Alteration of the N-glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1) , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.     

Proliferative response of seminal vesicle cells to androgen and estrogen in neonatally castrated mice

Proliferation and death of androgen- and estrogen-responsive cells in seminal vesicles were compared between neonatally and adult (on Day 60 after birth) castrated mice. Daily injections of either testosterone propionate (TP) or estradiol-17 beta (E2) were started on Day 90 after birth; the incorporation of 5-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) into the whole seminal vesicles was used as an index for proliferation. Although the peak of [125I]IdUrd uptake was observed 3 days after starting TP injections in both neonatally and adult castrated mice, the peak was lower and the period of proliferation was much longer in the former than in the latter. When TP injections were stopped, the fraction of surviving cells that synthesized DNA on Day 3 of TP injections was much larger in neonatally than adult castrated mice. The difference was attributed to the presence of TP-induced proliferation of fibromuscular cells in the neonatally castrated mice but not in the adult castrated mice; only the fibromuscular cells but not epithelial cells survived after stopping TP injections. Although injections of E2 increased the proliferation of epithelial cells but did not the weight of seminal vesicles in adult castrated mice, the same procedure increased the proliferation of both epithelial and fibromuscular cells and the weight in neonatally castrated mice. The E2-induced fibromuscular cells seemed to survive in the presence or absence of E2. The present results seem to indicate that androgen- and estrogen-induced proliferation of fibromuscular cells is irreversible in seminal vesicles of neonatally castrated mice and that the depletion of androgen in the seminal vesicle during neonatal and prepubertal periods is at least in part compensated by the administration of androgen, even after 90 days of age.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

Production of Reactive Oxygen Species and Release of l-Glutamate During Superoxide Anion-Induced Cell Death of Cerebellar Granule Neurons

Abstract: Enhanced production of superoxide anion (O₂⁻) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O₂⁻ generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) for O₂⁻ and other ROS and hydroethidine (HEt) specifically for O₂⁻ by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO-induced cell death. Catalase effectively reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA fluorescence correlated with O₂⁻ and hydrogen peroxide, respectively. The NMDA antagonist MK-801 prevented the death. XA + XO induced an increase in l -glutamate release from cerebellar granule neurons. These results indicate that elevation of O₂⁻ induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l -glutamate.     

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH(2) terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulmonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.     

Indoor environmental study and post occupancy evaluation in an office environment

1. 1. The objective of this paper is to investigate the indoor environment from the viewpoint of interaction between physical environment and the human responses. The field survey has been conducted over 1 year.

2. 2. A continuous measurement has been carried out for 1 week and distribution of variables have been measured for 1 day.

3. 3. The attitude of workers was investigated by a questionnaire.

4. 4. As the result, average luminance represented more than 1000 lx in the new building, in contrast with less tha 300 lx in the existing building.

5. 5. There was a significant difference of the occupants' response to the light environment between the two buildings.

6. 6. Measured thermal conditions are on the edge of the ASHRAE comfort envelope in summer, and in the neighborhood of the lower dry limit of the envelope in spring.

7. 7. The occupants' evaluations were remarkably changed before and after the moving. The office environment is better than that of the factory.