Fabio Forlani

• Biochemistry - Biology
• Professor (Assistant) at University of Milan

This study starts from the need to remove a mix of proteins, oils and natural resin, called beverone in the Italian literature, from the back of canvas paintings. The aim of this study is to develop and evaluate the effectiveness of two different agarose/enzyme gels containing, respectively, a trypsin derived from porcine pancreas and a lipase from...

Biofilm-dwelling cells endure adverse conditions, including oxidative imbalances. The NADH:quinone oxidoreductase enzyme WrbA has a crucial role in the mechanism of action of antibiofilm molecules such as ellagic and salicylic acids. This study aimed to exploit the potential of the WrbA scaffold as a valuable target for identifying antibiofilm comp...

Bacterial biofilm is a major contributor to the persistence of infection and the limited efficacy of antibiotics. Antibiofilm molecules that interfere with the biofilm lifestyle offer a valuable tool in fighting bacterial pathogens. Ellagic acid (EA) is a natural polyphenol that has shown attractive antibiofilm properties. However, its precise anti...

The fragrance industry is increasingly turning to biotechnology to produce sustainable and high-quality fragrance ingredients. Microbial-based approaches have been found to be particularly promising, as they offer a more practical, economical and sustainable alternative to plant-based biotechnological methods for producing terpene derivatives of pe...

The fulfilment of the European “Farm to Fork” strategy requires a drastic reduction in the use of “at risk” synthetic pesticides; this exposes vulnerable agricultural sectors—among which is the European risiculture—to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds n...

The effects of natural compounds on biofilm formation have been extensively studied, with the goal of identifying biofilm formation antagonists at sub-lethal concentrations. Salicylic and cinnamic acids are some examples of these compounds that interact with the quinone oxidoreductase WrbA, a potential biofilm modulator and an antibiofilm compound...

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia...

Purpose The main goal of the present work was to assess the effectiveness of zosteric acid (ZA) in hindering Escherichia coli biofilm formation on a mineral surface. Methods Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) flow system was used to probe in situ the biochemical changes induced by ZA on E. coli sessile cells growing...

This study investigated in-vitro the non-lethal effects of N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated from the olive trees affected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 µM. Cell...

Crop disease management often implies repeated application of fungicides. However, the increasing emergence of fungicide-resistant pathogens requires their rotation or combined use. Tank-mix combinations using fungicides with different modes of action are often hard to manage by farmers. An alternative and unexploited strategy are bifunctional fung...

The hypoglycemic effect in humans of Moringa oleifera (MO) leaf powder has, to date, been poorly investigated. We assessed the chemical composition of MO leaf powder produced at Saharawi refugee camps, its in vitro ability to inhibit α-amylase activity, and its sensory acceptability in food. We then evaluated its effect on postprandial glucose resp...

The present work concerns an efficient strategy to obtain novel medical devices materials able to inhibit biofilm formation. The new materials were achieved by covalent grafting of p-aminocinnamic or p-aminosalicylic acids on low density polyethylene coupons. The polyethylene surface, previously activated by oxygen plasma treatment, was functionali...

A straightforward procedure based on dot-blot immunoassay is proposed as an effective diagnostic tool suitable for detecting and quantifying milk casein in cultural heritage samples. A polyclonal primary antibody, denaturing conditions and the standard addition method were used to overcome barriers common to the traditional analysis of protein-base...

In this research, salicylic acid is proposed as an alternative biocide-free agent suitable for a preventive or integrative anti-biofilm approach. Salicylic acid has been proved to: (1) reduce bacterial adhesion up to 68.1 ± 5.6%; (2) affect biofilm structural development, reducing viable biomass by 97.0 ± 0.7% and extracellular proteins and polysac...

Abstract Sulfurtransferases (Strs) and thioredoxins (Trxs) are members of large protein families. Trxs are disulfide reductases and play an important role in redox-related cellular processes. They interact with a broad range of proteins. Strs catalyze the transfer of a sulfur atom from a suitable sulfur donor to nucleophilic sulfur acceptors in vit...

The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to...

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorim...

The goal was to investigate whether the diverse glucosinolate (Gl) profiles described for different Arabidopsis thaliana (L.) Heynh. ecotypes are at least partially shaped by the kinetic properties of sulfotransferases (SOTs) (EC 2.8.2.-) catalyzing the final step in Gl core structure biosynthesis. This study focuses on only one of the three SOTs t...

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g...

Effect of RhdA lack on levels of cysteine and l-glutamate, and on glutathione disulfide reductase (GR) activity in A. vinelandii. Significant differences between the strains at the level of P<0.05 and P<0.01 (Student's t test) are indicated as * and **, respectively. All values represent means ± standard deviation (SD) for three independent determi...

Supporting methods and references for Table S1. (DOC)

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the ant...

Zosteric acid sodium salt is a powerful antifouling agent. However, the mode of its antifouling action has not yet been fully elucidated. Whole cell proteome of Escherichia coli was analysed to study the different protein patterns expressed by the surface-exposed planktonic cells without and with sublethal concentrations of the zosteric acid sodium...

The tandem domain rhodanese-homology protein RhdA of Azotobacter vinelandii shows an active-site loop structure that confers structural peculiarity in the environment of its catalytic cysteine residue. The in vivo effects of the lack of RhdA were investigated using an A. vinelandii mutant strain (MV474) in which the rhdA gene was disrupted by delet...

Mobilization of the L-cysteine sulfur for the persulfuration of the rhodanese of Azotobacter vinelandii, RhdA, can be mediated by the A. vinelandii cysteine desulfurases, IscS and NifS. The amount of cysteine was higher in mutant strains lacking rhdA (MV474) than in wild type. The diazotrophic growth of MV474 was impaired. Taking into account the f...

The significance of the presence of anti-gliadin antibodies in patients affected by celiac disease is still unclear. It is hypothesized that gliadin deamidation, catalysed by transglutaminase, plays a role in favoring the antigen presentation. To determine the immunoreactivity of anti-gliadin antibodies from untreated celiac patients to transglutam...

The effects of chemical (acid-heating treatment) and enzymatic (microbial transglutaminase, TGase) modification (deamidation) of gluten proteins on their physicochemical and celiac disease-related properties were studied. Ammonia release, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sample solubility analyses were employed to chec...

The maize gene b-32, normally expressed in the maize (Zea mays) endosperm, encodes for a RIP (Ribosome Inactivating Protein) characterised by antifungal activity. Transgenic wheat plants were obtained via biolistic transformation, in which the b-32 gene is driven by the 35SCaMV promoter in association with the bar gene as a selectable marker. Plant...

Sulfurtransferases/rhodaneses (Str) are enzymes widely distributed in archaea, prokaryota and eukaryota, and catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. In this reaction, Str cycles between the sulfur-free and the sulfur-substituted form. Two-domain Str consist of two globular domains of nearly identica...

Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body f...

To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns. We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure (by means of light microscopy and scanning electron microscopy), and the effect of gliadin on actin...

After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate incre...

Sulfurtransferase are enzymes involved in the formation, conversion and transport of compounds containing sulfane-sulfur atoms. Although the three-dimensional structure of the rhodanese from the nitrogen-fixing bacterium Azotobacter vinelandii is known, the role of its two domains in the protein conformational stability is still obscure. We have ev...

3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vi...

The Azotobacter vinelandii rhodanese is a 31kDa sulfurtransferase protein that catalyzes the transfer of sulfur atom from thiosulfate to cyanide in the detoxification process from cyanide and is able to insert sulfur atom in the iron-sulfur cluster. A study of the uniformly 15N isotopic labeling by high resolution NMR, before obtaining the backbone...

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and ch...

We developed a competitive enzyme-linked immunosorbent assay (cELISA) based on bread wheat flour gliadin as coated antigen and human serum IgA as primary antibody in order to quantify the immunoreactivity of untreated celiac patient IgA antigliadin antibodies (AGAs) to different food proteins. No significant AGA immunoreactivity to bovine serum alb...

The Azotobacter vinelandii rhodanese is a sulfurtransferase enzyme that catalyzes the transfer of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the (15)N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur transfer from the enzyme to the substrate. The...

Nitric oxide (NO) is a versatile regulatory molecule that affects enzymatic activity through chemical modification of reactive amino acid residues (e.g., Cys and Tyr) and by binding to metal centers. In the present study, the inhibitory effect of the NO-donors S-nitroso-glutathione (GSNO), (+/-)E-4-ethyl-2-[E-hydroxyimino]-5-nitro-3-hexenamide (NOR...

In an attempt to clarify the role of gliadin toxicity in the pathogenesis of gluten intolerance (celiac disease), previous in vitro studies have been based on two-dimensional human cell cultures. However, the specific morphological and biochemical properties of in vivo tissue are better maintained in three-dimensional cell cultures (multicellular s...

SseA, the translation product of the Escherichia coli sseA gene, is a 31 kDa protein endowed with 3-mercaptopyruvate:cyanide sulfurtransferase activity in vitro. As such, SseA is the prototype of a sulfurtransferase subfamily distinguished from the better known rhodanese sulfurtransferases, which display thiosulfate:cyanide sulfurtransferase activi...

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemi...

The pathogenesis of celiac disease is not completely understood but, although the initial step of the process is still unclear, an altered immune response seems to play a major role. Previous studies of the biological properties of gliadin have highlighted its cytotoxic effects, and the aim of this study was to develop an in vitro technique to stud...

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfu...

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercapt...

The accessibility of primary amino groups to an external probe in durum wheat gluten proteins in the flour itself, and after the extraction and separation of gluten proteins, was studied by labeling the free amino groups with the hemisuccinate of 2(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl) propanol (FF18) and evidencing the label immunochemical...

Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Ly...

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feed...

A modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buff...

The maize b-32 protein is a functional ribosome-inactivating protein (RIP), inhibiting in vitro translation in the cell-free reticulocyte-derived system and having specific N-glycosidase activity on 28S rRNA. Previous results indicated that opaque-2 (o2) mutant kernels, lacking b-32, show an increased susceptibility to fungal attack and insect feed...

The maize Opaque-2 (O2) protein is a transcription factor of the basic/leucine-zipper class, involved in the regulation of endosperm proteins including the 22kDa alpha-zein storage proteins and b32 protein. In this study we have focussed our attention on the relationship between O2 and the cyPPDK1 gene, which encodes a cytoplasmic pyruvate orthopho...

Transient β-glucuronidase (GUS) expression was utilized to test the strength of a novel promoter system based on the maize (Zea mays) endosperm- specific b-32 gene promoter and its physiological activation Opaque 2 (O2). Up to ten O2 binding sites were taken together in the same promoter either separately, or in combination with the CaMV (Cauliflow...

Cross-reactivity between the different components in Parietaria judaica pollen extract has been investigated by polyclonal as well as monoclonal antibodies before and after chemical deglycosylation obtained by trifluoromethanesulphonic acid (TFMS) treatment of the extract. In western blotting a polyclonal rabbit antiserum, obtained by injecting pur...

A competitive enzyme-linked immunosorbent assay (ELISA) for some triazole fungicides was developed using a polyclonal antibody generated against the hemisuccinate of 2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl) propanol (DTP) conjugated to bovine serum albumin. In a typical standard curve of DTP the linear range of the test system extended from...

Polyamine oxidase was purified 4.8-fold from the cell wall extract of maize seedlings using only two chromatographic steps. The enzyme (Mrca 53 000) which had a specific activity of 700 nkat/mg at 37° showed a similar pH optimum (6.5) with both spermidine and spermine as substrates. For spermine and spermidine the Kms were 18 and 22 μM respectively...

Polyamine oxidase from oat shoots was purified to homogeneity by the criteria of polyacrylamide gel electrophoresis in native and denaturing conditions. The purified yellow enzyme had an Amax at 276, 370 and 452 nm. The A452 and A370 decreased upon addition in anaerobiosis of spermine and spermidine or dithionite, but not putrescine. The enzyme had...