The unit impulse response procedure for the pharmacokinetic evaluation of drug entry into the central nervous system

The unit impulse response theory has been adapted to characterize the transport profile of drugs into the central nervous system (CNS). From the obtained input function, the cumulative plasma volume (V) cleared by transport into the CNS in time can be calculated. Simulation studies demonstrated that transport governed by passive diffusion resulted in a linear relationship between V and time, while the slope of the line, the blood- brain barrier (BBB) clearance, proved to be an adequate and model independent parameter to characterize drug transport into the CNS. The error in the result of the numerical procedure could be limited to less than 10% of the theoretically predicted value. Superposition of 5 or 10% random noise on simulated data did not result in significant differences between the calculated and theoretically predicted clearance values. Simulations of carrier-mediated transport resulted in nonlinear transport curves; the degree of nonlinearity, and thus the detectability, was dependent on the initial degree of saturation of the system, the rate of desaturation, as caused by drug elimination processes and the noise level on the data. In vivoexperiments in the rat were performed, using atenolol, acetaminophen, and antipyrine as model drugs. Linear transport relationships were obtained for all drugs, indicating that transport was dependent on passive diffusion or a low affinity carrier system. BBB- clearance values were 7±1 l/min for atenolol, 63±7 ul/min for acetaminiphen and 316±25 l/min for antipyrine. These experiments validate the applicability of the presented technique in in vivostudies.     

The effects of short-term smokeless tobacco deprivation on performance

Several past studies have reported reliable changes in reaction time performance and self-rated withdrawal scores as a consequence of cigarette deprivation. The purpose of the present study was to determine, prospectively, the effect 24 h of smokeless tobacco deprivation in regular users has on performance and the associated withdrawal symptomology. Forty smokeless tobacco users (Copenhagen brand) were randomly divided evenly into two groups gen brand) were randomly divided evenly into two groups (N=20)24 h of deprivation and no deprivation. A third group of ten nonchewers was added as another control group. The results indicated that behavioral, subjective and physiological changes are associated with smokeless tobacco deprivation in regular users. These include increased craving scores, reaction time, self-rated withdrawal symptoms and decreased heart rate.     

Genetic mapping ofPim-1 putative oncogene to mouse chromosome 17

Pim-1 is a putative oncogene activated in T-cell lymphomas induced by Moloney and AKR mink cell focus forming (MCF) viruses. We have determined the chromosomal localization of the Pim-1gene in mice by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The Pim-1gene was localized on chromosome 17, a chromosome frequently aberrant in T-cell lymphomas. Two chromosomal regions, containing sequences homologous to regions within the Pim-1locus, were localized on chromosome 6 and 16.     

High frequency recombination betweenloxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination betweenloxP sites in human chromosomes. G418 resistant cells with oneloxP site, generated by transfection with a plasmid containingloxP between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containingloxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked byloxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.     

Antibodies to PAI-1 alter the invasive and migratory properties of human tumour cells in vitro

Recent reports suggest that elevated levels of plasminogen activator inhibitor-1 (PAI-1) may contribute to tumour progression. The studies reported here were designed to help elucidate PAI-1's contribution to the invasive and migratory phenotype. Antibodies to PAI-1 dose-dependently, and significantly, inhibited the invasive and migratory potential of human HT1080 fibrosarcoma cells, as did an antibody to uPA and the plasmin inhibitor aprotinin. Invasion of the human melanoma cell line, BLM, was also attenuated by the anti-PAI-1 monoclonal antibody MAI-12. The non-invasive human melanoma cell line, IF6, which does not express uPA, provided further confirmation of PAI-1 and uPA's role as, upon transfection with uPA, this cell line attained an invasive phenotype, which was again attenuated by MAI- 12. Although antibodies to PAI-1 did not affect the adhesion of HT1080 cells to vitronectin, the antibody to uPA reduced their attachment. Addition of exogenous PAI-1, however, prevented HT1080 cell adhesion (IC₅₀ 180nM) and promoted cell detachment from vitronectin. Furthermore melanoma cells transfected with a uPA variant, which had an impaired interaction with PAI-1, were not invasive and had impaired binding to vitronectin. These data highlight the importance of a balanced proteolysis and suggest an additional role for PAI-1 distinct from its role in proteolysis. These data also suggest that uPA and PAI-1 may co-operate in the migratory process by respectively facilitating the attachment to, and subsequent detachment from, vitronectin in the extracellular matrix. These results support the clinical findings and indicate that modulation of PAI-1 activity may be of therapeutic benefit for the treatment of cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multiblock poly(ether-ester-amide)s based on polyamide-6 and poly(ethylene glycol), 2 Effect of composition and soft-segment length on the structure of poly(ether-ester-amide)s as revealed by X-ray scattering

Isotropic unannealed and annealed melt-pressed and quenched samples of segmented multiblock poly(ether-ester-amide)s based on polyamide-6 and poly(ethylene glycol)s (PEGs) of molecular weights 400 and 1000 with various hard/soft segment weight ratios were investigated. The effect of composition, segment length and annealing temperature on the crystal structure was studied by differential scanning calorimetry, wide-angle and small-angle X-ray scattering. In addition to the γ-phase, one of the unannealed samples having relatively long hard and soft segments exhibits a high amount of α-phase. This is assumed to be due to the high flexibility of the PEG segments, linking the polyamide blocks thus enhancing their mobility. The lack of hydrogen bonds between the PEG segments facilitates crystallization of the polyamide segments in the more perfect α-modification, which is the most pronounced in the above mentioned sample. Part of the α-phase transforms into the α-phase upon annealing in all copolymer samples studied, similarly to polyamide-6.     

Effect of a new synthetic serum replacement on insulin and somatostatin secretion from isolated rat pancreatic islets in long-term culture

Summary Serum contains insulin degrading components. We have evaluated the insulin and somatostatin secretion from isolated rat pancreatic islets during a 2-wk culture period using three different serum-containing media, and one serum-free medium with a synthetic serum replacement. Islets incubated in serum-free medium elicited significantly higher daily insulin and somatostatin secretions than islets incubated in the serum-containing media. After a 2-wk culture period, islets from the serum-free medium secreted significantly more insulin and somatostatin than islets cultured in other media when stimulated with 25 mmol/liter glucose together with 15 mmol/liter theophylline. We conclude that the serum-free medium is superior for long-term culture of rat pancreatic islets.     

Züchtung des Variolavirus in der infantilen Maus

Zusammenfassung Das Variolavirus konnte aus menschlichem Pustel- und Krustenmaterial intraperitoneal in infantilen Mäusen angezüchtet und in fortlaufenden Passagen weitergeführt werden. Es blieb innerhalb von 20 Passagen in bezug auf seine biologischen Eigenschaften im Hühnerembryo stabil. Eintägige Mäuse waren empfänglicher als dreitägige Tiere. Innerhalb verschiedener Mäusefamilien bestand eine unterschiedliche Resistenz gegenüber der Variolainfektion. Bei resistenteren Tieren vermehrte sich das Virus, ohne klinische Erscheinungen zu verursachen.Ein besonders bevorzugtes Organ für die Vermehrung des Variolavirus war die Lunge.Bei infizierten, klinisch aber gesunden Mäusen ließ sich aus den Lungen der Tiere in einem Zeitraum von 45 Tagen p. i. infektiöses Variolavirus züchten.     

Rapid,non-genomic actions of progesterone and estradiol on steady-state calcium and resting calcium influx in lens epithelial cells

The effects of steroids on the steady-state intracellular [Ca(2+)] ([Ca(2+)](i)) and resting Ca(2+) influx in Fura-2-loaded bovine lens epithelial cells were examined to identify potential rapid, non-genomic actions. When administered in the presence of 1-2 mM extracellular Ca(2+) ([Ca(2+)](o)), 100 micro M progesterone produced large (up to 12-fold) and transient (5 min) increases in [Ca(2+)](i). These effects were abolished in EGTA-containing solutions, and were associated with large increases in the rate at which extracellularly administered Mn(2+) quenched the intracellular Fura signal. Lower concentrations of progesterone (10-100 micro M) produced smaller increases in [Ca(2+)](i) that were concentration dependent, and 17beta-estradiol induced large, rapid and brief increases in [Ca(2+)](i) at 100 nM and smaller oscillations in [Ca(2+)](i) at 10 nM. In cells pretreated with thapsigargin, 100 micro M progesterone produced slower increases in [Ca(2+)](i) that were maintained for several minutes. These results demonstrate rapid non-genomic actions of progesterone and estradiol on resting Ca(2+) influx and [Ca(2+)](i) that may involve specific interactions with a recently discovered steroid-binding protein in the plasma membrane of lens epithelial cells.     

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.