Effects of genetic backgrounds on hyperbilirubinemia in radixin-deficient mice due to different expression levels of Mrp3

ERM (ezrin/radixin/moesin) proteins are organizers of apical actin cortical layer in general. We previously reported that the knockout of radixin resulted in Rdx(-/-) mice with displacement/loss of the canalicular transporter Mrp2, giving rise to Dubin-Johnson syndrome-like conjugated hyperbilirubinemia in the mixed genetic background (C57BL/6-129/Sv) (Kikuchi, et al. (2002) Nature Genetics 31, 320-325). However, when these mice were kept under mixed genetic background for years (late mixed backgrounds; LMB), the conjugated hyperbilirubinemia gradually became inconspicuous, while evidence of liver injury increased. We examined the effect of genetic background by backcrossing LMB Rdx(-/-) mice to C57BL/6 and 129/Sv wild type mice with the result that the Rdx(-/-) congenic mice regained hyperbilirubinemia with reduced hepatocellular damage. As revealed by immunofluorescence and western blots, the localization/expression of apical transporters, Mrp2, CD26, P-gps, and Bsep were not influenced by backcrossing, though those of a basolateral transporter, Mrp3, were strikingly increased by backcrossing.     

Mitogen-activated protein kinase involves neutrophil elastase-induced morphological changes in human bronchial epithelial cells.

Neutrophil elastase (NE) promotes the detachment of airway epithelial cells; however, changes in overall morphology of NE-stimulated bronchial epithelial cell (BEC) monolayer are different from trypsin stimulation. Ras/Raf-initiated-mitogen activated protein kinase (MAPK) also known as extracellular signal-regulated kinase, pathway regulates integrin functions which participate in regulating attachment and detachment of cell and cellular morphology. However, little is known about the role of MAPK in NE-induced changes in overall morphology of BEC. In the present study, we examined the role of MAPK in NE-induced changes in overall morphology of BEC monolayer. To this end, we examined changes in cellular morphology and MAPK activation in NE-stimulated BEC monolayer, and the effect of PD 98059 as the specific inhibitor for MAPK kinase-1 (MEK-1, the upstream regulator of MAPK) on NE-induced changes in cellular morphology and MAPK activation. The results showed that in stimulation of NE, BECs detached and gaps developed, and MAPK activation was observed. PD 98059 attenuated NE-induced changes in cellular morphology as well as MAPK activation. These results indicated that in addition to proteolytic activity of NE on extracellular matrix (ECM), NE-activated MAPK pathway, at least in part, is involved in NE-induced changes in overall morphology and the detachment of BEC monolayer.     

Fine structure of micropylar region during late oogenesis in eggs of the hagfish Eptatretus burgeri (Agnatha)

Using the hagfish, Eptatretus burgeri, the fine structure of formation of the micropylar region in hagfish eggs during the late stages of oogenesis was investigated for the first time, focusing on the bottom region of the micropyle and the egg surface. During these stages, many cells penetrated through the chorion and reached a pit of the egg surface, forming a shovel-like structure in two-dimensional sections. The cells, which we called micropylar cells, were separated from the chorion by a wall of amorphous material. In the pit, another fibrous layer filled the space between the egg surface and the anterior portion of the shovel-like structure. Microvilli coming from the egg surface were embedded in this layer. In later stages, the stack of micropylar cells loosened, and a space appeared between the anterior region of the shovel-like structure and the layer on the egg surface. Microvilli decreased in length and number. The pit region appeared likely to have a role in fertilization. The structures associated with the forming micropyle were markedly different from those observed in the same region of teleost fishes. A hypothesis that hagfish might show transitional structures in gametes from protochordates to teleosts is suggested.     

Metazoan cellulase genes from termites: intron/exon structures and sites of expression.

Endogenous endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) cDNAs were cloned from representatives of the termite families Termitidae and Rhinotermitidae. These EGases are all composed of 448 amino acids and belong to glycosyl hydrolase family 9 (GHF9), sharing high levels of identity (40-52%) with selected bacterial, mycetozoan and plant EGases. Like most plant EGases, they consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. Using a PCR-based strategy, the entire sequence of the coding region of NtEG, a gene putatively encoding an EGase from Nasutitermes takasagoensis (Termitidae), was determined. NtEG consists of 10 exons interrupted by 9 introns and contains typical eukaryotic promoter elements. Genomic fragments of EGase genes from Reticulitermes speratus (Rhinotermitidae) were also sequenced. In situ hybridization of N. takasagoensis guts with an antisense NtEG RNA probe demonstrated that expression occurs in the midgut, which contrasts to EGase expression being detected only in the salivary glands of R. speratus. NtEG, when expressed in Escherichia coli, was shown to have in vitro activity against carboxymethylcellulose.     

The Observation of an Ultradian Rhythm of Heart Rate in Low Birth Weight Infants

The relationship between ultradian rhythm of heart rate and schedules of body contact or feeding was studied in five low birth weight infants of conceptional ages of 34-36 weeks. The differential contribution of body contact and feeding to the formation of the ultradian rhythm of heart rate was evaluated by applying two different schedules of two- and three-hour periods for feeding with a single schedule of three hours for body contact during an observation period of seven days. A chi-square periodogram was used to calculate the period of ultradian rhythm. As a result, a three-hour ultradian rhythm of heart rates was detected in all subjects, which seems to correspond to either schedule of body contact or of feeding. However, no clear changes in the ultradia n rhythm of heart rate were observed corresponding to changes in feeding schedules. The ultradian rhythm of heart rate seems to correspond more to body contact than to feeding.     

Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the Rho family of small G proteins.

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane-associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999) Oncogene 18, 1435-1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease-mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and -independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA-induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.