Distribution and sequence of pyknotic cells in rat fetuses exposed to busulfan

Busulfan, an antineoplastic bifunctional-alkylating agent, is known to induce developmental anomalies. In the present study, we examined the distribution and sequence of pyknotic cells in rat fetal tissues exposed to busulfan. Pregnant rats on gestation day 13 were administered intraperitoneally 30 mg/kg of busulfan, and fetal tissues were examined at 6, 12, 24, 36, 48, 72 and 96 hours after treatment (HAT). Pyknosis of component cells was observed markedly in the brain, moderately in the eyes and spinal cord and mildly in the craniofacial tissue, mandible, limb buds, tail bud, ganglions, alimentary tract, lungs, kidneys, pancreas and liver. In the brain, mitotic inhibition was also detected. Most of the pyknotic cells were considered to be apoptotic cells judging from the results of TUNEL staining and electron microscopic examination. Commonly in the above-mentioned tissues, pyknotic cells began to increase at 24 HAT, peaked at 36 or 48 HAT and disappeared at 96 HAT, which is when the histological picture returned to normal in most tissues except for the brain, spinal cord and eyes. The present study clarified the outline of busulfan-induced apoptosis in rat fetuses.     

Real-time imaging of nitrogen fixation in an intact soybean plant with nodules using 13N-labeled nitrogen gas

Real-time images of nitrogen fixation in an intact nodule of hydroponically cultured soybean ( Glycine max [L] Merr.) were obtained. In the present study, we developed a rapid method to produce and purify ¹³N-labeled radioactive nitrogen gas (half life: 9.97 min). ¹³N was produced from a ¹⁶O (p, α) ¹³N nuclear reaction. The target chamber was filled with CO₂ and irradiated for 10 min with protons at an energy of 18.3 MeV and an electric current of 5 μA, which was delivered from a cyclotron. All CO₂ in the collected gas was absorbed and removed with powdered soda-lime in a syringe and replaced with helium gas. The resulting gas was injected into gas chromatography and separated and a 35 mL fraction, including the peak of [¹³N]-nitrogen gas, was collected by monitoring the chromatogram. The obtained gas was mixed with 10 mL of O₂ and 5 mL of N₂ and used in the tracer experiment. The tracer gas was fed into the underground part of intact nodulated soybean plants and serial images of the distribution of ¹³N were obtained non-invasively using a positron-emitting tracer imaging system (PETIS). The rates of nitrogen fixation of the six test plants were estimated to be 0.17 ± 0.10 μmol N₂ h⁻¹ from the PETIS image data. The decreasing rates of assimilated nitrogen were also estimated to be 0.012 ± 0.011 μmol N₂ h⁻¹. In conclusion, we successfully observed nitrogen fixation in soybean plants with nodules non-invasively and quantitatively using [¹³N]N₂ and PETIS.     

Effects of a combination of hinokitiol (β‐thujaplicin) and an organic acid mixture on ruminal fermentation in heifers fed a high‐grain diet

This study evaluated the effects of hinokitiol (a natural antibacterial compound extracted from Thujopsis dolabrata var. hondai) and an organic acid mixture (citrate content 50%) on ruminal fermentation. Antibacterial properties were examined by measuring minimal inhibitory concentration. Hinokitiol at 1.56 µg/mL or an organic acid mixture at 1600 µg/mL inhibited Streptococcus bovis growth. The combination of 0.78 µg/mL hinokitiol and 200 µg/mL of an organic acid mixture also inhibited S. bovis growth. Both hinokitiol and the hinokitiol and an organic acid mixture combination showed strong antibacterial properties on Gram‐positive bacteria such as S. bovis, but relatively weak antibacterial activities on Gram‐negative bacteria such as Megasphaera elsdenii. Three ruminally cannulated heifers were fed a bloat‐producing diet containing barley, pelleted alfalfa meal, soybean meal and salt without long‐cut roughage to investigate the ruminal characteristics in vivo. Feeding to heifers a bloat‐producing diet containing 7.8 mg/kg hinokitiol and 0.2% of an organic acid mixture significantly decreased the increase in stable ingesta volume. Hinokitiol or an organic acid mixture did not affect ruminal volatile fatty acids, protozoa and bacteria. These results suggest that a combination of hinokitiol and an organic acid mixture might reduce frothy bloat in cattle fed high‐grain diets.     

Diversity of fimbrillin among Porphyromonas gulae clinical isolates from Japanese dogs

Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.     

The 2010 foot-and-mouth disease epidemic in Japan

Foot-and-mouth disease (FMD) occurred recently for the first time in a decade in Japan. The index case was detected on a beef-breeding farm in Miyazaki Prefecture, Southern Japan, on April 20, 2010. After confirmation of this first case, control measures such as stamping out, movement restriction and disinfection were implemented. However, these strategies proved insufficient to prevent the spread of FMD and emergency vaccination was adopted. Up until the last outbreak on July 4, 2010, a total of 292 outbreaks had been confirmed, with about 290,000 animals having been culled. The epidemic occurred in an area with a high density of cattle and pigs, making disease control difficult. Invasion of the disease into a high-density area aided its rapid spread and led to difficulties in locating suitable burial sites. Epidemiological investigations indicated that the disease was introduced into Japan approximately one month before detection. This delay in initial detection is considered to have allowed an increased number of outbreaks in the early stage of the epidemic. Nevertheless, the epidemic was contained within a localized area in Miyazaki Prefecture and was eradicated within three months because of intensive control efforts including emergency vaccination. Although this epidemic devastated the livestock industry in Japan, many lessons can be learnt for the future prevention and control of infectious diseases in animals.     

Isolation of genes showing increased expression during bovine adipocyte differentiation

The adipocyte is important not only for the storage of excess energy as fat, but also for the secretion of homeostatic factors. Gene expression profiles during adipocyte differentiation have been reported previously for mouse 3T3‐L1 cells. However, the profiles of adipogenic gene expression in mice and cattle may be different because several metabolic pathways of the ruminant adipose tissue are different from those of non‐ruminants. The gene expression profile in a clonal bovine intramuscular preadipocyte cell line during adipogenesis was examined using the polymerase chain reaction‐subtraction method. Six hundred and twenty‐one clones, which were expressed at an early stage of differentiation, from the preadipocyte to adipocyte, were isolated and characterized. Further detailed studies were carried out for 86 selected genes using northern blotting. Ten genes were found to be highly expressed after differentiation of bovine intramuscular preadipocyte cells. In particular, the expression profiles of genes for stearoyl CoA desaturase and FK506 binding protein were quite different from the time course of differentiation of that seen in the 3T3‐L1 cells reported previously. In addition, these genes were assigned to bovine chromosomes using a bovine/hamster somatic cell hybrid panel and public database.     

Evaluation of smothering effect of four legume covers on Pennisetum polystachion ssp. setosum (Swartz) Brunken

The smothering effect of four legume covers, Canavalia ensiformis , Lablab purpureus, Mucuna pruriens and Calopogonium caeruleum , on Pennisetum polystachion ssp. setosum , a troublesome weed in rubber plantations in southern Thailand, was evaluated. Growing legume covers with Pennisetum , M. pruriens , C. ensiformis and L. purpureus caused strong suppression of Pennisetum in this order. Pennisetum grew little under 10% relative light intensity (RLI), but almost normally under 40% RLI. The three legume species generally attained lower RLI than C. caeruleum . The high shading ability of these three species was thought to contribute to the suppression of Pennisetum growth.     

Microscopic Studies on Attachment and Differentiation of Zoospores of the Phytopathogenic Fungus <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis>

The mode of aggregation, attachment and differentiation of zoospores of the phytopathogenic fungus Aphanomyces cochlioides when interacting with the host and a host-specific attractant and a G-protein activator, mastoparan, was studied by light and scanning electron microscopy. When a zoospore approached very close to the host root, it seemed to halt, then coiled its anterior flagellum on its body. The halted zoospore appeared to contact the host surface with its posterior flagellum, which gradually drew the encysting zoospore onto the root surface. The spore then docked precisely on the root surface at its ventral face with the help of the posterior flagellum and anchored itself by releasing some adhesive materials. The adherent spore became a spherical after shedding its flagella and rapidly turned into an expanded cyst forming a smooth cyst coat around it, and finally changed into a smaller cystospore covered with a wrinkled surface. In contrast, the mastoparan- or cochliophilin A-stimulated zoospores on artificial membranes aggregated by using their posterior flagella before encystment. These contrasting phenomena suggest that A. cochlioides zoospores may use their posterior flagella for successful docking on the host surface or for aggregation of encysting spores in the absence of the host. Received 30 August 2001/ Accepted in revised form 8 November 2001     

Deleted in colorectal cancer (netrin‐1 receptor) antibodies and limbic encephalitis in a cat with hippocampal necrosis

A 7‐year‐old neutered female domestic shorthaired cat born in Poland and then moved to Japan presented to the local clinic with recent onset of convulsive cluster seizures and status epilepticus. Magnetic resonance imaging revealed bilateral swelling of the hippocampus with T2 hyperintensity and contrast enhancing image, suggesting hippocampal necrosis. The cat completely recovered after treatment with antiepileptic drugs (AED) and administration of prednisolone (1 mg/kg PO q24h for 4 days and tapered). However, cluster seizures reoccurred and developed into status epilepticus despite increasing doses of AED. Although the convulsions were resolved by other AEDs, stupor and renal failure developed, and the cat was euthanized. Pathological findings were consistent with hippocampal necrosis. Immunological analysis for leucine‐rich glioma inactivated 1 (LGI1) autoantibodies was negative, but antibodies against DCC (deleted in colorectal carcinoma) known as netrin‐1 receptor were found. This report describes a case of feline autoimmune limbic encephalitis and hippocampal necrosis that were presumably associated with DCC autoantibodies.