Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.     

Malignant pinealoma observed in the deep cerebral parenchyma of a male Wistar rat

This report describes a case of spontaneous malignant pinealoma in a 90-week-old male Wistar rat. The tumor mass occurred in the deep cerebral parenchyma and no intact pineal gland was observed in the area between the posterior-dorsal median line of the cerebrum and the cerebellum. The tumor was characterized by a large nodular proliferation occupying the central area of the brain, extending from the dorsal surface to the base of the brain, corresponding to the thalamus. The tumor cells had round to irregular oblong nuclei approximately 5–17 μm in diameter and showed faintly or moderately eosinophilic cytoplasm and indistinct cell boundaries. Immunohistochemically, the tumor cells were positive for synaptophysin and partially positive for neuron-specific enolase (NSE). The tumor showed malignant features including cellular pleomorphism, high mitotic index, necrotic foci, and invasive and extensive growth and was, therefore, diagnosed as an extremely rare malignant pinealoma in the deep cerebral parenchyma.     

Recommended doses of medetomidine-midazolam-butorphanol with atipamezole for preventing hypothermia in mice

A non-narcotic anesthetic combination (Me/Mi/Bu) of medetomidine (Me), midazolam (Mi), and butorphanol (Bu) has been recommended as the injectable anesthesia in mice. An original dose of Me/Mi/Bu (0.3/4.0/5.0 mg/kg) has provided sufficient anesthetic duration of 40–50 min in mice. In addition, atipamezole is available for reversal of Me/Mi/Bu anesthesia. As an adverse effect of Me/Mi/Bu anesthesia, however, severe hypothermia has been also observed in mice. In the present study, we investigated 1) the main agent in Me/Mi/Bu to cause of hypothermia, 2) the effects of the differential doses of atipamezole on hypothermia induced by Me/Mi/Bu anesthesia and on the plasma levels of creatinine phosphokinase and transaminases, and 3) those recommended doses for preventing hypothermia induced by Me/Mi/Bu anesthesia in mice. The results suggested that 1) the α₂-agonist medetomidine is most likely to induce hypothermia in mice under Me/Mi/Bu anesthesia, 2) the antagonism of atipamezole within proper dose range is effective in promoting the recovery from Me/Mi/Bu-induced hypothermia, and 3) Me/Mi/Bu at the recommended dose of 0.2/6.0/10.0 mg/kg enable to provide anesthetic effects for 40 min and is more considerable to prevent the hypothermia than that at the original dose of 0.3/4.0/5.0 mg/kg.     

Transformation of trifoliate orange with rice chitinase gene and the use of the transformed plant as a rootstock

RCC2, cDNA clone encoding rice class-I chitinase, was introduced into trifoliate orange (Poncirus trifoliata Raf.). Chitinase activity of the transformed plants was higher than that of the non-transformed plants. The introduced gene was not detected in the scions grafted onto the transformed plants. The effect of the introduction of foreign gene into rootstocks on the scions was discussed.     

Programmed Cell Death Induced by (?)-8,9-Dehydroneopeltolide in Human Promyelocytic Leukemia HL-60 Cells under Energy Stress Conditions

(+)-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−)-8,9-dehydroneopeltolide (8,9-DNP), a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD). Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose) polymerase (PARP). These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF), although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion.     

Identification of a telomere sequence type in three sponge species (Porifera) by fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization analysis

The telomere sequence type (TTAGGG)_n is known to be distributed in various phyla in the Animalia and in Mastigophora (Protista). However, the telomere type of Porifera (sponges), a phylum comprising the lowest multicellular animals, has not been reported. In this study, we examined the three sponge species Leucetta chagosensis, Halichondria japonica, and Halichondria panicea for the presence of the telomere type (TTAGGG)_n by fluorescence in situ hybridization (FISH). The oligonucleotide probe (TTAGGG)₇ clearly displayed signals on the interphase nuclei of all three sponges. In contrast, the (TTAGG)₇ probe, which has one base fewer than (TTAGGG)₇, did not display the signals. These results suggest that the telomeres of the three sponges consist of (TTAGGG)_n, which is identical to the sequence type found in many higher multicellular animals and in Mastigophora. Additionally, this is the first study to reveal a telomere sequence type for Porifera. Moreover, these results suggest that Porifera are phylogenetically related to Mastigophora, and supports the general theory that Porifera evolved from Mastigophora. Further, this study strongly suggests that the origin of the (TTAGGG)_n telomere sequence is to be found in a common ancestor of either the Bilateria and Porifera, or the Protista.     

Effect of Angiotensin II Type 1 receptor blocker on cardiac angiotensin-converting enzyme and chymase-like activities, and cardiac fibrosis in cardiomyopathic hamsters

It has been reported that cardiac chymase has an effect on cardiac fibrosis through the Angiotensin (Ang) II formation and an Ang II-independent mechanism. In the present study, Ang II type 1 (AT1) receptor blocker (candesartan cilexetil) was administered to dilated cardiomyopathic (DCM; Bio TO2) hamsters for 4 weeks to study the effect of AT1 receptor blocker on cardiac chymase-like activity and cardiac fibrosis. Echocardiography, histological examination, and assessment of cardiac angiotensin-converting enzyme (ACE)/chymase-like activities were conducted. Hamsters showed cardiac dysfunction due to increased left ventricular dimensions and decreased ventricular wall thickness, significant increase in cardiac chymase-like activity, and fibrosis. This result indicates that the cardiac chymase-like activity is responsible for cardiac fibrosis. When candesartan cilexetil was administered to Bio TO2 hamsters, cardiac chymase-like activity increased significantly, whereas cardiac fibrosis decreased significantly. Cardiac ACE and chymase-like activities were unchanged in non-DCM hamsters with candesartan cilexetil. This suggests that the cardiac Ang II formation mechanism was stimulated by suppressing the effect of cardiac Ang II, and cardiac chymase-like activity could be increased. Moreover, this mechanism may be more highly activated if cardiac Ang II is activated in the heart. In conclusion, we demonstrated that AT1 receptor blocker reduced cardiac fibrosis, although cardiac chymase-like activity increased. Because the Ang II-forming pathway and the effect of chymase in hamsters is similar to that in dogs, the results of the present study may supplement the available information for dogs.