The time course of lymph drainage from the peritoneal cavity in beagle dogs

Lymph drainage routes from the abdominal and pelvic cavities in beagle dogs were observed serially by following the time course of India ink administered intraperitoneally. Four systems of lymph drainage routes from the peritoneal cavity were observed in this study. The earliest drainage returned to the cranial mediastinal lymph nodes via the sternal lymph vessels; subsequently, the sternal lymph nodes located along the internal thoracic artery became involved. Then, a drainage route via the lymph vessel along the left vagus nerve was observed. The final drainage route flowed into the lateral lymph vessel through the thoracic duct located on the vertebra. These results show that India ink is absorbed from the peritoneal cavity, and that the lymph drainage first flows mainly towards the cranial mediastinal lymph nodes through the ventral lymphatic channels. Our serial observations suggest that, over time, the lymph drainage routes changed from the ventral abdominal to the dorsal thoracic lymphatic channels in the thorax.     

Effects on flowering and seed yield of dominant alleles at maturity loci E2 and E3 in a Japanese cultivar,Enrei

‘Enrei’ is the second leading variety of soybean (Glycine max (L.) Merr.) in Japan. Its cultivation area is mainly restricted to the Hokuriku region. In order to expand the adaptability of ‘Enrei’, we developed two near-isogenic lines (NILs) of ‘Enrei’ for the dominant alleles controlling late flowering at the maturity loci, E2 and E3, by backcrossing with marker-assisted selection. The resultant NILs and the original variety were evaluated for flowering, maturity, seed productivity and other agronomic traits in five different locations. Expectedly, NILs with E2 or E3 alleles flowered later than the original variety in most locations. These NILs produced comparatively larger plants in all locations. Seed yields were improved by E2 and E3 in the southern location or in late-sowing conditions, whereas the NIL for E2 exhibited almost the same or lower productivity in the northern locations due to higher degrees of lodging. Seed quality-related traits, such as 100-seed weight and protein content, were not significantly different between the original variety and its NILs. These results suggest that the modification of genotypes at maturity loci provides new varieties that are adaptive to environments of different latitudes while retaining almost the same seed quality as that of the original.     

Estimation of glomerular filtration rate in calves using the contrast medium iodixanol

To develop a simple procedure for estimating glomerular filtration rate (GFR) in calves, a three-sample method using iodixanol was first compared to that using the standard agent inulin. Iodixanol and inulin were co-administered intravenously to calves at 40 mg I/kg and 40 mg/kg, respectively, and blood was collected 30, 60, 120, and 180 min later. Serum iodixanol and inulin concentrations were separately determined by high performance liquid chromatography and colorimetry. Serum urea nitrogen (UN) and creatinine concentrations were also measured. GFR estimated by iodixanol was consistent with that using inulin in clinically healthy calves. Based on GFR estimations in healthy calves and those renal-loaded with iodixanol, it was found that the serum creatinine concentrations became elevated when GFR decreased to 60% of the reference value. In contrast, serum UN concentrations fluctuated widely, presumably due to extra-renal factors. When GFR was estimated using the three-sample method and compared with the single-blood-sample method, 62/69 (90%) of samples tested were within the agreement plots. The results demonstrated that the single-blood-sample method using iodixanol may be useful in monitoring GFR in calves.     

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 10⁴ clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Photosensitivity in mice caused by pyropheophorbide in the midgut gland of the scallop <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis> observed in diarrhetic shellfish poisoning mouse bioassays

Photosensitivity was observed in the mice used in bioassays of diarrhetic shellfish poisoning. The mice were intraperitoneally injected with the extracts from the midgut glands of cultured scallops Patinopecten yessoensis gathered in early spring in Funka Bay, Hokkaido, Japan. The injections induced atypical symptoms in the mice within 24 h. The symptoms included piloerection, substantial eye discharge, swelling of the ears and head, and ear scratching. They were similar to those reported in studies of photosensitivity caused by pyropheophorbide in the midgut glands of abalones, which are herbivorous gastropods. The problematic scallops also accumulated a large amount of pyropheophorbide-a. The amount determined with high-performance liquid chromatography was 300–530 μg per 1 g of the homogenate of the midgut glands. The dose of pyropheophorbide sufficient to cause photosensitivity in a mouse is estimated to be 0.99–2.3 mg per mouse with 20-g body weight. Moreover, comparative tests in mice showed that the onset of the symptoms required light and a substantial amount of pyropheophorbide. Therefore, we determined that the observed symptoms were photosensitivity caused by pyropheophorbide. Scallops, which are suspension-feeding bivalves, can accumulate a large amount of pyropheophorbide in the midgut glands depending on the habitat environment.     

Liquid-chromatographic hybrid triple–quadrupole linear-ion-trap MS/MS analysis of fatty-acid esters of dinophysistoxin-1 in bivalves and toxic dinoflagellates in Japan

Fatty-acid esters of dinophysistoxin-1 (DTX1) in scallops Patinopecten yessoensis, mussels Mytilus coruscus, and toxic dinoflagellate Dinophysis species, collected from Japanese seawater, were analyzed by liquid-chromatography mass spectrometry (LC–MS). Precursor ion monitoring, multiple reaction monitoring for 18 fatty-acid esters of DTX1, and full-scan MS/MS spectra obtained with a hybrid triple–quadrupole linear-ion-trap mass spectrometer showed that 14:0, 16:0, and 16:1 esters were the most abundant 7-O-acyl-DTX1 analogues in bivalves. Fatty-acids esters formed by conjugations at hydroxyl positions other than the 7-position of DTX1 were not detected in the bivalves. DTX1 and okadaic acid-16:0 fatty-acid esters have been reported as the most abundant ester in bivalves in several previous studies; however, we found that 7-O-16:1-DTX1 was the most abundant ester in some mussels in which 16:1 was more dominant than 16:0 in the free fatty-acid profile. Comparison between 7-O-acyl-DTX1 and free fatty-acid profiles in the same bivalve samples suggests that polyunsaturated fatty acids are selectively excluded in enzymatic acylation of DTX1. No 7-O-acyl-DTX1 was detected in any single-cell isolates of D. fortii, D. acuminata, D. mitra, D. norvegica, D. tripos, D. infundibulus, and D. rotundata.