Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Lupin seed γ-conglutin lowers blood glucose in hyperglycaemic rats and increases glucose consumption of HepG2 cells.

The aim of the present study was to evaluate the effect of a chronic oral γ-conglutin treatment in male Sprague-Dawley rats in which hyperglycaemia had been induced by supplying 10 % d-glucose in drinking-water. A γ-conglutin dosage of 28 mg/kg body weight was daily administered to animals for 21 d. Plasma glucose, insulin and glucose overloading were monitored. Chronic administration of glucose resulted in a statistically significant (P < 0·05) increase in fasting blood glucose (2·5-fold) and insulin (2·7-fold) v. the values recorded in control rats. Simultaneous treatment with γ-conglutin attenuated the rise in plasma glucose (1·9-fold) and insulin (1·8-fold) levels in the glucose-fed rats (P < 0·05). Fasting insulin and homeostasis model of insulin resistance were decreased by 34 and 48 % (P < 0·05), respectively, in the γ-conglutin-treated rats v. the values found in pair-fed animals. To confirm these results with a different approach, HepG2 cells, grown for 24 and 48 h in Dulbecco's minimum essential medium containing different glucose concentrations (5·5, 11·1 and 16·5 mmol/l), were exposed to 10 µmol/l γ-conglutin with or without 10 mmol/l metformin or 100 nmol/l insulin. γ-Conglutin increased glucose consumption (from 1·5- to 2·5-fold) in HepG2 cells, under all experimental conditions; this effect was more evident after 48 h incubation. Moreover, in this in vitro model, the addition of γ-conglutin potentiated the activity of insulin and metformin in cell glucose consumption. These findings extend the previous ones and suggest the potential use of lupin γ-conglutin in the control of glycaemia.

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 µM) were similar to those found in cells incubated with 1 µM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.

Uterine leiomyoma with tubules.

We report two cases of "uterine leiomyoma with tubules" as a new pathological entity. Since these are biphasic neoplasms (composed by epithelial and mesenchimal elements), the differential diagnosis is between mixed mullerian tumors and uterine tumors resembling ovarian sex cord tumors (UTROSCTs). In the differential diagnosis, the mixed mullerian tumors are easily excluded because of histological and immunohistochemical features. UTROSCTs are similar to the lesions we reported, and the differential diagnosis requires positivity for some immunohistochemical markers as inhibin, CD99, calretinin, Melan-A. Our conclusions are that to perform a diagnosis of UTROSCT at least two immunohistochemical marker have to be expressed; in the present case they didn't, so we call the lesion "leiomyoma with tubules".

Prevalence and significance of psammoma bodies in cervicovaginal smears in a cervical cancer screening program with emphasis on a case of primary bilateral ovarian psammocarcinoma.

BACKGROUND: The purpose of our study was to determine the prevalence and significance of psammoma bodies (PBs) in the cervicovaginal smears of the screening population of Trento district (Italy), with the description of the cytological presentation of an asymptomatic bilateral ovarian psammocarcinoma. METHODS: From 1993 to 2006, women with PBs detected on consecutively screened cervical smears were identified from the computerized pathology database of Rovereto Hospital. The follow-up period was set from the time of cytological diagnosis to May 31st, 2007. Clinical information was obtained from retrospective review of women's medical records. The source of PBs was identified with adequate diagnostic procedures. RESULTS: PBs were found in six of the 201,231 Papanicolaou screening smears (0.0029%). Benign conditions (intrauterine device, inclusion ovarian cysts and ovarian cystoadenofibroma with PBs) were found in four patients. In two cases, PBs were associated with malignant cells; a bilateral ovarian malignancy was diagnosed in both cases, a serous adenocarcinoma and a psammocarcinoma. CONCLUSION: PBs in the cervicovaginal smears are a rare finding, associated more often with benign conditions than with malignancies. Moreover, to our knowledge, our case of primary ovarian psammocarcinoma is the first report in which the presence of malignant cells and PBs in the cervicovaginal and endometrial smears represents the first manifestation of disease.