Caveolin-1 and polymerase I and transcript release factor: new players in insulin-like growth factor-I receptor signaling.

Caveolae are plasma membrane regions enriched in Caveolin proteins which regulate vesicular transport, endocytosis, and cell signaling. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates Caveolin-1 (Cav-1), the most represented caveolar protein. Cav-1 participates to IGF-IR internalization and signaling directly interacting with IGF-IR and its substrates. Recently, polymerase I and transcript release factor (PTRF) or Cavin-1, has been identified in the caveolar backbone. PTRF does not play a Cav-1 ancillary role and emerging data support a direct role of PTRF in IGF-IR signaling. PTRF and Cav-1 can bind IGF-IR and regulate IGF-IR internalization and plasma membrane replacement, mechanisms frequently deregulated in cancer cells. Although the exact roles of Cav-1 and IGF-IR in human cancer continue to be a matter of some debate, there is a strong evidence for an association between Cav-1 and IGF-IR in cancer development. With the discovery of IGF-IR interaction with PTRF in caveolae, new insight emerged to understand the growing functions of these domains in IGF-I action.

Changes in adiponectin and leptin concentrations during glucocorticoid treatment: a pilot study in patients with polymyalgia rheumatica.

This study is concerned with an evaluation of the effects of glucocorticoids (GC) on adiponectin and leptin concentrations in patients with polymyalgia rheumatica (PMR). Seven patients diagnosed with PMR were studied at baseline and after one and three months of prednisone treatment. Serum leptin and adiponectin, serum glucose and insulin, erythrocyte sedimentation rate, C-reactive protein, and IL-6 were all measured by commercial assays. The treatment with GC normalized inflammation markers and significantly increased adiponectin and leptin concentrations without any impairment of insulin sensitivity measured by HOMA-IR. Adiponectin significantly increased only between baseline and 1 month (P= 0.013). A significant correlation was found between adiponectin and leptin concentrations both at baseline and after 3 months of treatment (both rho = 0.89, P= 0.03). In addition, adiponectin correlated also with serum glucose at baseline (rho = 0.81, P= 0.047). According to our results, adiponectin concentrations seem to be driven by inflammation, whereas leptin seems to be increased directly by the use of steroids.

IGF-I induced rapid recruitment of integrin beta1 to lipid rafts is Caveolin-1 dependent.

Caveolin-1 (Cav-1) regulates both insulin like growth factor receptor (IGF-IR) and integrin beta1 function. However, the role of Cav-1 in IGF-IR/integrin beta1 cross talk remains to be established. In this study, we observed that IGF-I did not induce integrin beta1 internalization but its plasma membrane reorganization. In particular, we found a rapid and transient association between integrin beta1 and Cav-1 followed by the enrichment of integrin beta1 in lipid rafts. To determine the role of Cav-1 in this process, we transfected Hacat cells with small interfering RNA specific for Cav-1 (siRNA-Cav-1) and with a scrambled siRNA as control (siRNA-Ctr). Cav-1 down regulated Hacat cells were then stimulated with IGF-I and analyzed by immunofluorescence and flow cytometry. We found that Cav-1 silencing abolished the recruitment of integrin beta1 to lipid rafts in the presence of IGF-I. These data demonstrate that IGF-IR/integrin beta1 cross talk is followed by integrin beta1 lipid raft compartmentalization and that Cav-1 is required for this process.

Caveolin-1 is essential for glimepiride-induced insulin secretion in the pancreatic betaTC-6 cell line.

The K(ATP) channels play a pivotal role in the complex mechanism of insulin secretion. K(ATP) channels represent the target of sulphonylureas, a class of drugs widely used in type 2 diabetes to stimulate insulin secretion. We previously showed that caveolin-1 depletion impairs action of the sulphonylurea glimepiride in human endothelial cells. The aim of this work was to investigate the possible role of caveolin-1 in glimepiride-induced insulin secretion. Caveolin-1 was depleted using siRNA method in the pancreatic betaTC-6 cell line. Then stimulation of insulin secretion was performed with different secretagogues (glucose, KCl, and glimepiride). Here, we show that betaTC-6 caveolin-1 depleted cells maintained high rate of insulin secretion after KCl, but not after glucose and glimepiride stimulation. Moreover, we find a direct interaction between caveolin-1 and Kir6.2, one of the K(ATP) channel subunit. These results demonstrate that Cav-1 plays a critical role for glucose and sulfonylurea-stimulated insulin secretion.

Changes in aqueductal CSF stroke volume and progression of symptoms in patients with unshunted idiopathic normal pressure hydrocephalus.

BACKGROUND AND PURPOSE: Idiopathic normal pressure hydrocephalus (iNPH) represents a diagnostic challenge, given its variable presentation and progression. Stroke volume (SV), defined as the mean volume of CSF passing through the aqueduct during both systole and diastole, greater than or equal to 42 muL, serves as a selection criterion for patients with good probabilities of improvement after ventriculoperitoneal shunt surgery (VPS). In this study, we evaluated the changes in SV during the progression of clinical symptoms in patients with suspected NPH. MATERIALS AND METHODS: Nine patients who presented with clinical and radiologic evidence of NPH, but refused treatment with VPS, were evaluated every 6 months for up to 2 years for progression in their clinical symptoms and changes in their SV, as measured by phase-contrast cine MR imaging (PCCMR). RESULTS: SV seems to increase between the onset of the symptoms and the following 18 to 20 months, then seems to plateau, followed in the next 18 to 20 months by a slight decline, and finally to a more precipitous drop in the next 12 months. During this time, however, the patient's clinical symptoms progressively worsen. CONCLUSION: Patients with a low SV have not necessarily had brain atrophy and can show, in the following months, a progressive increase in SV, which qualifies them as good candidates for VPS. The progressive reduction of the SV in untreated patients with worsening clinical symptoms may be a sign of a progressive cerebral ischemic injury, which renders the NPH irreversible.

Newborn liver gene transfer by an HIV-2-based lentiviral vector.

Newborn gene therapy, because it can prevent the damage caused by the onset of a disease, deserves specific attention. To evaluate gene transfer in tissues of newborn mice, we used a human immunodeficiency virus (HIV)-2 based lentiviral vector pseudotyped with vesicular stomatitis virus G glycoprotein expressing the green fluorescent protein reporter gene under the control of the cytomegalovirus promoter. We found that very low doses of HIV-2 could infect and be expressed in newborn mice. Under these conditions, the virus was preferentially expressed in the liver and hepatocytes were the predominant target. The treatment was not toxic, the infected liver cells proliferated and the transduced gene was stably expressed. Adult mice could be infected by HIV-2, but the vector was detected in the liver only utilizing the sensitive method of polymerase chain reaction coupled with Southern blot. Direct comparison between newborn and adult recipients demonstrated a much greater efficiency of liver transduction in the newborn mouse. These results indicate that the combination of early intervention and low multiplicity of infection may be a strategy for preferentially and efficiently targeting newborn liver for gene therapy applications.

Autoinhibition mechanism of proto-Dbl.

The dbl oncogene encodes a prototype member of the Rho GTPase guanine nucleotide exchange factor (GEF) family. Oncogenic activation of proto-Dbl occurs through truncation of the N-terminal 497 residues. The C-terminal half of proto-Dbl includes residues 498 to 680 and 710 to 815, which fold into the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, respectively, both of which are essential for cell transformation via the Rho GEF activity or cytoskeletal targeting function. Here we have investigated the mechanism of the apparent negative regulation of proto-Dbl imposed by the N-terminal sequences. Deletion of the N-terminal 285 or C-terminal 100 residues of proto-Dbl did not significantly affect either its transforming activity or GEF activity, while removal of the N-terminal 348 amino acids resulted in a significant increase in both transformation and GEF potential. Proto-Dbl displayed a mostly perinuclear distribution pattern, similar to a polypeptide derived from its N-terminal sequences, whereas onco-Dbl colocalized with actin stress fibers, like the PH domain. Coexpression of the N-terminal 482 residues with onco-Dbl resulted in disruption of its cytoskeletal localization and led to inhibition of onco-Dbl transforming activity. The apparent interference with the DH and PH functions by the N-terminal sequences can be rationalized by the observation that the N-terminal 482 residues or a fragment containing residues 286 to 482 binds specifically to the PH domain, limiting the access of Rho GTPases to the catalytic DH domain and masking the intracellular targeting function of the PH domain. Taken together, our findings unveiled an autoinhibitory mode of regulation of proto-Dbl that is mediated by the intramolecular interaction between its N-terminal sequences and PH domain, directly impacting both the GEF function and intracellular distribution.

Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides.

The adjuvanticity of heat-labile enterotoxin (LT) of Escherichia coli and its non-toxic mutant LTK63 was assessed and compared for intranasal immunization of synthetic peptides. Mice immunized intranasally with LT, or its mutant LTK63, generated strong systemic proliferative and cytotoxic T-cell responses to co-administered synthetic peptides. The wild LT toxin promoted higher peptide-specific proliferative and cytotoxic T-cell responses than the LTK63 mutant. Moreover, the wild-type LT toxin was shown to promote peptide-specific memory CTL responses which were detectable 1 year after intranasal priming. Both LT and LTK63 molecules were shown to be immunogenic, with serum antibody subclasses being predominantly IgG1 and to a lesser extent IgG2a. These findings demonstrate that cellular immune responses to small synthetic peptide antigens administered by the intranasal route can be potentiated with the use of mucosal adjuvants. Moreover, the ability of LT and LTK63 to promote both CD4+ and CD8+ T-cell responses will have relevance to the design and production of future mucosal vaccines.

Is the telephone interview for cognitive status a valid alternative in persons who cannot be evaluated by the Mini Mental State Examination?

The Mini Mental State Examination (MMSE), a widely used tool for first-line assessment of cognitive function, cannot be fully administered to persons with severe visual or upper extremity impairments. This cross-sectional study, which was performed in a sample of patients admitted to the outpatient clinic of the INRCA Geriatric Department "I Fraticini" (Firenze, Italy) and their relatives, evaluated whether the Telephone Interview for Cognitive Status (TICS), a test originally created for telephone screening of cognitive impairment, is a valid alternative for assessment of cognition in persons who cannot provide valid responses to all the MMSE items. Fifteen subjects in each of seven MMSE strata (9-11, 12-14, 15-17, 18-20, 21-23, 24-26, 27-30) were consecutively selected. Inclusion criteria were: age > or = 60 years; Italian as the main language; education > or = 3 years; medical stability; and ability to complete the MMSE. At admission, participants were examined for dementia by an expert clinician according to the DSM IV criteria. Italian versions of the TICS and the MMSE were administered face-to-face by two trained interviewers. The TICS was readministered face-to-face one week later by the same interviewer that had previously administered the test. The TICS showed good test-retest reproducibility. Assessments taken one week apart differed on average by 0.52 +/- 1.9 points (p < 0.01), indicating a systematic improvement. The TICS explained 96% of the variance in the MMSE, and this association was independent of age, gender and education. More than 90% of the cases "screening positive" (i.e., scoring below a certain cut-off) by the TICS, were also identified as "screening positive" when comparable MMSE cut-off scores were used. Using comparable cut-off scores, the sensitivity and specificity of TICS and MMSE in detecting a standard clinical diagnosis of dementia largely overlapped. In persons who cannot be evaluated with the MMSE in its full form, the face-to-face administration of the TICS is a reliable and valid alternative.

Constant hierarchic patterns of physical functioning across seven populations in five countries.

This research was aimed at identifying critical steps in the decline in physical function that often parallels aging. Six basic and nine instrumental activities of daily living (ADLs) were classified into four domains of disability characterized by specific underlying physical impairment. The hierarchical order of this classification was verified in two random samples representative of the older home-dwelling population. The concordance level of disability and results of performance-based measures of physical function were also tested. Finally, the cross-cultural reliability of the model was verified in seven population-based samples of older persons living in five European countries. In older persons the disabling process follows a general pattern of progression based on a typical sequence of impairments.

Formal education as an effect modifier of the relationship between Mini-Mental State Examination score and IADLs disability in the older population.

The objective of this retrospective, case-series study was to examine the relationship between disability in Instrumental Activities of Daily Living (IADLs) and cognitive impairment measured by the Mini-Mental State Examination (MMSE), and how this relationship was influenced by different levels of formal education. The setting of the study was the Cognitive Disorders Center of the Local Sanitary Unit (USL) No. 4 in Prato (Italy). Independence in IADL was strongly related to MMSE scores. A score of less than 24 at the MMSE was predictive of disability in at least one IADL with 0.82 sensitivity and 0.73 specificity. However, when only people with a very low level of formal education were considered, the best threshold predicting IADL disability was 17 (sensitivity 0.76, specificity 0.75). Thus, scholastic achievement is an effect modifier of the relationship between cognitive status, as measured by the MMSE, and IADL disability. Subjects with low education level and poor performance at the MMSE are often independent in activities making demand on cognitive function, such as IADLs. These results suggest that a lower MMSE cut-off score should be considered when screening for cognitive impairment in these persons.

Epidemiology of atypical melanocytic naevi: an analytical study in a Mediterranean population.

Atypical naevi are markers for increased risk of malignant melanoma, providing additional information about melanoma risk beyond that given by common melanocytic naevi. Little is known about the epidemiology of atypical naevi (AN), and available data are limited to predominantly fair-skinned populations. By using a case-control study that included 705 subjects with atypical naevi and 1,782 controls, we have analysed the aetiology of AN in a Mediterranean population, paying particular attention to the role of sunburn. After adjusting for age, sex and years of formal education, the presence of atypical naevi was significantly related to frequent sunburn before the age of 20 (odds ratio, OR, = 1.8; 95% CI, 1.3-2.5). Although less evident, this relationship was maintained by also taking into account the sun sensitivity, expressed as phototype, as a cofounder (OR = 1.5; 95% CI, 1.0-2.0). Concerning phenotypical features, an increased risk of having atypical naevi was found for all the parameters included in so-called 'fair complexion', i.e. blue eyes, blond or red hair, fair skin, phototype I-II, and a tendency to freckle. The greatest difference between subjects with and without atypical naevi related to the number of common melanocytic naevi: more than 30 common naevi were found in 41.5% of cases, but only in 9% of controls (OR = 8.0; 95% CI, 6.3-10.3). Overall, the six variables entered in the multivariate model that best predicted the risk of atypical naevi, were: young age, male sex, high educational level, presence of a large number of common naevi, phototype I-II, and a history of painful sunburn. In conclusion, the variables predicting the risk of developing atypical naevi in Mediterranean people are identical to those observed in predominantly fair-skinned populations. The aetiologic role of sunlight has been pointed out and shows a statistically significant relationship between frequent sunburn and the development of atypical naevi also after controlling for the subject's phototype.

Does the Clock Drawing Test predict cognitive decline in older persons independent of the Mini-Mental State Examination? The FINE Study Group. Finland, Italy, The Netherlands Elderly.

OBJECTIVE: To evaluate the value of the Clock Drawing Test (CDT) in predicting cognitive deterioration over a 4-year period, independent of baseline cognitive status evaluated by the Mini-Mental State Examination (MMSE). DESIGN: A preplanned analysis of data collected during the second (1991) and the third (1995) follow-up of the Italian rural cohorts of the FINE Study (Finland, Italy, the Netherlands Elderly). SUBJECTS: Of the 427 men (mean age 77.6 +/- 4.1 years; range 72-90 years) interviewed in 1991, 264 survived and were reinterviewed in 1995. The study population included 247 persons who were interviewed and received a complete cognitive evaluation in both 1991 and in 1995. MEASUREMENTS: Cognitive assessment in 1991 included the MMSE, the Dementia Rating Scale (DRS), and the CDT. The CDT was classified as normal or pathological, based on previously established criteria. The MMSE and the DRS were repeated in 1995. RESULTS: Independent of age and baseline MMSE, score, subjects with pathological CDT compared with normal CDT had lower MMSE scores at follow-up (P < .01). These results were also confirmed by evaluating cognitive decline through its impact on change over time in daily life autonomy, as measured by the DRS (P < .01). Among persons scoring more than 21 on the MMSE, compared with persons with a normal CDT, those with pathological CDT performance were 5.4 (95% CI: 2.1-14.2) and 5.5 (95% CI: 1.6-19.6) times more likely to have a MMSE score below 21 and 18, respectively, 4 years later, independent of age and baseline MMSE score. CONCLUSIONS: Findings suggest that the CDT identifies older persons at high risk of cognitive decline and adds prognostic information that supplements the standard MMSE test.

Age-related changes in the pharmacodynamics of intravenous glyceryl trinitrate.

Comparable hemodynamic effects were obtained administering a much lower intravenous dose of glyceryl trinitrate (GTN) in elderly than in younger patients. The pharmacodynamics and kinetics of GTN were thus assessed in 2 groups of patients with acute myocardial infarction (group A: less than or equal to 65 years, 6 patients; group B: greater than or equal to 75 years, 6 patients). The arterial and venous dose-concentration relationship and the associated hemodynamic changes at end-point (EP: 10% reduction in mean systemic arterial pressure) were similar in the 2 groups. However, in older subjects EP was reached at a lower GTN infusion rate (0.11 +/- 0.04 vs 0.33 +/- 0.11 micrograms.kg-1.min-1, mean +/- S.D.; p less than 0.001), and with lower arterial and venous drug concentrations (arterial [GTN]: 1.2 +/- 0.1 vs 4.6 +/- 1.2 ng.ml-1; p less than 0.01; venous [GTN]; 0.09 +/- 0.05 vs 0.35 +/- 0.15 ng.ml-1; p less than 0.05), whereas overall GTN kinetics appeared to be substantially independent of age. Thus, the enhanced efficacy of GTN in advanced age seems to stem mainly from pharmacodynamic changes, which may be the consequence of dampened baroreceptor reflexes, as suggested by a lower heart rate increase per unitary fall in systolic arterial pressure observed in group B (0.12 +/- 0.07 vs 0.41 +/- 0.29 b.min-1.mmHg-1; p less than 0.05).

Improved exercise tolerance by i.v. fructose-1,6-diphosphate in chronic, stable angina pectoris.

The effect of IV fructose-1,6-diphosphate (FDP) on transient, reproducible myocardial ischemia was evaluated in ten patients, aged 50 to 66 years, with chronic, stable exertional angina. FDP or placebo (glucose) were administered between basal and posttreatment ergometric stress testing; an identical procedure was repeated in each patient with the second treatment on the following day according to a single-blind, cross-over design. FDP improved exercise tolerance and total work capacity, significantly delaying the onset of ST-segment depression and angina. Nevertheless, the critical level of the rate x pressure (R X P) product, causing appearance of myocardial ischemia, was not remarkably changed. However, the R X P product at same workload was significantly lower after FDP. These results suggest that improved exercise tolerance might have resulted from peripheral (increased oxygen delivery to skeletal muscle) rather than from central (cardiac) effects of FDP.

Age-related hemodynamic effects of intravenous nitroglycerin for acute myocardial infarction and left ventricular failure.

Acute hemodynamic effects of intravenous nitroglycerin (NTG) were assessed in 24 patients with acute myocardial infarction and left ventricular failure, and results were compared between 2 groups of different age (group A--65 years or less, n = 12; group B--75 years or more, n = 12). Overall, hemodynamic effects of NTG consisted of an increase in stroke volume index and left ventricular stroke work index (+21 and +23%), coupled with a 16% reduction in systemic vascular resistance, and of a marked decrease in right atrial and pulmonary artery (PA) pressures. The hemodynamic end-point (5 to 10% reduction in mean systemic arterial pressure) used for NTG titration was achieved with a significantly lower dose in group B, in which a greater percent reduction in mean PA and mean PA wedge pressures was also observed. However, because effects of NTG on systemic vascular resistance were similar in groups A and B, it was concluded that the vasodilating action of NTG is maintained in advanced age, as opposed to what has been demonstrated for beta-adrenergic agents.

Prediction of left ventricular function in acute anterior myocardial infarction by serum creatine kinase activity and precordial ECG mapping.

Right cardiac catheterization, serial determination of creatine phosphokinase (CPK) and of CPK-MB activities, and precordial 35-lead ECG mapping were performed in 17 consecutive patients with a first anterior acute myocardial infarction (AMI) within 6 hours of the onset of symptoms. Left ventricular function as determined by the stroke index (SI, ml/m2) to mean pulmonary capillary wedge pressure (PCWP, mmHg) ratio inversely correlated either with the total CPK and CPK-MB released and with peak enzymatic activity, or with the entity of early (6th hour ECG) ST-segment elevation. Conversely, a direct correlation was found between the SI/PCWP ratio and the ratio of R to Q+S waves from late (48th hour) ECG. Residual left ventricular function after a first anterior AMI can therefore be estimated noninvasively in the individual patient by early precordial ECG mapping and by peak enzymatic activity.