Protective effects of sarpogrelate, a 5-HT2A antagonist, against postischemic myocardial dysfunction in guinea-pig hearts

The protective effects of sarpogrelate (SG), a 5-HT_2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca²⁺ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and ³¹P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5×10^-7 M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca²⁺ ([Ca²⁺_i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (T_NO) level at the end of ischemia compared with the control, but [Ca²⁺]_i during ischemia decreased. Meanwhile, mitochondrial Ca²⁺ uptake on acidification or Ca²⁺ content changes of the perfusate was suppressed by pre-treatment with SG or the K_ATP channel opener diazoxide, but not the K_ATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT_2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT_2A receptor induced in ischemia–reperfusion plays an important role in the mitochondrial K_ATP channel of hearts in close relation with NO and active oxygen radicals.     

Generation of reactive oxygen species and activation of NF-kappaB by non-Abeta component of Alzheimer's disease amyloid

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.     

Effects of Aging on the ATP- and Pyrophosphate-dependent Pumping of Protons across the Tonoplast Isolated from Pumpkin Cotyledons

Tonoplast vesicles were isolated from 7- to 26-day-old pumpkincotyledons by an improved floating method, and the activitiesof pyrophosphatase (PPase) and adenosine triphosphatase (ATPase)in tonoplast vesicles, as well as rates of PPase- and ATPase-dependentpumping of protons across tonoplast vesicles, were measured.PPase activity and the rate of pyrophosphate-dependentproton-pumpingdecreased more rapidly than loss of chlorophyll from cotyledons,and the pumping on day 14 was only 10% of that on day 7, whilePPase activity was still more than 30% of that on day 7. Bycontrast, ATPase activity and the rate of ATP-dependent proton-pumpingincreased until day 14. In this latter case, the changes inboth activity and pumping were not major and were parallel toone another until day 21. However, a rapid decrease was observedonly in the rate of pumping on day 26, at which time an apparentloss of fresh weight was observed in cotyledons. The relationshipbetween the aging of pumpkin cotyledons and functional changesin vacuoles is discussed in terms of ATP- and pyrophosphate-dependentproton-pumping across the tonoplast. The two proton pumps inthe tonoplast, H⁺-ATPase and H⁺-pyrophosphatase, appear to playdifferent roles during the growth and senescence of pumpkincotyledons. ¹Plant EcoPhysiology Laboratory, Tohoku National AgriculturalExperiment Station, Shimo-Kuriyagawa, Morioka, Iwate, 020-01Japan.     

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.     

Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism

Kinetics of refolding and unfolding of staphylococcal nuclease and its six mutants, each carrying single or double amino acid substitutions, are studied by stopped-flow circular dichroism measurements. A transient kinetic intermediate formed within 10 ms after refolding starts possesses a substantial part of the N-domain core β-structure, whereas helices are formed at the later stages. The structure of the kinetic intermediate is less organized than the structure that is known to be formed by a nuclease 1-136 fragment. Only the refolding kinetics are affected by the mutations in all the mutants except two in which the mutations have changed the native structure. From this result and also from the locations of the mutation sites, the major N-terminal domain of the nuclease in the transition state of folding has a structure nearly identical to the native one. On the other hand, the minor C-terminal domain has previously been shown to be still disorganized in the transition state. The effects of the amino acid substitutions on the stability of the native and the transition states are in good agreement with the changes in the hydration free energy, expected for the corresponding amino acid replacements in the unfolded polypeptide. Since side chains of all the mutated residues are not accessible to solvent in the native structure, the result suggests that it is the unfolded state that is mainly affected by the mutations. © 1995 Wiley-Liss, Inc.     

Changing taxonomic and functional β-diversity of cladoceran communities in Northeastern and South Brazil

Hydrobiologia - Disturbances caused by both natural and anthropogenic forces can drive changes in alpha and beta taxonomic diversity and be accompanied by losses or gains in freshwater ecosystem...     

Biological monitoring of occupational exposure to 1-bromopropane by means of urinalysis for 1-bromopropane and bromide ion

The purposes of the present study are (1) to develop a sensitive analytical method to measure 1-bromopropane (1-BP) in urine, (2) to examine if 1-BP or bromide ion (Br) in urine is a useful biomarker of exposure to 1-BP, and (3) to identify the lowest 1-BP exposure concentration the method thus established can biomonitor. A factory survey was carried out on Friday, and 33 workers (all men) in cleaning and painting workshops participated; each worker was equipped with a diffusive sampler (carbon cloth KF-1500 as an adsorbent) to monitor 1-BP vapour for an 8-h shift, and offered a urine sample at the end of the shift for measurement of 1-BP and Br in urine. In addition, 10 non-exposed men offered urine samples as controls. The performance of the carbon cloth diffusive sampler was examined to confirm that the sampler is suitable for monitoring time-weighted average 1-BP vapour exposure. A head-space GC technique was employed for analysis of 1-BP in urine, whereas Br in urine was analysed by ECD-GC after derivatization to methyl bromide. The workers were exposed to vapours of seven other solvents (i.e. toluene, xylenes, ethylbenzene, acetone, etc.) in addition to 1-BP vapour; the 1-BP vapour concentration was 1.4 ppm as GM and 28 ppm as the maximum. Multiple regression analysis however showed that 1-BP was the only variable that influenced urinary 1-BP significantly. There was a close correlation between 1-BP in urine and 1-BP in air; the correlation coefficient (r) was >0.9 with a narrow variation range, and the regression line passed very close to the origin so that 2 ppm 1-BP exposure can be readily biomonitored. The correlation of Br in urine with 1-BP in air was also significant, but the r (about 0.7) was smaller than that for 1-BP, and the background Br level was also substantial (about 8 mg l^-1). Thus, it was concluded that 1-BP in end-of-shift urine is a reliable biomarker of occupational exposure to 1-BP vapour, and that Br in urine is less reliable.     

Novel asymmetrically engineered antibody Fc variant with superior FcγR binding affinity and specificity compared with afucosylated Fc variant

Fc engineering is a promising approach to enhance the antitumor efficacy of monoclonal antibodies (mAbs) through antibody-dependent cell-mediated cytotoxicity (ADCC). Glyco- and protein-Fc engineering have been employed to enhance FcγR binding and ADCC activity of mAbs; the drawbacks of previous approaches lie in their binding affinity to both FcγRIIIa allotypes, the ratio of activating FcγR binding to inhibitory FcγR binding (A/I ratio) or the melting temperature (T_M) of the C_H2 domain. To date, no engineered Fc variant has been reported that satisfies all these points. Herein, we present a novel Fc engineering approach that introduces different substitutions in each Fc domain asymmetrically, conferring optimal binding affinity to FcγR and specificity to the activating FcγR without impairing the stability. We successfully designed an asymmetric Fc variant with the highest binding affinity for both FcγRIIIa allotypes and the highest A/I ratio compared with previously reported symmetrically engineered Fc variants, and superior or at least comparable in vitro ADCC activity compared with afucosylated Fc variants. In addition, the asymmetric Fc engineering approach offered higher stability by minimizing the use of substitutions that reduce the T_M of the C_H2 domain compared with the symmetric approach. These results demonstrate that the asymmetric Fc engineering platform provides best-in-class effector function for therapeutic antibodies against tumor antigens.     

Elevated expression of proto-oncogenes during interleukin-5-induced growth and differentiation of murine B lineage cells

Interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. To elucidate IL-5-mediated intracellular mechanisms, we have established IL-5-dependent and -independent murine early B cell lines, J6 and MJ88-1, respectively, and examined the effect of IL-5 on the expression of proto-oncogenes during proliferation. Two- to 3.5-fold increases in the levels of c-myb, c-myc, c-fos, and c-fms mRNA were observed in J6 cells, compared with those in MJ88-1 cells. Further, a role of IL-5 in the proto-oncogene expression during differentiation was examined by using thymidine-treated murine B-cell chronic leukemia BCL1-B20 cells with growth arrest. After 4-day culture, the amount of IgM secreted from BCL1-B20 cells was augmented 4-6 fold in the presence of IL-5. Although expression of c-myb, c-fos, and c-fms mRNA did not change, only c-myc mRNA expression was elevated within 30 min of stimulation with IL-5 and reached a maximal level by 1 hr. Addition of phorbol 12-myristate 13-acetate (PMA) or IL-4 to the culture of BCL1-B20 cells inhibited both the IL-5-mediated augmentation of IgM secretion and the elevated expression of c-myc mRNA. These findings suggest that the IL-5 signal may be associated with the up-regulation of c-myc expression.