Monitoring Preparation of Derivative Protein Crystals via Raman Microscopy

This chapter deals with the application of Raman confocal microscopy to monitor the preparation of chemically modified protein crystals. We first introduce the peculiar properties of protein single crystals and related crystallization techniques. Then we move to present experimental sampling and theoretical background of Raman microscopy. Finally we show some examples of applications of Raman microscopy to protein derivative crystal

Utilizzo di una Superossidodismutasi modificata ottenuta in forma ricombinante (rMnSod) come agente antineoplastico e marker tumorale per neoplasie mammarie canine estrogeno-positive

Nel presente lavoro viene studiato l'utilizzo diagnostico di un’isoforma ricombinante di una superossidodismutasi isolata da liposarcoma umano (rMnSod) utilizzabile come marker tumorale per neoplasie mammarie canine sovraesprimenti recettori per estrogeni. Le capacità citostatiche e selettive Er+ di tale proteina sono state valutate in esperimenti “in vitro” prima su colture cellulari “MCF-7”e “in vivo” poi. In questo lavoro vengono descritte le pratiche diagnostiche e l’utilizzo della proteina marcata con Gallio 68 in cinque cagne sottoposte ad indagine PET di cui si è voluto dimostrare la captazione neoplastica della molecola

Biophysical and biochemical characterization of a liposarcoma-derived recombinant MnSOD protein acting as an anticancer agent

A recombinant MnSOD (rMnSOD) synthesized by specific cDNA clones derived from a liposarcoma cell line was shown to have the same sequence as the wild-type MnSOD expressed in the human myeloid leukaemia cell line U937, except for the presence of the leader peptide at the N-terminus. These results were fully confirmed by the molecular mass of rMnSOD as evaluated by ES/MS analysis (26662.7 Da) and the nucleotide sequence of the MnSOD cDNA. The role of the leader peptide in rMnSOD was investigated using a fluorescent and/or 68Gallium-labelled synthetic peptide. The labelled peptide permeated MCF-7 cells and uptake could be inhibited in the presence of an excess of oestrogen. In vivo it was taken up by the tumour, suggesting that the molecule can be used for both therapy and diagnosis. The in vitro and in vivo pharmacology tests confirmed that rMnSOD is only oncotoxic for tumour cells expressing oestrogen receptors. Pharmacokinetic studies in animals performed with 125I- and 131I-labelled proteins confirmed that, when administered systemically, rMnSOD selectively reached the tumour, where its presence was unambiguously demonstrated by scintigraphic and PET scans. PCR analysis revealed that Bax gene expression was increased and the Bcl2 gene was down regulated in MCF7 cells treated with rMnSOD, which suggests that the protein induces a pro-apoptotic mechanism

Interaction of rat liver glucocorticoid receptor with heparin.

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin

Imaging Modalities for the Diagnosis of Vascular Graft Infections:A Consensus Paper amongst Different Specialists

Vascular graft infection (VGI) is a rare but severe complication of vascular surgery that is associated with a bad prognosis and high mortality rate. An accurate and prompt identification of the infection and its extent is crucial for the correct management of the patient. However, standardized diagnostic algorithms and a univocal consensus on the best strategy to reach a diagnosis still do not exist. This review aims to summarize different radiological and Nuclear Medicine (NM) modalities commonly adopted for the imaging of VGI. Moreover, we attempt to provide evidence-based answers to several practical questions raised by clinicians and surgeons when they approach imaging in order to plan the most appropriate radiological or NM examination for their patients

Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P212121, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date

Mirabegron relaxes arteries from human visceral adipose tissue through antagonism of α1-adrenergic receptors

Aim: As inadequate perfusion has emerged as a key determinant of adipose tissue dysfunction in obesity, interest has grown regarding possible pharmacological interventions to prevent this process. Mirabegron has proved to improve insulin sensitivity and glucose homeostasis in obese humans via stimulation of β3-adrenoceptors which also seem to mediate endothelium-dependent vasodilation in disparate human vascular beds. We characterized, therefore, the vasomotor function of mirabegron in human adipose tissue arteries and the underlying mechanisms. Methods: Small arteries (116-734 μm) isolated from visceral adipose tissue were studied ex vivo in a wire myograph. After vessels had been contracted, changes in vascular tone in response to mirabegron were determined under different conditions. Results: Mirabegron did not elicit vasorelaxation in vessels contracted with U46619 or high-K+ (both P > 0.05). Notably, mirabegron markedly blunted the contractile effect of the α1-adrenergic receptor agonist phenylephrine (P < 0.001) either in presence or absence of the vascular endothelium. The anti-contractile action of mirabegron on phenylephrine-induced vasoconstriction was not influenced by the presence of the selective β3-adrenoceptor blocker L-748,337 (P < 0.05); lack of involvement of β3-adrenoceptors was further supported by absent vascular staining for them at immunohistochemistry. Conclusions: Mirabegron induces endothelium-independent vasorelaxation in arteries from visceral adipose tissue, likely through antagonism of α1-adrenoceptors

Bacterial Lipopolysaccharide Rapidly Inhibits Expression of C–C Chemokine Receptors in Human Monocytes

The present study was designed to investigate the effect of bacterial lipopolysaccharide (LPS) on C–C chemokine receptors (CCR) expressed in human mononuclear phagocytes. LPS caused a rapid and drastic reduction of CCR2 mRNA levels, which binds MCP-1 and -3. CCR1 and CCR5 mRNAs were also reduced, though to a lesser extent, whereas CXCR2 was unaffected. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced from 1.5 h to 45 min. As expected, LPS-induced inhibition of CCR2 mRNA expression was associated with a reduction of both MCP-1 binding and chemotactic responsiveness. The capacity to inhibit CCR2 expression in monocytes was shared by other microbial agents and cytokines (inactivated Streptococci, Propionibacterium acnes, and to a lesser extent, IL-1 and TNF-α). In contrast, IL-2 augmented CCR2 expression and MCP-1 itself had no effect. These results suggest that, regulation of receptor expression in addition to agonist production is likely a crucial point in the regulation of the chemokine system