants, and increased resistance to oxidative stress-induced apoptosis. Accordingly, a series of studies defined the pathophysiological role of p66^Shc in cardiovascular disease where ROS represent a substantial triggering component. As p66^Shc modulates the production of cellular ROS, it represents a proximal node through which high glucose exerts its deleterious effects on different cell types; indeed, several studies tested the hypothesis that deletion of the p66^Shc gene may confer protection against diabetes-related cardiovascular complications. The present review focuses on the reported evidence linking p66^Shc signaling pathway to high glucose-associated endothelial dysfunction, atherogenesis, nephropathy, and cardiomyopathy.