Binding of mammalian and avian ferritins with biotinylated hemin: demonstration of preferential binding of the H subunit to heme

The binding of ferritin to heme has been well studied using commercial horse spleen apoferritin, which is almost entirely composed of the L subunit, suggesting that mammalian ferritins bind heme. The present study revealed that both mammalian holoferritins (commercial horse spleen ferritin and purified horse spleen, bovine spleen and canine liver ferritins with L/H subunit ratios of 4.0, 1.1, and 2.3, respectively) and their apoferritins bound biotinylated hemin; apoferritins had higher binding activity than holoferritins, except for canine holo- and apoferritins, which showed the same binding. Bovine ferritin H subunit homopolymers expressed by a baculovirus expression system showed heme binding and had higher binding activity to biotinylated hemin than the L subunit homopolymer expressed by the same system. These bindings were inhibited by heme but not by iron-free or Zn-protoporphyrin IX (Zn-PPIX). Purified chicken liver holoferritin was found to be composed of only H subunits and showed the highest binding activity with biotinylated hemin compared with mammalian holoferritins. The binding of chicken liver holoferritin to biotinylated hemin was also inhibited by heme but not by PPIX or Zn-PPIX. These results indicate that mammalian and avian ferritins bind heme and that the H subunit preferentially recognizes heme.     

Improvement of porcine oocytes with low developmental ability after fusion of cytoplasmic fragments prepared by serial centrifugation

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.     

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.     

Comparative distribution of immunoglobulin-containing cells in stomach, intestine and associated lymph nodes of cattle

Immunoglobulin (Ig)-containing cells were investigated immunohistochemically in the stomach, intestine and associated lymph nodes of clinically normal calves, cows and fetuses to examine mucosal immune responses in the forestomach to rumen microbial flora. IgG-containing cells were observed in the mucosal propria of the forestomach of a 32 day-old, a 37 day-old, two 90 day-old calves, and of all 5 cases of cows. However, no IgA- and IgM-containing cells were observed. Further, no Ig-containing cells were detected in the associated lymph nodes of all calves and cows. Various numbers of Ig-containing cells, predominantly IgG, were observed in the mucosal propria of the abomasum, small intestine and cecum, and in the mesenteric lymph nodes of aged calves and all cows. No Ig-containing cells were observed in any tissues of fetuses. The results suggest that the development of mucosal immune responses in the forestomach may be incomplete as compared with that of the intestine.     

Effects of ligation of the ductus deferens on the fowl epididymal region

The effects of ligation of the ductus deferens on the epididymis in the fowl were studied histochemically and immunohistochemically to reveal the mechanisms of sperm disposal. At one week post-ligation, the lumina of the rete testes (RT) and the efferent ductules (ED) were distended and filled with densely accumulated spermatozoa. Macrophages and foreign-body giant cells were aggregated in and around the accumulations. The epithelium regressed in the initial portion of the RT with the invasion of fibroblasts and heterophiles into the lumen. The other part of the epithelium was penetrated by many spermatozoa. Numerous lymphocytes and plasma cells infiltrated into the interstitium. At 4 weeks, larger number of spermatozoa agglutinated in the lumen, and large masses of foamy cells and proliferated connective tissue protruded into the lumen. At 8 weeks, large masses of foamy cells were noted. The connecting ductules or the epididymal duct showed no marked changes after ligation. The epithelium of the ED showed weaker or no acid phosphatase activity after ligation. Immunoglobulin G-containing cells increased in number in the interstitium. These results showed that ligation of the ductus deferens in the fowl causes granuloma in the RT and ED, and that epithelial cells, macrophages and granuloma are engaged in the removal of spermatozoa. The participation of antibody is suggested in the sperm disposal processes.     

Relative Contribution of Endocrine-disrupting Chemicals to the Estrogenic Potency of Marine Sediments of Osaka Bay,Japan

Water, Air, &; Soil Pollution - Karst systems harbor large groundwater resources for human consumption and represent an important habitat for rare and unprotected specialized animals, the...