Effects of cueing on visuospatial processing in unilateral spatial neglect

Patients with typical left unilateral spatial neglect bisected lines after cueing to the left end-point, the fixation point being monitored with an eye camera. They persisted with the point of initial fixation made after cueing and placed the mark there without searching leftwards again. The rightward shift of fixation to the initial point of fixation thus determined the location of the subjective midpoint. We consider that rightward attentional bias increased the amplitude of this shift that was planned on the basis of the perception of the whole line while cueing. This hypothesis may explain smaller but obvious rightward bisection errors found in the cueing condition.     

Metalloproteinase activity is present in rat urine and derived from the renal cortex

Summary: Matrix metalloproteinases (MP) are important candidates for the degradation of extracellular matrix, but the role of MP in the diseased kidney remains poorly understood. to examine the significance of urinary MP, we first investigated the characteristics of MP in normal rat urine and renal cortex, and then evaluated the urinary MP activity in anti-thymocyte induced glomerulonephritis (Thy.1 GN). Metalloproteinase activity was measured as the EDTA-inhibitable degradation of [³H] gelatin. the enzyme was purified from urine and the renal cortex homogenate in normal Wistar rats by using several chromatographic and gel filtration methods. Both materials contained the identical molecular weight (Mr 126 kDa by gel permeation method) of gelatin-degrading enzymes, the activity of which was inhibited by metal chelating agents and reactivated by ZnC1₂ but not by other proteinase inhibitors. Thy.1 GN was induced by intravenous injection of rabbit anti-thymocyte serum into rats, and daily urine was collected at sequential time points. Urinary MP activity was markedly reduced soon after the serum injection, and returned to the control level in 9 weeks. Conversely, urinary MP-inhibitor activity (Mr 30 kDa), determined as inhibiting activity against MP derived from renal cortex, showed serial changes strikingly reflected as urinary MP activity. These findings suggested that rat urine contained the MP which seemed to be derived from the renal cortex, and the urinary MP activity was decreased in Thy.1 GN model, probably due to the presence of MP-inhibitor. As urinary MP is likely to reflect intra-renal MP, the evaluation of urinary MP may be useful to search metabolic alteration of extracellular matrix in the diseased kidney.     

Molecular design of hybrid tumour necrosis factor alpha with polyethylene glycol increases its anti-tumour potency.

This study was conducted to increase the anti-tumour potency and reduce the toxic side-effects of tumour necrosis factor alpha (TNF-alpha). Natural human TNF-alpha was chemically conjugated with monomethoxy polyethylene glycol (PEG) using succinimidyl coupling of lysine amino groups of TNF-alpha. The number-average molecular weight of PEG-modified TNF-alpha (PEG-TNF-alpha) increased with an increase in the reaction time and the initial molar ratio of PEG relative to TNF-alpha. The resulting modified TNF-alpha was separated into fractions of various molecular weights. The specific activity of separated PEG-TNF-alpha s relative to that of native TNF-alpha gradually decreased with an increase in the degree of PEG modification, but the plasma half-life was drastically increased with the increase in molecular weight of modified TNF-alpha. PEG-TNF-alpha s, in which 29% and 56% of lysine residues were coupled to PEG, had anti-tumour activity approximately 4 and 100 times greater than unmodified TNF-alpha in the murine Meth-A fibrosarcoma model. Extensive PEG modification did not increase its in vivo activity. A high dose of unmodified TNF-alpha induced toxic side-effects, but these were not observed with the modified TNF-alpha s. Optimal PEG modification of TNF-alpha markedly increased its bioavailability and may facilitate its potential anti-tumour therapeutic use.     

Activity-dependent survival and enhanced turnover of calcium in cultured rat cerebellar granule neurons

Neurons survive when their activity is maintained. An influential hypothesis on the cellular mechanism underlying this phenomenon is that there is an appropriate range of intracellular Ca²⁺ concentration ([Ca²⁺]i) for survival. The rat cerebellar granule neuron in culture serves as the most often used model system for the analysis of activity-dependent survival, since it does not survive unless an excitant (KCl or glutamate) is added to the culture medium. Against the above-mentioned hypothesis, we found in our previous examination no difference between steady-state [Ca²⁺]i in granule neurons cultured under high KCl (i.e., survival) and low KCl (i.e., death) conditions. In this report, we present the quantitative background of unchanged [Ca²⁺]i between the two culture conditions. Influx of Ca²⁺ due predominantly to L-type voltage-dependent calcium channels was higher in high KCl cultures than in low KCl cultures. At the same time, efflux of Ca²⁺ due to the activity of Ca²⁺/Na⁺ antiport was also higher in high KCl cultures. Additionally, we found that the endocytotic activity was greater in high KCl cultures than in low KCl cultures, as monitored by the rate of uptake of horseradish peroxidase added to medium. Since the uptake was blocked by an internal Ca²⁺ chelator, the increased endocytotic activity in high KCl cultures might be a consequence of the enhanced Ca²⁺ turnover.     

A new HPLC fluorimetric method to monitor urinary delta-aminolevulinic acid (ALA-U) levels in workers exposed to lead

Summary A new sensitive HPLC method for the determination of urinary delta-aminolevulinic acid (ALA-U) was used to evaluate the relationship between blood-lead (Pb-B) and ALA-U levels in male workers exposed to lead. The differences between the ALA-U levels determined by this method (ALAU-HP) and by a colorimetric method (ALA-U-CL) are discussed. The HPLC method gave values similar to the ALA-U-CL values at high ALA-U level. However, at low blood-lead levels (58 ± 22 g/l, n = 23), the mean ALA-U-HP level corrected by urinary creatinine level was one-third of the corrected ALA-UCL level (0.83 ± 0.14 and 2.4 ± 0.5 mg/g creatinine, respectively). A significant increase of the mean corrected ALA-U-HP level was observed at 162 ± 22 g/l Pb-B (P < 0.05, n = 26), while that of ALA-UCL was observed at 245 ± 30 g/l Pb-B (P < 0.01, n = 37). The regression equation based on the logistic model fitted well to the relationship data between the Pb-B level and the percentage of the subjects with corrected ALA-U-HP above the cut-off point (1.12 mg/g creatinine) and the expected Pb-B level for 50% response was 270 g/l Pb-B, while it did not fit well to the relationship data between Pb-B level and the percentage of the subjects with corrected ALAU-CL above the cut-off point (3.5 mg/g creatinine). The maximum responses for the two sets of corrected ALA-U levels were both observed at 625 ± 25 g/l. The corrected ALA-U level by HPLC method seems to be a useful indicator for biological monitoring of exposure to lead at low levels (< 400 g/l Pb-B = health-based biological limit, WHO) as well as high ones.     

In vivo activity on murine tumors of a novel antitumor compound,N-β-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]phenazine-6-carboxamide sodium salt (NC-190)

Summary A novel antitumor compound, N--dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxybenzo[a]-phenazine-6-carboxamide sodium salt (NC-190) was evaluated for its antitumor activity in experimental murine tumor systems. In the initial studies with P388 leukemia (i.p.-i.p.), NC-190 led to an increase of >200% in life span (ILS), and 75% of the mice were alive on day 30, when the optimal dose (50 mg/kg, days 1–5) was given. Additionally, the compound had significant activities against i.p. inoculated mouse L1210 leukemia, B16 melanoma, M5076 reticulum cell sarcoma, sarcoma 180, mouse hepatoma MH134, and rat Yoshida sarcoma and Yoshida ascites hepatoma AH130. The optimal dose resulted in a >280% ILS with a 30-day survival of 50% in mice with L1210 leukemia (100 mg/kg, days 1–5), a 156% ILS in mice with B16 melanoma (50 mg/kg, days 1–5), a 98% ILS with a 90-day survival of 25% in mice with M5076 reticulum cell sarcoma (25 mg/kg, days 1, 5, 9, and 13), a >300% ILS with a 60-day survival of 50% in mice with sarcoma 180 (50 mg/kg, days 3–10), a 148% ILS with a 60-day survival of 25% in mice with MH134 (25 mg/kg, days 1–5), a 129% ILS with a 60-day survival of 12.5% in rats with Yoshida sarcoma (12.5 mg/kg, day 3–10), and a >161% ILS with a 60-day survival of 50% in rats with AH130 (6.3 mg/kg, days 3–10). In the experiments with s.c. inoculated tumors, NC-190 not only inhibited tumor growth, but also increased the life span of mice with Lewis lung carcinoma or B16 melanoma. The 60-day survivors accounted for 60% and 30% in mice with Lewis lung carcinoma and B16 melanoma, respectively. The compound significantly inhibited the spontaneous lung metastasis of Lewis lung carcinoma by more than 90% when eight daily i.v. injections were given. NC-190 was active by the i.p., s.c., and i.v. routes. Five consecutive daily i.p. doses (days 1–5) were more effective than a single dose (day 1), two doses (days 1 and 5), or three doses (days 1, 5, and 9). NC-190 warrants further study as a potential antineoplastic agent against human neoplasms, as it has a broad spectrum of antitumor activity and inhibits metastasis.Abbreviations ILS increase in life span - MST median survival time - MMC mitomycin C - ADM adriamycin - CPA cyclophosphamide - 5-FU 5-fluorouracil     

Reversal of multidrug resistance by new dihydropyridines with lower calcium antagonistic activity

Summary BS compounds, a series of new dihydropyridines, successfully overcame multidrug resistance in P388/ADR cells in vitro. These agents synergistically potentiated the cytotoxicity of Adriamycin to P388/ADR cells at a concentration of 1–2 M, whereas they showed hardly any synergistic effect in the parental cell line (P388/S) at the same concentration. They inhibited the active drug efflux in P388/ADR cells as well as the binding of [G-³H]-vinblastine to membrane vesicles from P388/ADR, which was increased in resistant P388 cells as compared with parental cells. Besides, unlike the activity of clinically used calcium antagonists, the calcium antagonistic activity associated with BS compounds was very weak: their arterial relaxation activity was <21% of that of verapamil. These data suggest that BS compounds specifically overcome multidrug resistance without the serious hypotensive side effects that accompany the use of verapamil orother calcium antagonists.     

Divergent expression and localization of aquaporin 5, an exocrine-type water channel,in the submandibular gland of Sprague-Dawley rats

By Western blot analysis, the expression level of aquaporin (AQP) 5 in the submandibular gland (SMG) was found to be different among individual rats of the Sprague-Dawley (SD) strain. Such differences were observed for AQP5 but not for AQP1 and consequently the SD strain was divided into two groups, one expressing a high level of AQP5 and the other a low one. The difference in average intensity of expression between the two groups was more than twofold. Immunohistochemical analysis of the SMG demonstrated that the AQP5 protein was localized in the basal and apical/lateral plasma membrane of acinar cells in rats expressing the high level of AQP5. In the rat expressing the low level, however, this channel protein was localized strongly in the apical/lateral plasma membrane, but only very weakly in the basal membrane of the acinar cells. Such a diverse localization of AQP5 was confirmed by Western blotting as well. Breeding between brother and sister was repeated for two times within high expressers and low expressers to obtain the third generation progenies (F2); the AQP5 level of the SMG in the third generation (F2 rats) from high expressers was significantly higher than the F2 from low expressers. Our present study suggests the existence of genetic variation in the expression of a water channel protein, AQP5, in rats.     

Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6J, as defined by BAC-end sequence-SNP analysis

MSM/Ms is an inbred strain derived from the Japanese wild mouse, Mus musculus molossinus. It is believed that subspecies molossinus has contributed substantially to the genome constitution of common laboratory strains of mice, although the majority of their genome is derived from the west European M. m. domesticus. Information on the molossinus genome is thus essential not only for genetic studies involving molossinus but also for characterization of common laboratory strains. Here, we report the construction of an arrayed bacterial artificial chromosome (BAC) library from male MSM/Ms genomic DNA, covering approximately 1x genome equivalent. Both ends of 176,256 BAC clone inserts were sequenced, and 62,988 BAC-end sequence (BES) pairs were mapped onto the C57BL/6J genome (NCBI mouse Build 30), covering 2,228,164 kbp or 89% of the total genome. Taking advantage of the BES map data, we established a computer-based clone screening system. Comparison of the MSM/Ms and C57BL/6J sequences revealed 489,200 candidate single nucleotide polymorphisms (SNPs) in 51,137,941 bp sequenced. The overall nucleotide substitution rate was as high as 0.0096. The distribution of SNPs along the C57BL/6J genome was not uniform: The majority of the genome showed a high SNP rate, and only 5.2% of the genome showed an extremely low SNP rate (percentage identity = 0.9997); these sequences are likely derived from the molossinus genome.