Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson's disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson's disease.     

Determining heart muscle perfusion by magnetic resonance tomography progressing to clinical application

Magnetic resonance imaging detects the flow of contrast-enhanced blood and even allows the quantitative assessment of myocardial perfusion. The clinical application of this method is being held back by the difficulties in image evaluation and the limitation of standard techniques to the acquisition of a single slice per heart beat cycle. Recent developments in scanner hardware as well as in image acquisition techniques open up the possibility of assessing myocardial perfusion over the entire heart with a spatial resolution in the range of 2 mm. As an example of such a new scanning strategy, a segmented gradient-echo recalled echo planar imaging sequence with preceding saturation is discussed and results in a patient with an infarction are presented. The clinical use of perfusion assessment covering the entire heart for the diagnosis of coronary artery disease is enhanced by the flexibility of magnetic resonance imaging for the assessment of functional cardiac parameters.     

The role of a lymphoid-restricted, Grb2-like SH3-SH2-SH3 protein in T cell receptor signaling

We have characterized an SH3-SH2-SH3 linker protein that is prominently expressed in lymphoid tissues. This protein has 58% sequence identity to Grb2. An identical protein called Grap has been found in hematopoietic cells. In Jurkat cells, T cell receptor activation leads to the association of Grap with phosphoproteins p36/38 and, to a lesser degree, Shc. This interaction is mediated by the Grap SH2 domain, which has similar binding specificity to the Grb2 SH2 domain. Grap also associates via its SH3 domains with Sos, the Ras guanine nucleotide exchange factor; with dynamin, a GTPase involved in membrane protein trafficking; and with Sam68, a nuclear RNA-binding protein that serves as a substrate of Src kinases during mitosis. T cell activation effects an increase in Grap association with p36/38, Shc, Sos, and dynamin. Sam68 binding is constitutive. Phospholipase C-gamma1 and Fyn are also found in activated Grap signaling complexes, although these interactions may not be direct. We conclude that Grap is a prominent component of lymphocyte receptor signaling. Based on the known functions of bound effector molecules, Grap-mediated responses to antigen challenge may include endocytosis of the T cell receptor, cellular proliferation, and regulated entry into the cell cycle.     

Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis

The pathogenesis of neuronal degeneration in both sporadic and familial amyotrophic lateral sclerosis (ALS) associated with mutations in superoxide dismutase may involve oxidative stress. A leading candidate as a mediator of oxidative stress is peroxynitrite, which is formed by the reaction of superoxide with nitric oxide. 3-Nitrotyrosine is a relatively specific marker for oxidative damage mediated by peroxynitrite. In the present study, biochemical measurements showed increased concentrations of 3-nitrotyrosine and 3-nitro-4-hydroxyphenylacetic acid in the lumbar and thoracic spinal cord of ALS patients. Increased 3-nitrotyrosine immunoreactivity was observed in motor neurons of both sporadic and familial ALS patients. Neurologic control patients with cerebral ischemia also showed increased 3-nitrotyrosine immunoreactivity. These findings suggest that peroxynitrite-mediated oxidative damage may play a role in the pathogenesis of both sporadic and familial ALS.     

Rotavirus virus-like particles administered mucosally induce protective immunity

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Prolactin inhibits EGF-induced DNA synthesis in mammary epithelium via early signaling mechanisms: possible involvement of protein kinase C

Prolactin and prolactin agonists inhibited EGF-induced DNA synthesis in mammary epithelium, whereas other pituitary hormones had no effect on EGF-induced DNA synthesis. The inhibitory effect of prolactin was seen for EGF and TGF-alpha, but not for IGF-I or cholera toxin. Autoradiography indicated that prolactin decreased the ability of EGF to induce cells to progress to S phase of the cell cycle, and time course studies indicated that the effects of prolactin were not due to an altered timing of DNA synthesis induction. Prolactin addition within 30 min of adding EGF was necessary to inhibit EGF-induced DNA synthesis. Conditioned media from prolactin-treated cells from which prolactin had been neutralized with the extracellular domain of the prolactin receptor had no effect on EGF-induced DNA synthesis, suggesting that the effect was due to prolactin, not an autocrine factor induced by prolactin. Prolactin induced a rapid association of protein kinase C with the membrane fraction of NMuMG cells, as well as increased threonine phosphorylation of the EGF receptor. Protein kinase C inhibitors eliminated most of the inhibitory effect of prolactin on EGF-induced DNA synthesis. The protein kinase C inhibitor Calphostin C restored high-affinity EGF binding in prolactin-treated cells and reversed the inhibitory effect of prolactin on EGF-induced EGF receptor tyrosine phosphorylation.     

14C]7-ethoxycoumarin metabolism by precision-cut rat hepatic slices

It is recognized that iodine-123-labelled 15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (123I-BMIPP) slowly washes out of the myocardium. The mechanism for the washout was investigated in normal rat hearts by analyses of the subcellular distribution and lipid classes based on the BMIPP metabolism. Rat hearts were excised at 1-120 min after intravenous injection of 123I-BMIPP. After counting the radioactivity, the hearts were digested with Nagarse and homogenized, and then fractionated into the cytosolic, mitochondrial, microsomal and crude nuclear fractions by centrifugations. The radioactivity of each fraction was counted, and the lipid classes were analysed by radio-thin-layer chromatographic and high-performance liquid chromatographic methods. The heart uptake of 123I-BMIPP was maximal at 5 min (6.81%+/-0.36% ID/g), and 41% of the radioactivity disappeared within 120 min. The myocardial radioactivity was immediately distributed into the cytosolic, mitochondrial, microsomal and crude nuclear fractions. The distribution (%) of each fraction was almost identical from 5 min through 120 min. The cytosolic fraction was always the major site of radioactivity deposition (60%), and the time-activity curve of the cytosolic fraction paralleled that of the whole heart throughout the 120-min study period. In the cytosolic fraction, most of the radioactivity was incorporated into the triglyceride class, and the rest was present in the free fatty acid, phospholipid (phosphatidylcholine) and diglyceride classes. In the mitochondrial fraction, the radioactivity was mostly incorporated into the phospholipid class (phosphatidylethanolamine), followed by free fatty acids. The final metabolite of 123I-BMIPP, 123I-p-iodophenylacetic acid (123I-PIPA), initially appeared in the mitochondrial fraction as early as 1 min, and subsequently in the cytosolic fraction at 5 min. Another intermediary metabolite, 123I-p-iodophenyldodecanoic acid (123I-PIPC12), was found only in the mitochondrial fraction after 5 min. In conclusion, the slow washout kinetics of 123I-BMIPP from the myocardium mainly reflects the turnover rate of the triglyceride pool in the cytosol. The BMIPP metabolism, i.e. initial alpha-oxidation followed by subsequent cycles of beta-oxidation, was confirmed in vivo. The participation of the mitochondria in the metabolism was also proven.