Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine,thiol and aspartyl proteases

We synthesized short chromogenic peptidyl–Arg–p-nitroanilides containing either (Galβ)Ser or (Glcα,β)Tyr at P₂ or P₃ sites as well as O-acetylated sugar moieties and studied their hydrolysis by bovine trypsin, papain, human tissue kallikrein and rat tonin. For comparison, the susceptibility to these enzymes of Acetyl–X–Arg–pNa and Acetyl–X–Phe–Arg–pNa series, in which X was Ala, Phe, Gln and Asn were examined. We also synthesized internally quenched fluorescent peptides with the amino acid sequence Phe⁸–His–Leu–Val–Ile–His–Asn¹⁴ of human angiotensinogen, in which [GlcNAcβ]Asn was introduced before Phe⁸ and/or after His¹³ and ortho-aminobenzoic acid (Abz) and N-[2-, 4-dinitrophenyl]–ethylenediamine (EDDnp) were attached at N- and C-terminal ends as a donor/receptor fluorescent pair. These peptides were examined as substrates for human renin, human cathepsin D and porcine pepsin. The chromogenic substrates with hydrophilic sugar moiety increased their susceptibility to trypsin, tissue kallikrein and rat tonin. For papain, the effect of sugar depends on its position in the substrate, namely, at P₃ it is unfavorable, in contrast to the P₂ position that resulted in increasing affinity, as demonstrated by the higher inhibitory activity of Ac–(Galβ)Ser–Arg–pNa in comparison to Ac–Ser–Arg–pNa, and by the hydrolysis of Ac–(Glcα,β)Tyr–Arg–pNa. On the other hand, the acetylation of sugar hydroxyl groups improved hydrolysis of the susceptible peptides to all enzymes, except tonin. The P^′₄ glycosylated peptide [Abz–F–H–L–V–I–H–(GlcNAcβ)N–E–EDDnp], that corresponds to one of the natural glycosylation sites of angiotensinogen, was shown to be the only glycosylated substrate susceptible to human renin, and was hydrolysed with lower K_m and higher k_cat values than the same peptide without the sugar moiety. Human cathepsin D and porcine pepsin are more tolerant to substrate glycosylation, hydrolysing both the P^′₄ and P₄ glycosylated substrates.     

The status of dopamine nerve terminals in Parkinson's disease and essential tremor: a PET study with the tracer [11-C]FE-CIT

Neuroimaging studies of the striatal dopamine transporter (DAT) are useful in the assessment of the dopaminergic system in Parkinson's disease (PD). We used positron emisson tomography (PET) and the tracer [11-C]FE-CIT to measure DAT binding in the caudate nucleus and putamen of 31 patients with PD, 5 with essential tremor and 8 healthy control subjects. Of the patients with PD, 17 were drug naive, while the others were either on levodopa or dopamine agonist monotherapy. DAT binding was significantly reduced in the caudate nucleus and to a greater extent in the putamen of PD patients compared to both healthy controls and essential tremor individuals. No overlap was observed between putamen values in PD and normals. No differences were found between controls and essential tremor subjects. These data confirm that measurements of DAT binding can provide an accurate and highly sensitive measure of degeneration in the dopamine system in PD.     

Autoerythrocyte sensitization syndrome (Gardner–Diamond syndrome): review of the literature**

A review of the literature concerning psychogenic purpura is presented. The diagnosis is usually based on typical anamnestic data, clinical presentation (painful inflammatory skin lesions, which progressed to ecchymoses during the next 24 h) and positive diagnostic tests with intracutaneous injections of 80% solution of washed autologous erythrocytes. No pathological findings of blood coagulation parameters are usually detected. Histopathological evaluations of lesional biopsies revealed non-specific changes. Taking into account the high frequency of psychic disorders and stress dependence of skin symptoms, therapy with psychotropic drugs (according to indications) and psychotherapy are pathogenetically grounded methods of treatment in psychogenic purpura, and should be provided together with symptomatic therapy.     

The early systemic and gastrointestinal oxygenation effects of hemorrhagic shock resuscitation with hypertonic saline and hypertonic saline 6% dextran-70: a comparative study in dogs

The smaller volemic state from hypertonic (7.5%) saline (HS) solution administration in hemorrhagic shock can determine lesser systemic oxygen delivery and tissue oxygenation than conventional plasma expanders. In a model of hemorrhagic shock in dogs, we studied the systemic and gastrointestinal oxygenation effects of HS and hyperoncotic (6%) dextran-70 in combination with HS (HSD) solutions in comparison with lactated Ringer's (LR) and (6%) hydroxyethyl starch (HES) solutions. Forty-eight mongrel dogs were anesthetized, mechanically ventilated, and subjected to splenectomy. A gastric air tonometer was placed in the stomach for intramucosal gastric CO(2) (Pgco(2)) determination and for the calculation of intramucosal pH (pHi): The dogs were hemorrhaged (42% of blood volume) to hold mean arterial blood pressure at 40-50 mm Hg over 30 min and were then resuscitated with LR (n = 12) in a 3:1 relation to removed blood volume; HS (n = 12), 6 mL/kg; HSD (n = 12), 6 mL/kg; and HES (mean molecular weight, 200 kDa; degree of substitution, 0.5) (n = 12) in a 1:1 relation to the removed blood volume. Hemodynamic, systemic, and gastric oxygenation variables were measured at baseline, after 30 min of hemorrhage, and 5, 60, and 120 min after intravascular fluid resuscitation. After fluid resuscitation, HS showed significantly lower arterial pH and mixed venous Po(2) and higher systemic oxygen uptake index and systemic oxygenation extraction than LR and HES (P < 0.05), whereas HSD showed significantly lower arterial pH than LR and HES (P < 0.05). Only HS and HSD did not return arterial pH and pHi to control levels (P < 0.05). In conclusion, all solutions improved systemic and gastrointestinal oxygenation after hemorrhagic shock in dogs. However, the HS solution showed the worst response in comparison to LR and HES solutions in relation to systemic oxygenation, whereas HSD showed intermediate values. HS and HSD solutions did not return regional oxygenation to control values.     

Evolving concepts in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) represents a particular aetiology of systolic heart failure that frequently has a genetic background and usually affects young patients with few co‐morbidities. The prognosis of DCM has improved substantially during the last decades due to more accurate aetiological characterization, the red‐flag integrated approach to the disease, early diagnosis through systematic familial screening, and the concept of DCM as a dynamic disease requiring constant optimization of medical and non‐pharmacological evidence‐based treatments. However, some important issues in clinical management remain unresolved, including the role of cardiac magnetic resonance for diagnosis and risk categorization and the interaction between genotype and clinical phenotype, and arrhythmic risk stratification. This review offers a comprehensive survey of these and other emerging issues in the clinical management of DCM, providing where possible practical recommendations.     

Unsuspected strongyloidiasis in hospitalised elderly patients with and without eosinophilia

The prevalence and associated factors of chronic uncomplicated strongyloidiasis were estimated among 200 consecutive elderly patients (aged >or= 60 years) admitted to a general hospital in northern Italy. One-hundred patients had a peripheral eosinophil concentration >or= 500 cells/microL (group A), and 100 were age- and gender-matched controls (group B). Measurements included serum IgG anti-Strongyloides antibody titre by an indirect immunofluorescence assay, combined with faecal culture for Strongyloides stercoralis. Anti-Strongyloides antibodies were detected in 28 patients (at high titre in 11 patients). Seropositivity was significantly more common among group A than among group B patients (OR 4.85). Strong seropositivity for anti-Strongyloides antibodies was associated with farm work (p < 0.001), but not with other patient characteristics or with signs and symptoms of strongyloidiasis. In conclusion, strongyloidiasis was relatively common among elderly in-patients; eosinophilia and a history of farm work were the most useful indications for this diagnosis.     

Chromosomal gains in the sarcomatoid transformation of chromophobe renal cell carcinoma.

The hallmark of chromophobe renal cell carcinoma is multiple chromosomal losses from among chromosomes 1, 2, 6, 10 and 17. Chromophobe renal cell carcinoma with distant metastases or sarcomatoid transformation are uncommon and little is known about their chromosomal abnormalities. We collected six sarcomatoid chromophobe renal cell carcinomas and three primary chromophobe renal cell carcinomas with distant metastases. A cytogenetic analysis by fluorescent in situ hybridization on paraffin-embedded tissue was performed using centromeric probes for chromosomes 1, 2, 6, 10 and 17. We found more than one signal in four of six (66%) sarcomatoid chromophobe renal cell carcinomas, in both sarcomatoid and adjacent epithelial components. Both primary chromophobe renal cell carcinomas and matched metastases showed single signals for all chromosomes studied in two cases and no abnormalities in the remaining case. We concluded that: (1) both epithelial and sarcomatoid components of sarcomatoid chromophobe renal cell carcinoma show different genetic abnormalities from those characteristic of chromophobe renal cell carcinoma; (2) sarcomatoid chromophobe renal cell carcinomas frequently have multiple gains (polysomy) of chromosomes 1, 2, 6, 10 and 17; (3) distant metastases show the same genetic patterns, usually chromosomal losses (monosomy), found in the primary tumors.     

A PET study with [11-C]raclopride in Parkinson's disease: preliminary results on the effect of amantadine on the dopaminergic system

Amantadine has been proved to be beneficial in Parkinson's disease. Although it is still uncertain which neurochemical events are modified at therapeutic doses, an increase in dopaminergic tone secondary to NMDA receptor blockade and a direct inhibition of the glutamatergic overactivity have been suggested to be involved in its clinical effects. The aim of this study was to evaluate the effects of amantadine on the dopaminergic system by measuring the in vivo binding of [11-C]raclopride to D2 dopamine receptors in the basal ganglia of 6 patients with idiopathic Parkinson's disease. Each patient underwent a PET study, before and after 14 days of treatment with amantadine (200 mg/day). Repeated treatment with therapeutic doses of amantadine induced a moderate increase in the in vivo binding of [11-C]raclopride in the putamen of PD patients. This observation indicates that in PD patients, 200 mg/day amantadine does not produce an increase in extracellular levels of dopamine sufficiently to inhibit raclopride binding or that, if present, is it masked by a concurrent increase in receptor availability, as recently reported in rat striatum.