Spinocerebellar ataxia types 2 and 3 segregating simultaneously in a single family.

Spinocerebellar ataxia (SCA) types 2 and 3 are autosomal-dominant neurodegenerative disorders caused by mutations in two different genes. We identified mutations for SCA2 and SCA3 segregating simultaneously in a single Brazilian family. The index patient had SCA2, whereas her two second-degree cousins had SCA3. Disease was more rapidly progressive in the SCA2 patient, who presented severe brainstem and pancerebellar atrophy, as opposed to the two SCA3 patients, who had only mild cerebellar vermian atrophy. In such situations, molecular confirmation of all patients may avoid misdiagnosis of SCA subtypes and eventual errors in predictive testing of unaffected family members.     

Wortmannin-mediated inhibition of phosphatidylinositol 3-kinase activity triggers apoptosis in normal and neoplastic B lymphocytes which are in cell cycle

The B cell functional response following ligation of surface(s) lgM is dependent upon the differentiation stage of the populationstudied: cross-linking slgM promotes proliferation of restingtonsillar follicular mantle (FM) B lymphocytes but induces apoptosisin the susceptible Epstein- Barr virus genome-negative Burkittlymphoma (BL) cell line Ramos (Ramos-BL). This study investigateswhether phosphatidylinositol-3-kinase (Pl3-kinase), which hasbeen reported to be intimately involved in the regulation ofcellular growth, plays a role in the regulation of these sig-promoted B cell responses, and uses the selective and irreversibleinhibitor of Pl3-kinase activity, wortmannin (Wm). In Ramos-BLB cells, at 8 h post-treatment, Wm triggers a transient increasein apoptosis of 16 ± 6.9% with a concomitant cellularloss of 16 ± 6.1% from the G₁ phase of cell cycle; [³H]thymidineincorporation also decreases by 33 ± 5.0%, from 37,274c.p.m. ± 10% to 25,127 c.p.m. ± 4.0%. Moreover,at 72 h culture, Wm inhibits anti-lgM-induced FM B lymphocytelevels of [³H]thymidine incorporation typically by 47% and triggers80% apoptosis from the G₀G₁ phase of cell cycle. Ramos-BL Bcells exhibit high basal levels of Pl3-kinase activity, as determinedby immunoprecipitation with antibody to the p85 regulatory subunitof Pl3-kinase and ³²P incorporation into phosphatidylinositol,which is not significantly affected by anti-lgM stimulation;by contrast, anti-lgM stimulates significant Pl3-kinase activityover negligible basal levels in FM B lymphocytes. Pre-treatmentwith Wm inhibits Pl3-kinase activity in both cell types. Takentogether these data indicate that in Ramos-BL B cells slgM-triggeredgrowth arrest and apoptosis is Pl3- kinase independent, whereasPl3-kinase activity is critical for slgM-triggered mitogenesisof FM B lymphocytes. Thus Pl3-kinase plays a pivotal role inthe regulation of both normal and neoplastic B lymphocyte progressionthrough the cell cycle, such that if this Pl3-kinase-dependentpathway is inhibited these cells default to apoptosis.     

“Vinylogs” and “Acetylenylogs” of β-Adrenergic Agents

Vinylogous (Groups III and V ) and acetylenologous (Group IV ) analogs of the classical β-adrenergic agents — stimulants and blockers — were prepared in order to evaluate the effect of degree of saturation, position of unsaturation and rigidity of the chain linking the aromatic ring and the amino containing functional group on biological activity. Derivatives from Group III , which represent 4-aryl-3-butenyl-2-ol-amine analogs of Group II , retained β₁-adrenoceptor antagonist activity albeit substantially less potent (50–200-fold) than that possessed by their aryloxy counterparts. Consistent with the SAR for Group II compounds, substitution at position 2 of the aromatic ring yielded the most potent antagonists ( 5a, 5d, 5g ), with K_B's ranging from 73–93 nM while 3,4-dichloro substitution ( 5e ) markedly reduced antagonist potency (K_B = 2,400 nM). Agonist activity was also noted for 5b and 5d , suggesting that these compounds may be best classified as partial agonists. Representatives from Groups IV and V were inactive as antagonists at the β₁-adrenoceptor confirming the importance of the spatial relationship between the hydroxyl and the amino nitrogen.     

Release of cholecystokinin from rat cerebral cortex in vivo: role of GABA and glutamate receptor systems

Using cortical cups in chloralose-urethanized rats, the in vivo release of cholecystokinin-like immunoreactivity (CCK-LI) from cerebral cortex was examined. Resting levels of cholecystokinin-like immunoreactivity ranged from 20 to 30 pg/20 min sample. The addition of potassium (40 mM) in excess, resulted in a highly significant elevation in the levels of CCK-LI in the cortical superfusate. Deletion of calcium and the substitution of cobalt (10 mM), resulted in a significant reduction in both resting release and the release otherwise evoked by the addition of potassium. Focal electrical stimulation of the cortex (20 Hz), resulted in a significant (1.9 +/- 0.2-fold, n = 8) increase in the levels of CCK-LI. The addition of glutamate (10(-6)-10(-4) M) of kainic acid (10(-8)-10(-6) M), also resulted in significant elevations in the levels of CCK-LI. The co-administration of a putative glutamate receptor antagonist, kynurenic acid (10(-4) M) resulted in a significant reduction in the levels of release otherwise evoked by the addition of glutamate, but not by electrical stimulation. The addition of GABA (10(-5)-10(-3) M) resulted in a dose-dependent decrease in the resting release of CCK-LI, and the release evoked by glutamate. Picrotoxin (10(-6)-10(-4) M), resulted in a highly significant increase in the levels of CCK-LI in the cortical effluent. These results are consistent with a tonic GABAergic inhibition of CCK-releasing neurons. The treatment of the animal with diazepam (30 mg/kg, i.p.) also resulted in a significant reduction in resting release and the release otherwise evoked by focal cortical stimulation.     

1 alpha(OH)D3 (ETALPHA) treatment and receptor studies in 16 patients with chronic and myeloproliferative disorders

10 patients with CLL and 2 with CML were treated with gradually increasing doses of 1 alpha(OH)D3, up to 4 micrograms daily during 6 wk. 3 patients with preleukemia and 1 with myelofibrosis were treated with 2 micrograms daily of 1 alpha(OH)D3 for a prolonged period up to 17 wk. The treatment with 1 alpha (OH)D3 did not result in changes of disease parameters in any of the patients under study. Receptor studies for 1,25(OH)2D3 were performed in 8 CLL patients and revealed only 1 patient with increased specific receptor binding capacity. The maximum tolerable dose of 1 alpha(OH)D3 varied individually, but was in the range of 2-4 micrograms daily.     

Dopaminergic transmission in the hypothalamic arcuate nucleus to produce acupuncture analgesia in correlation with the pituitary gland

Acupuncture analgesia (AA) caused by low frequency stimulation of the acupuncture point (AP) was abolished by hypophysectomy and adrenalectomy. Termination of the AA producing pathway from the AP to the pituitary gland was in the medial hypothalamic arcuate nucleus (M-HARN). The origin of the descending pain inhibitory system associated with AA was in the posterior HARN (P-HARN). AA in the hypophysectomized rats, and enhanced neuronal activity in the P-HARN that were abolished during acupuncture stimulation, were both restored by intraperitoneal microinjection of 0.5 mg/kg morphine or 0.1 micrograms beta-endorphin into the P-HARN during acupuncture stimulation. Of the analgesia produced by dopamine or beta-endorphin injected into the P-HARN, that caused by beta-endorphin disappeared after denervation of the M-HARN. The P-HARN neurons that responded to acupuncture stimulation also responded to iontophoretic dopamine, but not to iontophoretic morphine nor ultramicroinjected beta-endorphin. The transmission between the M-HARN and P-HARN may be dopaminergic, and beta-endorphin might presynaptically modulate this transmission. Reduction of sodium ions may have been the reason for abolition of AA after adrenalectomy.     

Folding and function of the myelin proteins from primary sequence data.

To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular. In this program, propensities for the secondary structure conformations as well as physical-chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two-dimensional map of the correlation coefficients. Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the alpha or beta conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of beta propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed alpha-beta-alpha folding. To trace the immunoglobulin fold along the P0 sequence, we compared the beta propensity curve of P0 with that of the immunoglobulin M603, whose three-dimensional structure has been determined. We propose that the flat beta-sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra beta-fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x-ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N-glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2',3'-nucleotides into corresponding 2'-nucleotides.     

Multidrug transport in human glioblastoma cells is inhibited by transforming growth factors type beta 1, beta 2, and beta 1.2

The transforming growth factors type beta 1, beta 2, and beta 1.2 suppress multidrug transport in human pat-1 glioblastoma cells and even in cells that strongly over-express mdr genes and are resistant to inhibition of multidrug transport by chemosensitizers. Thus, inhibition of multidrug transport by cytokines might be a new approach to increase cellular accumulation of chemotherapeutic agents in multidrug resistant glial tumor cells. Interestingly, a member of the more distantly related decapentaplegic subgroup of transforming growth factors, the bone morphogenetic protein BMP 2, did not inhibit multidrug transport.     

Synthesis of deramciclane* labeled with tritium in various positions

[(1R)‐endo]‐(+)‐3‐bromocamphor was dehalogenated with tritium gas to [3‐³H]camphor and via [3‐³H]phenylborneol converted to [3‐³H]deramciclane isolated as the fumarate salt (specific activity 51.8 GBq/mmol). This three step synthesis from [3‐³H]camphor gave an overall yield of 22%. Benzyloxy‐acetic acid methyl ester was reduced with sodium‐borotritide to 2‐benzyloxy‐ethanol‐[1‐³H], and through a four step procedure was converted to 2‐dimethylaminoethyl‐[2‐³H] chloride. The latter was condensed with the sodium derivative of 2‐phenylborneol giving rise to [2‐dimethylamino‐[2‐³H]ethoxy]deramciclane isolated as the fumarate (specific activity 8.177 GBq/mmol). This six step synthesis from [³H]NaBH₄ gave an overall yield of 6%. Copyright © 2005 John Wiley & Sons, Ltd.