Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Transplant of polymer-encapsulated cells genetically engineered to release nerve growth factor allows a normal functional development of the visual cortex in dark-reared rats.

Visual experience is necessary for the normal development of the visual system. Dark-reared mammals show abnormal vision when reintroduced into a normal environment. The absence of visual experience during the critical period results in reduced and/or inappropriate neural responses in visual cortical neurons. The change in electrical activity induced by dark rearing is probably reflected by the modulation of specific unknown molecules. Neurotrophins are present in the developing visual cortex and their production depends on visually driven electrical activity. Recent findings support the possibility that an important link between electrical activity in the visual pathway and correct development of visual properties is represented by neurotrophins. We advance the hypothesis that the visual abnormalities present in dark-reared animals could be due to a decreased production of a neurotrophin secondary to the lack of visual stimulation. We report that some properties of visual cortical response such as receptive field size, orientation selectivity, adaptation to repeated stimulation, response latency and visual acuity are virtually normal in dark-reared rats transplanted with polymer-encapsulated baby hamster kidney cells genetically engineered to release nerve growth factor.

Synergistic but transient rescue effects of BDNF and GDNF on axotomized neonatal motoneurons.

Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), members of distinct families of polypeptide growth factors, have been shown to support motoneurons under various in vitro and in vivo conditions. We used a model of motoneuron cell death induced by sciatic nerve section in newborn rats and compared the efficacy of BDNF and GDNF administered alone or simultaneously in order to determine whether combinations of neurotrophic proteins can produce more potent motoneuron rescue than individual factors. The factors were administered by different methods, including (i) a single dose on to the transected nerve, (ii) continuous delivery from implanted slow-release polymer rods (BDNF) or encapsulated cells (GDNF), and (iii) repeated systemic injections (BDNF). Irrespective of the method of administration, either factor alone produced rescue effects which dramatically declined at two weeks as compared to one week post-lesion. In contrast, this decrease was significantly reduced when BDNF and GDNF were used simultaneously provided that one factor was applied on to the nerve while the other was continuously released from the rods or capsules. Other combinations in which GDNF was replaced by ciliary neurotrophic factor or axokine-1 failed to reproduce such additive activity. Two conclusions can be made from these experiments. First, when BDNF and GDNF are administered simultaneously but by distinct routes of delivery, their survival-promoting effects on the injured developing motoneurons are potentiated; second, even continuous delivery of each of these trophic factors alone cannot completely abrogate the time-dependent decline in rescue effects in this model of motoneuron cell death.

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry.

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains ß-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS).BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds.For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H](+), of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.

Neurturin protects dopaminergic neurons following medial forebrain bundle axotomy.

To determine whether neurturin (NTN), a recently identified homologue of glial cell line-derived neurotrophic factor (GDNF), is able to preserve tyrosine hydroxylase immunoreactivity (TH-IR) in a rat model of Parkinson's disease, polymer encapsulated cells genetically engineered to release NTN were implanted near the substantia nigra 1 week before a unilateral medial forebrain bundle axotomy. Animals were allowed to survive for 1 week post-axotomy. Upon sacrifice, animals that received a NTN capsule had a significantly higher percentage of TH-IR (lesioned side vs non-lesioned side) than animals that had received a capsule containing non-transfected parent cells. However, in contrast to GDNF, no reduction of turning was observed upon amphetamine rotation with NTN. Nevertheless, these results suggest that NTN might have a therapeutic value for the treatment of Parkinson's disease.

Beta-endorphin-containing proteins in the human pituitary.

Two new proopiomelanocortin (POMC)-derived beta-endorphin (BE)-containing proteins were detected in the human pituitary, using HPLC, trypsin digestion, and a high sensitivity search with liquid secondary ion mass spectrometry (LSIMS) for the protonated molecule ion, (M + H)+, of tryptic peptides that are unique to BE. Proteins were extracted from pituitary tissues and were purified by solid phase extraction (SPE) chromatography and RP-HPLC. Each HPLC fraction was treated with trypsin, and each unseparated peptide mixture was analyzed by LSIMS to detect the two selected marker peptides (BE 20-24 and BE 10-19) that have excellent LSIMS desorption-ionization properties. The detection of both of those peptides indicated the presence of BE-containing proteins in two HPLC fractions (number 47 and 51). Tandem MS determined the amino acid sequence of the marker peptide BE 20-24 (NAIIK), and those sequence data optimized the specificity of the method. The two new BE-containing proteins derive from the C-terminal region of POMC, and were minor components in the two HPLC fractions. The major component in fraction 51 derived from the vasopressin-neurophysin 2-copeptin precursor.

Dynorphin A(1-8) in human placenta: amino acid sequence determined by tandem mass spectrometry.

Presence of the kappa receptor-preferring neuropeptide dynorphin A(1-8) in human placenta has been demonstrated by mass spectrometry to establish rigorously the appropriate molecular weight and amino acid sequence. Liquid secondary ionization mass spectrometry produced the protonated molecule ion, (M + H)+, at m/z 981 of the endogenous peptide, and tandem mass spectrometry collected the product ion spectrum that contained the appropriate amino acid sequence-determining fragment ions produced from the precursor ion (M + H)+. The amino acid sequence of the peptide is YGGFLRRI.

Opioid and tachykinin neuropeptides in prolactin-secreting human pituitary adenomas.

Two opioid neuropeptides, methionine enkephalin (ME) and beta-endorphin (BE), and one tachykinin neuropeptide, substance P (SP), were quantified in 10 prolactin (PRL)-secreting human pituitary adenomas and in 10 control human pituitaries. Immunohistochemical techniques provided appropriate staining for PRL. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to purify these three neuropeptides before their analysis, radioimmunoassay (RIA) was used for the quantification of SP-like immunoreactivity (SP-LI), and liquid secondary-ion mass spectrometry (LSIMS) was used for the qualitative and quantitative analysis of ME and a tryptic peptide of BE. This study shows that, for 90% of the cases studied here (excluding one hypothyroidism case), the tachykinin A neuropeptide SP-LI level is decreased, the POMC peptide BE level is not altered, and the proenkephalin A neuropeptide ME level is increased in these PRL-secreting tumors.

Mass spectrometric detection of preproenkephalin A-derived peptides in bovine pituitary.

A new analytical system, including chromatography separation, enzyme digestion, radioimmunoassay detection, and mass spectrometry characterization, has been designed to detect native preproenkephalin A-derived peptides in the bovine pituitary. The direct evidence of these intact peptides was provided by the mass spectrometric detection of the (M+H)+ ions of ME-Lys at m/z 702 and of ME-Arg at m/z 730, following trypsin digestion and the detection of ME-like immunoreactivity in HPLC fractions.

Encapsulated cell implants as a novel treatment for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic cells of the substantia nigra pars compacta (SNPc). Systemic levodopa therapy has proved to be an effective initial treatment for this disorder. However, resistance to this therapy inevitably develops with time, necessitating other approaches including surgery. Current experimental surgical treatments for this disorder include pallidal stimulation, pallidal lesion, subthalamic stimulation, and dopaminergic cell transplants. The current limitation of these approaches is that they all treat the symptoms but not the cause, that is, the progressive degeneration of the SNPc goes unabated.

Interfacial study of benzenesulfinate chemisorbed on silver.

The oxidative stability of self-assembled monolayer (SAM) of thiols on silver was examined with matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). A benzenesulfinate (BS) monolayer on silver was also prepared and investigated with MALDI-TOF-MS and surface-enhanced Raman scattering (SERS). The presence of sulfinate, sulfonate, and thiosulfonate fragment ions reveals that thiolate monolayers oxidize in the air. The relative abundance of C6H5SO2- and C6H5SO3- species in the mass spectra of the monolayer with different air-exposure times provides an estimation of the oxidation progress. In the vibrational spectrum, the large red shift of v(SOO-), combined with the lack of a shift of v(C-S) upon adsorption, indicates its bidentate O-coordination. The orientation of the confined molecules, based on the Raman surface selection rules, was derived from the preferential enhancement of the different functional groups. The benzene ring of BS was found almost normal to the metal surface as a result of the intermolecular forces; that geometry excludes the possibility of the pi-system of the benzene ring from participating as another binding site. The SER spectra of BS obtained via a selective etching process confirms these observations.

Tandem mass spectrometry analysis of synthetic opioid peptide analogs.

Five synthetic opioid peptides that were designed to have specific opioid receptor-binding properties were studied by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The MS/MS data are required for the analysis of those peptides in ovine plasma in a study to determine the placental transfer of the peptide to the fetus. The synthetic enkephalin-related peptides were: Tyr-D-Arg-Phe-Lys-NH2, (DALDA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, (ICI 174,864), Tyr-D-Thr-Gly-Phe-Leu-Thr, (DTLET), Tyr-D-Pen-Gly-Phe-D-Pen-OH, (DPDPE), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, (CTAP). Liquid secondary ion mass spectrometry (LSIMS) was used for sample desorption-ionization, and a hybrid (E1BE2qQ) tandem mass spectrometer was used to collect the product-ion spectra. A protonated molecule ion, [M + H]+, was observed for each peptide. Amino acid sequence-determining fragment ion were produced by CID and collected by MS/MS for the three linear peptides, and also for the two disulfide-bond-containing peptides in their unreduced and dithiothreitol (DTT)-reduced forms. The detection level for the [M + H]+ ion of DTLET was ca. 3 pmol; and the stabilities of the CTAP and ICI analogs in plasma were studied.

Preparative and analytical capillary zone electrophoresis analysis of native endorphins and enkephalins extracted from the bovine pituitary: mass spectrometric confirmation of the molecular mass of leucine enkephalin.

One proopiomelanocortin peptide [beta-endorphin (BE)] and two proenkephalin A peptides [methionine enkephalin (ME) and leucine enkephalin (LE); LE derives also from proenkephalin B] were searched for in a bovine pituitary extract by capillary zone electrophoresis (CZE) and liquid secondary-ion mass spectrometry (LSIMS). A bovine pituitary homogenate was subjected to acid precipitation/centrifugation and solid-phase extraction of peptides using an octadecyl-silyl disposable cartridge. The peptide-enriched fraction was subjected to CZE at pH 2.5 and at pH 5.5., and fractions were collected under preparative CZE conditions within defined time windows where synthetic BE, ME, and LE migrate. The resolving power of CZE was demonstrated by collecting biological fractions at pH 5.5 under preparative conditions and by subsequently analyzing these fractions at pH 2.5 under analytical conditions. Preparative CZE was further performed at pH 2.5 for fractions collected at pH 5.5. LSIMS analysis of this second-dimensional CZE fraction revealed the appropriate protonated molecule ion [(M + H)+, m/z 556.4] of LE.

Neuronal GDNF expression in the adult rat nervous system identified by in situ hybridization.

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor which has been purified on the basis of its ability to promote the survival of dopaminergic neurons in vitro. GDNF has subsequently been cloned and its sequence shown to be distantly related to transforming growth factor-beta (TGF-beta). To identify GDNF expressing cells in the adult rat brain, in situ hybridization using a digoxygenin (DIG)-labelled riboprobe has been performed. Our results show that GDNF mRNA is mainly expressed in neurons and that its synthesis is not restricted to dopaminergic areas. It is widely expressed in the cortex, the hippocampus, the striatum, the substantia nigra, the thalamus, the cerebellum and the spinal cord. Neuronal GDNF expression varies among brain regions as determined by the intensity of the in situ signal. Double labelling of the substantia nigra using tyrosine hydroxylase immunohistochemistry, associated with GDNF in situ hybridization, show that the majority of dopaminergic neurons express GDNF. The widespread expression of GDNF throughout the adult brain suggests that its administration in Parkinson's disease should be restricted to the altered structures, in order to avoid possible deleterious side effects.

GDNF reduces drug-induced rotational behavior after medial forebrain bundle transection by a mechanism not involving striatal dopamine.

Parkinson's disease (PD) is characterized by the progressive loss of the substantia nigra (SN) dopaminergic neurons projecting to the striatum. Neurotrophic factors may have the potential to prevent or slow down the degenerative process occurring in PD. To that end, we examined whether low amounts of glial cell line-derived neurotrophic factor (GDNF) continuously released from polymer-encapsulated genetically engineered cells are able to prevent the loss of tyrosine hydroxylase immunoreactivity (TH-IR) in SN neurons and ameliorate the amphetamine-induced rotational asymmetry in rats that have been subjected to a unilateral medial forebrain bundle (MFB) axotomy. Baby hamster kidney (BHK) cells transfected with the cDNA for GDNF were encapsulated in a polymer fiber and implanted unilaterally at a location lateral to the MFB and rostral to the SN. ELISA assays before implantation show that the capsules release approximately 5 ng of GDNF/capsule per day. One week later, the MFB was axotomized unilaterally ipsilateral to the capsule placement. Seven days later, the animals were tested for amphetamine-induced rotational asymmetry and killed. The striatum was excised and analyzed either for catecholamine content or TH-IR, while the SN was immunostained for the presence of TH-IR. GDNF did not prevent the loss of dopamine in the striatum. However, GDNF significantly rescued TH-IR neurons in the SN pars compacta. Furthermore, GDNF also significantly reduced the number of turns per minute ipsilateral to the lesion under the influence of amphetamine. Improvement of rotational behavior in the absence of dopaminergic striatal reinnervation may reflect neuronal plasticity in the SN, as suggested by the dendritic sprouting observed in animals receiving GDNF. These results illustrate that the continuous release of low levels of GDNF close to the SN is capable of protecting the nigral dopaminergic neurons from an axotomy-induced lesion and significantly improving pharmacological rotational behavior by a mechanism other than dopaminergic striatal reinnervation.

Mass spectrometric quantification of the mu opioid receptor agonist Tyr-D-Arg-Phe-Lys-NH2 (DALDA) in high-performance liquid chromatography-purified ovine plasma.

The mu opioid receptor agonist Tyr-D-Arg-Phe-Lys-Amide (D-Arg2-Lys4-Dermorphin(1-4)amide=DALDA) was infused continuously for 2 h into sheep. The presence of DALDA in ovine plasma was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) in plasma samples that were obtained at different times during and following that infusion. A stable isotope-incorporated internal standard, deuterated DALDA (d5-DALDA), was used for the MS quantification of DALDA via the protonated molecule ion, (M+H)+, of DALDA and of d5-DALDA. Time-course data (microg DALDA ml(-1) plasma vs. time) were obtained. Tandem MS (MS-MS) provided the product-ion spectrum of the (M+H)+ ion of DALDA in one of the samples to confirm the amino acid sequence of DALDA.

Seizure suppression in kindled rats by intraventricular grafting of an adenosine releasing synthetic polymer.

Adenosine, an endogenous inhibitory neuromodulator in the central nervous system, exerts anticonvulsant activity that is largely based on the inhibition of the release of excitatory amino acids. As a novel approach to treat pharmacoresistant partial epilepsies, the grafting of adenosine-releasing cells is foreseen to provide a local and sustained source of adenosine. The feasibility of this cell-based therapy was investigated in the present study by the intraventricular implantation of synthetic polymers that release adenosine. Kindled rats with a ventricular implant of an adenosine-releasing polymer showed a profound reduction of seizure activity. This was demonstrated not only by a 75% reduction of grade 5 seizures but also by a reduction of the amplitude and duration of afterdischarges in electroencephalographic (EEG) recordings. Kindled control rats that were implanted with bovine serum albumin (BSA)-containing polymers or were sham operated, continued to show their presurgery seizure pattern. Adenosine displayed antiepileptic activity when released in an amount of 20-50 ng per day. This finding sets the target for the required amount of adenosine to be released from future adenosine-releasing cells for antiepileptic therapy. The present results clearly support the feasibility of a novel therapy for epilepsy based on adenosine-releasing cells.

Effect of aging the hollow cathode by sputtering on the analytical precision of the hollow cathode discharge emission source.

Sputtering changes the analytical performance of the hollow cathode as an emission source. Proper conditioning (aging) of the hollow with sputtering greatly improves precision. Conditioning decreases the blank emission signal and changes the shape of the hollow. Micrographs which show the evolution of the bottom of the hollow into a stable bulb shape are presented. The blank emission signal decreases with aging of the hollow and approaches a constant value when the hollow is properly conditioned. A short-term stability (n = 5) of approximately 1.8% RSD has been observed frequently. An average long-term stability over several days (n = 45) of 4.5% for Li and 3.5% for Na in the emission signal from microsamples (less than 50 nL) deposited in the hollow cathode discharge source is reported here. Instrumentation, operation, and sample preparation procedures are described.