Nuclear Phosphoinositides: Their Regulation and Roles in Nuclear Functions.

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.

Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.

Sleep disturbances in elderly subjects: an epidemiological survey in an Italian district.

OBJECTIVES: Disturbed sleep is common in elderly people and has been related to comorbidities. The aim of this study was to evaluate the prevalence of sleep problems and their relationship with chronic disease in an elderly population. MATERIALS AND METHODS: The whole population of subjects aged more than 65 years, in the municipality of Vecchiano, Pisa was considered as eligible and underwent a clinical interview and a questionnaire about insomnia, sleepiness, snoring and sleep apnea. A model of logistic regression was applied to the data. RESULTS: The participation rate was 60.3% (1427 subjects). Insomnia was observed in 44.2% of our population, while sleepiness in 31.3%, snoring in 47.2% and sleep apnea in 9.0%. The most common diseases associated with sleep symptoms were depression, cognitive decline and diabetes. CONCLUSIONS: Our results confirm that sleep problems are very common in elderly subjects and closely related to medical and psychiatric illnesses.

The anti-tumoral potential of the saporin-based uPAR-targeting chimera ATF-SAP.

The development of personalized therapies represents an urgent need owing to the high rate of cancer recurrence and systemic toxicity of conventional drugs. So far, targeted toxins have shown promising results as potential therapeutic compounds. Specifically, toxins conjugated to antibodies or fused to growth factors/enzymes have been largely demonstrated to selectively address and kill cancer cells. We investigated the anti-tumor potential of a chimeric recombinant fusion protein formed by the Ribosome Inactivating Protein saporin (SAP) and the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), whose receptor has been shown to be over-expressed on the surface of aggressive tumors. ATF-SAP was recombinantly produced by the P. pastoris yeast and its activity was assessed on a panel of bladder and breast cancer cell lines. ATF-SAP resulted to be highly active in vitro, as nano-molar concentrations were sufficient to impair viability on tumor cell lines. In contrast to untargeted toxins, the chimeric fusion protein displayed a significantly improved toxic effect in uPAR-expressing cells, demonstrating that the selective activity was due to the presence of the targeting moiety. Fibroblasts were not sensitive to ATF-SAP despite uPAR expression, indicating that cell-specific receptor-mediated internalization pathway(s) might be considered. The in vivo anti-tumor effect of the chimera was shown in a bladder cancer xenograft model. Current findings indicate ATF-SAP as a suitable anti-tumoral therapeutic option to cope with cancer aggressiveness, as a single treatment or in combination with traditional therapeutic approaches, to appropriately address the intra- and inter- tumor heterogeneity.

Saporin as a novel suicide gene in anticancer gene therapy.

We used a non-viral gene delivery approach to explore the potential of the plant saporin (SAP) gene as an alternative to the currently employed suicide genes in cancer therapy. Plasmids expressing cytosolic SAP were generated by placing the region encoding the mature plant ribosome-inactivating protein under the control of cytomegalovirus (CMV) or simian virus 40 (SV40) promoters. Their ability to inhibit protein synthesis was first tested in cultured tumor cells co-transfected with a luciferase reporter gene. In particular, SAP expression driven by CMV promoter (pCI-SAP) demonstrated that only 10 ng of plasmid per 1.6 x 10(4) B16 cells drastically reduced luciferase activity to 18% of that in control cells. Direct intratumoral injection of pCI-SAP complexed with either lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) in B16 melanoma-bearing mice resulted in a noteworthy attenuation of tumor growth. This antitumor effect was increased in mice that received repeated intratumoral injections. A SAP catalytic inactive mutant (SAP-KQ) failed to exert any antitumor effect demonstrating that this was specifically owing to the SAP N-glycosidase activity. Our overall data strongly suggest that the gene encoding SAP, owing to its rapid and effective action and its independence from the proliferative state of target cells might become a suitable candidate suicide gene for oncologic applications.

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate.

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.

High prevalence of restless legs syndrome in multiple sclerosis.

Despite the fact that multiple sclerosis (MS) patients often include leg restlessness as a sensory symptom, MS is not mentioned amongst symptomatic restless legs syndrome (RLS) forms. The aim of this study was to estimate RLS prevalence in a large population of MS patients, comparing clinical and MRI findings between patients with and without RLS. Each of the 156 MS patients (100 females, 56 males, mean age 40.7 +/- 10.4) enrolled in a prospective study underwent a medical history interview, a neurological examination with the assessment of the Expanded Disability Status Scale (EDSS), and a structured questionnaire to verify the presence and features of RLS. Conventional brain-spinal MRIs of 99 subjects were also evaluated and compared between patients with and without RLS. Fifty-one subjects (32.7%) (mean age 43.8 +/- 12.8) met the criteria for RLS. In a few patients (8.5%), the RLS preceded clinical MS onset, whilst in the remaining cases the RLS was followed by or was simultaneous with clinical MS onset. Comparing the RLS group with the group without RLS, no significant differences were found in MS duration, gender, and referred sleep habits. The primary progressive MS course was more represented in the RLS group, which also showed a higher EDSS score. RLS is a very common finding in MS patients and should be considered amongst the symptomatic RLS forms. RLS is also associated with higher disability.

Mismatch negativity analysis in drug-resistant epileptic patients implanted with vagus nerve stimulator.

It is well known that some epileptic patients does not respond to conventional treatments, despite multiple combination of antiepileptic drugs, and they are therefore considered drug-resistant. For these patients, vagal nerve stimulation (VNS) represents a successful alternative to traditional therapy, and it is generally well tolerated; beside benefits on seizure frequency, VNS showed positive effects on cognition and mood. Aim of this study was to investigate short-term memory changes in a group of 12 patients implanted with VNS, through Mismatch Negativity wave (MMN). After 1 year of follow-up, MMN latencies and amplitudes did not show significant changes following VNS implantation, independently on current intensity, as compared with pre-implantation values. In two patients, MMN values, which were abnormal before VNS implantation, showed a major reduction in latency and an increase in amplitude after implantation, suggesting a likely positive effect of VNS on pre-attentive processes investigated by MMN.

A low-cost interface for control of computer functions by means of eye movements.

Human-computer interactions (HCI) have become an important area of research and development in computer science and psychology. Appropriate use of computers could be of primary importance for communication and education of those subjects which could not move, speak, see or hear properly. The aim of our study was to develop a reliable, low-cost and easy-to-use HCI based on electrooculography signal analysis, to allow physically impaired patients to control a computer as assisted communication. Twenty healthy subjects served as volunteers: eye movements were captured by means of four electrodes and a two-channel amplifier. The output signal was then transmitted to an "Analog to Digital" (AD) converter, which digitized the signal of the amplifier at a rate of 500 Hz, before being sent to a laptop. We designed and coded a specific software, which analyzed the input signal to give an interpretation of eye movements. By means of a single ocular movement (up, down, left and right) the subjects were then able to move a cursor over a screen keyboard, passing from one letter to another; a double eye blink was then necessary to select and write the active letter. After a brief training session, all the subjects were able to confidently control the cursor and write words using only ocular movements and blinking. For each subject we presented three series of randomized words: mean time required to enter a single character was about 8.5s, while input errors were very limited (less than 1 per 250 characters). Our results confirm those obtained in previous studies: eye-movement interface can be used to properly control computer functions and to assist communication of movement-impaired patients.

Quantitative EEG analysis in post-traumatic anosmia.

Many objective and quantitative methods have been developed to create a procedure or a device to prove, describe and quantify olfactory deficit and anosmia, especially after a head trauma. Electrophysiological testing throughout olfactoelectroencephalography (olfactoEEG) is based on brain activity desynchronisation, and on the subsequent disappearance of alpha activity on the posterior regions after an olfactory stimulus. Yet traditional evaluation of EEG can be difficult, because of little or hardly detectable alpha activity on the posterior regions ('alpha rare'). The aim of this study was to evaluate the Olfactory Stop Reaction (OSR) by means of frequency band power calculation and subsequent topographical mapping in patients with post-traumatic anosmia, who presented 'alpha rare' EEG. Twenty-five consecutive patients, affected by anosmia caused by head trauma, were submitted to an EEG recording with olfactory stimulation. After signal processing and analysis, an Olfactory Stop Reaction was detected in 17 out of 25 patients; moreover, in these patients we detected a significant decrease in alpha band power in the occipital regions and an increase in theta band power on midline frontal and central regions after olfactory stimulation. In the remaining eight patients, no significant variation in band power was observed. In conclusion, an objective evaluation of the olfactory function with this method of automatic EEG signal analysis allows the limits given by psychophysical methods and traditional EEG to be overcome and attempts to fulfil the requirements for standardization of olfactory function evalution.

Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells.

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site.

1-Deoxymannojirimycin inhibits Golgi-mediated processing of glycoprotein in Xenopus oocytes.

We prepared in vitro an mRNA transcript coding for the erythroagglutinating subunit of the kidney bean glycoprotein phytohemagglutinin, E-PHA. The mRNA, injected into Xenopus oocytes, synthesized E-PHA carrying two Asn-linked carbohydrate chains, one of which was processed and acquired resistance to endo-beta-N-acetylglucosaminidase H, as occurs in the native bean cells. When the mannose analog 1-deoxymannojirimycin, an inhibitor of mammalian Golgi mannosidase I, was included in the oocyte culture medium, the acquisition of endo-beta-N-acetylglucosaminidase H resistance was abolished, indicating that also in an amphibian cell the inhibitor blocks a key reaction in Golgi-mediated processing.

The signal peptide of human preproendothelin-1.

Synthetic mRNAs were produced using either the complete coding sequence of a human preproendothelin-1 cDNA clone or a truncated form in which the portion encoding the first 17 amino acids, representing a putative signal peptide for insertion into the endoplasmic reticulum, was replaced with a methionine codon. The mRNAs were translated in vitro in the presence or in the absence of microsomal membranes. Protection from trypsin digestion demonstrated that the full-length polypeptide, but not the truncated form, could be inserted into the membranes. Sequence analysis revealed that membrane insertion is accompanied by removal of the first 17 amino acids. These results indicate that the first 17 amino acids of human preproendothelin-1 are a functional signal peptide which allows the protein to enter the secretory pathway.

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Pyrrolobenzoxazepinone derivatives as non-nucleoside HIV-1 RT inhibitors: further structure-activity relationship studies and identification of more potent broad-spectrum HIV-1 RT inhibitors with antiviral activity.

Pyrrolobenzoxazepinone (PBO) derivatives represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTs) whose prototype is (+/-)-6-ethyl-6-phenylpyrrolo[2,1-d][1,5]benzoxazepin-7(6H)- one (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis and biological evaluation of novel derivatives and analogues of 6 featuring a meta-substituted phenyl or a 2-thienyl ring at C-6 and a pyridine system in place of the fused-benzene ring to yield pyrrolopyridooxazepinones (PPOs). Compared with the lead 6 and nevirapine, several of the synthesized compounds (PBOs 13a-d and PPOs 13i-k) displayed higher inhibitory activity against wild-type RT and clinically relevant mutant RTs containing the single amino acid substitutions L100I, K103N, V106A, Y181I, and Y188L. The most potent inhibitors were further evaluated for in vitro antiviral activity on lymphocytes and monocyte-macrophages, for cytotoxicity on a panel of cell lines, and for potential synergistic antiviral activity with AZT. Pharmacokinetic studies performed on 13b, 13c, and 13i showed that these compounds achieve high concentrations in the brain. The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of analogues such as 13b-d, 13i, and 13j, in combination with nucleoside RT inhibitors, against strains of HIV-1 bearing those mutations that confer resistance to known NNRTI.

Non-nucleoside HIV-1 reverse transcriptase inhibitors: synthesis and biological evaluation of novel quinoxalinylethylpyridylthioureas as potent antiviral agents.

New heterocyclic derivatives of ethylpyridylthiourea, quinoxalinylethylpyridylthiourea (QXPT) and analogues, inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and prevented HIV-1 cytopathogenicity in T4 lymphocytes. Several of these novel non-nucleoside RT inhibitors, with a substituted pyrroloquinoxalinone heteroaromatic skeleton, showed inhibitory activity against wild-type RT as well as against mutant RTs containing the single amino acid substitutions L1001, K103N, V106A, Y1811 and Y188L that was much greater than other non-nucleoside inhibitors such as nevirapine. Maximum potency in enzymatic assays was achieved with a fluoropyrroloquinoxaline skeleton linked to the ethylpyridylthiourea moiety (FQXPT). In cell-based assays on different cell lines and on human monocyte-macrophages, 6-FQXPT exhibited EC50 values in the nanomolar range, with a promising selectivity index. Moreover, 6-FQXPT showed synergistic antiviral activity with zidovudine.

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris.

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [(3)H]glucosamine or [(3)H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M(r) 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M(r) 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [(3)H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.