Mutations in BICD2 Cause Dominant Congenital Spinal Muscular Atrophy and Hereditary Spastic Paraplegia

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.     

The Background of Reduced Face Specificity of N170 in Congenital Prosopagnosia

Congenital prosopagnosia is lifelong face-recognition impairment in the absence of evidence for structural brain damage. To study the neural correlates of congenital prosopagnosia, we measured the face-sensitive N170 component of the event-related potential in three members of the same family (father (56 y), son (25 y) and daughter (22 y)) and in age-matched neurotypical participants (young controls: n = 14; 24.5 y±2.1; old controls: n = 6; 57.3 y±5.4). To compare the face sensitivity of N170 in congenital prosopagnosic and neurotypical participants we measured the event-related potentials for faces and phase-scrambled random noise stimuli. In neurotypicals we found significantly larger N170 amplitude for faces compared to noise stimuli, reflecting normal early face processing. The congenital prosopagnosic participants, by contrast, showed reduced face sensitivity of the N170, and this was due to a larger than normal noise-elicited N170, rather than to a smaller face-elicited N170. Interestingly, single-trial analysis revealed that the lack of face sensitivity in congenital prosopagnosia is related to a larger oscillatory power and phase-locking in the theta frequency-band (4–7 Hz, 130–190 ms) as well as to a lower intertrial jitter of the response latency for the noise stimuli. Altogether, these results suggest that congenital prosopagnosia is due to the deficit of early, structural encoding steps of face perception in filtering between face and non-face stimuli.     

Novel Method to Load Multiple Genes onto a Mammalian Artificial Chromosome

Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.     

Water bird guilds and their feeding connections in the Bodrogzug, Hungary

Thalli of the intertidal Phaeophyte Fucus spiralis L. and the subtidal Chlorophyte Ulva olivascens Dangeard were exposed to artificial UV-A, UV-B and photosynthetically active radiation (PAR) by combination of PAR + UV-A + UV-B (PAB), PAR + UV-A (PA) and PAR (P) treatments. UV-A enhanced photosynthesis and stimulated carbonic anhydrase (CA) and nitrate reductase (NR) in F. spiralis whilst PAR only had an inhibitory effect in this species. U. olivascens suffered chronic photoinhibition in all the treatments as evidenced by reduced maxima photosynthesis (P_max) and photosynthetic efficiency (α). Non stimulatory effect was observed upon CA and NR in this species. Our results showed that artificial UV radiation triggered opposite responses in both species. We suggest that differences shown by both species might be related to their location in the rocky shore and their ability to sense UV. We propose that the ratio UV:PAR acts as an environmental signal involved in the control of photosynthesis as shown by pronounced inhibition in samples exposed to only PAR. We also suggest that UV-regulated photosynthesis would be related to carbon (C) and nitrogen (N) cycles, regulating feedback processes that control C and N assimilation.     

The synthesis of 17-alkoxycarbonyl- and 17-carboxamido-13alpha-estra-1,3,5(10),16-tetraene derivatives via palladium-catalyzed carbonylation reactions

17-Alkoxycarbonyl- and 17-carboxamido-13alpha-estra-1,3,5(10),16-tetraenes were synthesized from the 17-iodo-13alpha-estra-1,3,5(10),16-tetraene derivative in palladium-catalyzed alkoxycarbonylation and aminocarbonylation reactions, respectively. The synthesis of the 17-iodo-16-ene derivative, used as substrate, is based on the transformation of the 17-keto derivative (epiestrone methyl ether) to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). 17-Carboxamides were obtained in good yields (up to 88%) not only with simple alkyl/aryl amines but also with amino acid methyl esters as N-nucleophiles. The use of alcohols as O-nucleophiles in alkoxycarbonylation resulted in the corresponding 17-esters; however, yields of synthetic interest were obtained only with methanol.     

Multiple treatment potentiates the anticonvulsive activity of cholecystokinin octapeptides

The dose-response curves for the anticonvulsive activity of sulfated and nonsulfated cholecystokinin octapeptide (CCK-8-SE and CCK-8-NS) against picrotoxin-induced (6 mg/kg SC) seizures were assessed either following or without pretreatment with a single high dose of CCK-8-SE or CCK-8-NS, to examine acute tolerance to the effect after IP injections in mice. As CCK-8-SE or CCK-8-NS pretreatment, a 1.6 μmole/kg dose was injected 2 hr prior to the second injection. No acute tolerance to the anticonvulsive activity was demonstrated, and CCK-8-NS pretreatment significantly potentiated its own anticonvulsive activity. Chronic (8-day) daily treatment with a 0.16 μmole/kg dose of CCK-8-SE or CCK-8-NS antagonized seizures by picrotoxin, presumably in a cumulative manner. To investigate the interactions of CCK octapeptides with other anticonvulsive agents, picrotoxin-induced seizures were antagonized with several doses of diazepam following or without acute, high-dose pretreatment with CCK-8-SE or CCK-8-NS. The two octapeptides only slightly modified the activity of diazepam: CCK-8-SE pretreatment displayed a tendency to antagonize it, while CCK-8-NS pretreatment to potentiate it. The results suggest that multiple treatment with CCK-8 induces sensitization of CCK receptors mediating anticonvulsive activity.     

Antisense oligonucleotides with antitumor activity

Antisense oligonucleotides (AONs) provide an efficient approach for developing target-selective anticancer drugs, because they can inhibit gene expression sequence specifically. To improve the therapeutic effenciency of AONs, two new types of the compounds have been developed. The first group of antisense oligodeoxynucleotides investigated contains base modified nucleotide units. Incorporation of 5-substituted pyrimidines into AONs increases cell membrane permeability (a), duplex stability (b), and nuclease resistance (c). These properties were studied using a large number of model oligonucleotides. The application of 5-(1-hexynyl)dU has been found to be the best modification. Application of MMP-9 collagenase inhibitor oligonucleotides (potential metastasis inhibitors) containing these nucleotide units instead of thymidines increased the collagenase inhibition potency by one order of magnitude compared to that of parental oligonucleotide including thymine bases. The second group of the compounds investigated represents a new type of antisense oligonucleotide synthesized by the antisense directed prodrug therapy (ADPT) conception. According to this principle, a telomerase inhibitor AON was conjugated with 5-fluoro-2'-deoxyuridine (FdU) and oligo-FdUs by phosphodiester bond at the 3'-terminus. The antitumor activities of conjugates in comparison with that of FdU were tested in HT1080 human fibrosarcoma and HT29 human colon adenocarcinoma cell lines. In HT29 cell culture the antiproliferative activity of prodrugs significantly increased with increasing length of the 3'-(FdU)n tail. The conjugate with one FdU unit was about 5 times, while the AON-(FdU)3 analogue was almost 19 times more active than FdU. Antitumor activity of the prodrug containing six FdU units was extremely high (relative efficiency = 26.6), therefore, in vivo testing of this analogue seems to be reasonable and promising. Antiproliferative activity of (FdU)n conjugated with a telomerase inhibitor increased by 5-13 times in HT1080 cells as compared to FdU administered in nucleoside form.