Tomi T. Tsuda

Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5′UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human MhcDRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.

Anorexigenic substance isolated from feces of rat and mouse

A substance was isolated from the feces of conventional rats and mice which were fed laboratory diets. Marked reduction in food intake occurred for a few hours after intraperitoneal administration of this substance, while water intake also decreased. Two hr after the injection, when the anorectic effect appeared to be the strongest, no change was found in body temperature or blood glucose, but free amino acids in plasma were decreased. A comparative study using germfree and conventional mice indicated that the anorexigenic substance was produced by gastrointestinal microflora, since the yields of the anorexigenic substance from germfree mice was less than one tenth of that from conventional mice. A partially purified form of the substance, with large molecular weight, was isolated by Sephadex G-150 fractionation. It contained protein but the anorexigenic activity was not diminished by protein digestion.

Impaired hypophagia induced by fecal anorexigenic substance in Zucker obese rats

Influence of fecal anorexigenic substance (FS-T) on feeding by Zucker obese rats was compared to that by their lean littermates and Wistar King A rats. FS-T, which has been found to suppress food intake mainly by activation of glucoreceptor neurons in the ventromedial hypothalamus, was injected intraperitoneally in a dose of 7 U/kg at 1930 hr, immediately before the dark period. Potency of FS-T in feeding suppression was much less in the obese rats than in their lean littermates or the Wistar King A rats. Meal size of the obese rats was decreased after the injection, but meal duration was unaffected. The suppressive effect on the lean rats and the Wistar King A rats included decrease of both size and duration of the meal. These results suggest that chemosensitivity in the ventromedial hypothalamus of Zucker obese rats may be impaired, which may be one explanation of the obesity in Zucker obese rats.

Feeding suppression induced by a fecal anorexigenic substance (FS-T)

Intraperitoneal (IP) injection of a fecal anorexigenic substance (FS-T) induced significant suppression of feeding and this suppression recovered on the second day. At 2 hr after IP injection, at the time of maximum feeding suppression, plasma glucose, insulin and free fatty acid (FFA) levels did not change but amino acid level decreased. Intra-third cerebral ventricle (ICV) infusion of FS-T induced parallel but more potent feeding suppression. Analysis of meal patterns demonstrated that suppression of feeding after ICV treatment continued into the second day. FS-T was applied electrophoretically to glucose-sensitive and non glucose-sensitive neurons in the lateral hypothalamic area (LHA) and to glucoreceptor and non glucoreceptor neurons in the ventromedial hypothalamic nucleus (VMH). It significantly inhibited glucose-sensitive neurons but not non glucose-sensitive neurons, and excited both neuron types in the VMH. FS-T might thus work directly through the hypothalamic feeding control centers to suppress feeding. Even after pronase treatment of FS-T, a non-dialysable fraction of large molecular weight, consisting of protein and carbohydrate, maintained the original anorexigenic activity.

Isolation and characterization of a high molecular weight type IV collagenase isolated from human carcinoma tissue

A proform of high molecular weight type IV collagenase was isolated and purified 1230‐fold from human metastatic carcinoma tissue. Like matrix metalloproteinases (MMPs), the enzyme was activated by trypsin and degraded type IV collagen and gelatin at a neutral pH, the activity was inhibited by EDTA and o‐phenanthroline. However, the molecular weight was much higher than MMPs which degraded type IV collagen, gelatinase A (MMP‐2; 72 kDa gelatinase/type IV collagenase) (EC 3.4.24.24), gelatinase B (MMP‐9; 92 kDa gelatinase/type IV collagenase) (EC 3.4.24.35), stromelysin‐1 (MMP‐3; 57 kDa) (EC 3.4.24.17) and stromelysin‐2 (MMP‐10; 57 kDa) (EC 3.4.24.22). The other significant difference from MMPs was that the enzyme was not activated by 4‐aminophenylmercuric acetate nor inhibited by TIMP. Taking together these results, this high molecular weight type IV collagenase might be a newly found enzyme different from MMPs or might have the same configuration as MMPs already reported.

Diversity of MHC class I alleles in Spheniscus humboldti

The major histocompatibility complex locus (MHC) is a gene region related to immune response and exhibits a remarkably great diversity. We deduced that polymorphisms in MHC genes would help to solve several issues on penguins, including classification, phylogenetic relationship, and conservation. This study aimed to elucidate the structure and diversity of the so far unknown MHC class I gene in a penguin species. The structure of an MHC class I gene from the Humboldt penguin (Spheniscus humboldti) was determined by using an inverse PCR method. We designed PCR primers to directly determine nucleotide sequences of PCR products from the MHC class I gene and to obtain recombinant clones for investigating the diversity of the MHC class I gene in Humboldt penguins. A total of 24 MHC class I allele sequences were obtained from 40 individuals. Polymorphisms were mainly found in exons 2 and 3, as expected from the nature of MHC class I genes in vertebrate species including birds and mammals. Phylogenetic analyses of MHC class I alleles have revealed that the Humboldt penguin is closely related to the Red Knot (Calidris canutus) belonging to Charadriiformes.