In vivo growth and genomic characterization of rickettsia-like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand.

A rickettsia-like organism, designated NZ-RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ-RLO2 was able to grow in CHSE-214, EPC, BHK-21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF-89T grew in all but BHK-21 and Sf21. NZ-RLO2 grew optimally in EPC at 15°C, CHSE-214 and EPC at 18°C. The growth of LF-89 T was optimal at 15°C, 18°C and 22°C in CHSE-24, but appeared less efficient in EPC cells at all temperatures. Pan-genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p-value = 94%). NZ-RLO2 was genetically different from previously described NZ-RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p-value = 88%), but were closely related to each other. TaqMan and Sybr Green real-time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ-RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.

Monitoring Theileria orientalis (Ikeda)-associated bovine anaemia in affected cattle over time.

The aim of the study was to observe changes in haematocrit (HCT) over time in a New Zealand South Island dairy herd affected by an outbreak of Theileria-associated bovine anaemia (TABA; Ikeda). A secondary aim was to relate individual cow HCTs to the amount of Theileria orientalis Ikeda DNA present in the blood, as measured by cycle threshold values, using a quantitative PCR (qPCR). Over a 6 month period, blood samples from 19 randomly selected cattle were monitored from a herd of 600 dairy cows. The sampling interval was approximately fortnightly for the first six weeks, followed by sampling at between four and six week intervals. At the initial report of the outbreak, two from six cattle were anaemic (HCT<0.25L/L). Blood collected from 14 cattle 11 days later showed that 57% (95% CI 33-77%) of the cattle sampled were anaemic. Of the 19 cattle that went on to be monitored, 12 (63% 95% CI=41-81%) developed anaemia at some point during the period of monitoring. One of the anaemic animals did not meet the case definition for TABA Ikeda. For individual cattle, the average number of days between when cattle were first detected as anaemic and when HCT returned to normal was 53days (median=47 days, range=6-92 days). At the point of notification the amount of T. orientalis Ikeda DNA in the blood of the six cattle tested was low (Cq median=36), but 11days later the amount of DNA in blood of 14 additional cows tested was relatively high (Cq median=24). Levels of all 19 cows monitored continued to remain moderately high through the period of testing (Cq median=29). This was despite a general improvement in the HCT of affected cattle. In four of the 15 cattle positive to T. orientalis Ikeda where blood fractions (plasma and whole blood) were tested, it appeared that T. orientalis Ikeda (as measured by qPCR) dropped more rapidly in plasma fractions than in whole blood at the point that HCT started to return to normal levels. Despite the assumption that tick populations were low in the Canterbury region of the South Island the impact of TABA (proportion of herd affected and the average period that animals remained anaemic) on the case herd was still relatively high.

Nocardiosis in freshwater reared Chinook salmon (Oncorhynchus tshawytscha).

CASE HISTORY: An investigation was conducted to identify the cause of mortalities in freshwater reared Chinook salmon (Oncorhynchus tshawytscha). Mortalities occurred in juvenile salmon, at a salmon rearing facility in the South Island of New Zealand. The affected fish were from a pen inside the facility with no surrounding pens or other year classes affected. CLINICAL FINDINGS: Clinically affected fish presented with skin lesions. The majority of skin lesions were unruptured, boil-like, raised circular masses up to 4 cm in diameter, particularly on the dorsolateral aspects and the flank. A number of fish presented with large ulcers resulting from rupturing of the raised lesions described above. This clinical presentation showed similarities to that of furunculosis caused by typical Aeromonas salmonicida, a bacterium exotic to New Zealand. LABORATORY FINDINGS: Samples were taken from two representative fish in the field for histopathology, bacterial culture and molecular testing. Histopathological findings included granulomatous lesions in the kidney, liver, spleen and muscle. When stained with Fite-Faraco modified acid fast stain filamentous branching rods were identified within these granulomas. Following bacterial culture of kidney swabs pure growth of small white matt adherent colonies was observed. This isolate was identified as a Nocardia species by biochemical testing and nucleotide sequencing of the partial 16S rRNA gene. All samples were negative for A. salmonicida based on bacterial culture and PCR testing. DIAGNOSIS: Nocardiosis caused by a Nocardia species. CLINICAL RELEVANCE: Nocardiosis in these fish was caused by a previously undescribed Nocardia species that differs from the species known to be pathogenic to fish: N. asteroides, N. salmonicida and N. seriole. This bacterium is likely to be a new or unnamed environmental species of Nocardia that has the potential to cause disease in Chinook salmon under certain conditions. The clinical presentation of this Nocardia species manifested as raised, boil-like skin lesions which has similarities to the presentation of furunculosis caused by the bacterium typical A. salmonicida, a species exotic to New Zealand.

The First Detection of Ornithobacterium rhinotracheale in New Zealand.

Ornithobacterium rhinotracheale (ORT) has been considered exotic to New Zealand and thus, any samples from poultry suspected of ORT infection are submitted as part of an exotic disease investigation managed by Ministry for Primary Industries (MPI) and subjected to standardized test protocols carried out in the physical containment level 3+ laboratory at MPI's Animal Health Laboratory (AHL). All previous exotic disease investigations concerning ORT produced negative results by bacterial culture and conventional PCR. Following the recent introduction of a real-time PCR for ORT at the AHL, several tracheal wash fluids from backyard chickens ( Gallus gallus domesticus ) were tested positive. This identification constituted the first detection of ORT in New Zealand poultry. As a result, a second premise was investigated with further samples testing positive for ORT by molecular assays. This paper describes the two exotic disease investigations associated with the first detection of ORT in New Zealand poultry and its implications.

First report and histological features of Chlamydia pecorum encephalitis in calves in New Zealand.

CASE HISTORY: Between September and October 2013, 40 of 150 crossbred Friesian dairy calves on a farm in the Manawatu region of New Zealand developed neurological signs when between 1 and 3 months of age. Calves were grazed in multiple mobs and calves from each mob were affected. A variable response was observed to initial treatment with thiamine, fluoroquinolone antibiotics and non-steroidal anti-inflammatory drugs. CLINICAL AND PATHOLOGICAL FINDINGS: Affected calves exhibited a range of neurological signs that included generalised depression, hind limb ataxia with a stiff gait, and knuckling of the fetlocks. In advanced cases, calves became recumbent with opisthotonous. Over a 4-week period, 13 calves died or were subject to euthanasia and a thorough necropsy was performed on three of these calves. Necropsy findings included fibrinous peritonitis, pleuritis and pericarditis, with no gross abnormalities visible in the brain or joints. Histology of the brain was possible in seven of the affected calves, with lesions ranging from lymphocytic and histiocytic vasculitis and meningoencephalitis, to extensive thrombosis and neutrophilic inflammation. Immunohistochemistry using an anti-chlamydial lipopolysaccharide antibody revealed positive immuno-staining in all seven cases, with no brain samples exhibiting immunostaining for Histophilus somni. DNA was extracted from a sample of fresh brain from one case and chlamydial DNA sequences were amplified by PCR and found to be identical to Chlamydia pecorum. PCR was also performed on formalin-fixed brain tissue from three of the other cases, but no chlamydial DNA was amplified. DIAGNOSIS: Chlamydia pecorum meningoencephalomyelitis (sporadic bovine encephalomyelitis). CLINICAL RELEVANCE: This is the first time that C. pecorum has been confirmed as a cause of clinical disease in New Zealand. Practitioners should be aware of this disease as a differential in calves with neurological signs, and submit samples of formalin-fixed brain as well as fresh brain to enable confirmation of suspected cases using PCR analysis. Furthermore, these cases illustrate that the histological lesions in the brains of calves with C. pecorum are more variable than previously reported, and pathologists should be aware that histological features may overlap with those traditionally ascribed to other organisms, such as H. somni.

Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR.

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis. METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results. RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR. CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.

Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand.

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations. CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara. LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA. DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates. CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.

Investigation of bovine haemoplasmas and their association with anaemia in New Zealand cattle.

CASE HISTORY AND CLINICAL FINDINGS: A dairy cow, from a herd in the Waikato region of New Zealand, was reported with regenerative anaemia on 12 September 2014. Testing of blood from the animal using PCR assays for Theileria orientalis produced a negative result for both Chitose and Ikeda types. LABORATORY FINDINGS: Using PCR and DNA sequencing, blood from the cow was positive for Candidatus Mycoplasma haemobos. Further testing of another 12 animals from the case herd, 27 days after the affected cow was first reported, showed 11 animals were positive for Candidatus M. haemobos or Mycoplasma wenyonii in the PCR. None of these cattle were clinically anaemic or positive for T. orientalis Ikeda type using PCR. A convenience sample of 47 blood samples from cattle throughout New Zealand, submitted to the Investigation and Diagnostic Centre (Ministry for Primary Industries) for surveillance testing for T. orientalis Ikeda, was selected for further testing for bovine haemoplasmas. Of these samples, 6/47 (13%) and 13/47(28%) were positive for M. wenyonii and Candidatus M. haemobos, respectively. There was no difference in the proportion of samples positive for the bovine haemaplasmas between cattle with anaemia that were negative for T. orientalis (6/20, 33%), or without anaemia or T. orientalis (10/18, 56%), or from cattle herds experiencing anaemia and infection with T. orientalis Ikeda type (3/9, 33%). DIAGNOSIS: Bovine haemoplasmosis. CLINICAL RELEVANCE: The presence of bovine haemoplasmas in blood does not establish causality for anaemia in cattle. Diagnosis of anaemia associated with haemoplasmosis would require exclusion of other causes of regenerative anaemia and an association of the agent with anaemia in affected cattle herds. The data collected in this study did not provide evidence that bovine haemoplasmas were associated with a large number of outbreaks of anaemia in cattle in New Zealand.

The seroprevalence of avipoxvirus and its association with avian malaria (Plasmodium spp.) infection in introduced passerine birds in the southern regions of the North Island of New Zealand.

Blood samples were collected from 65 free-ranging birds from six species in the southern North Island of New Zealand. Sera from the birds were tested for the presence of avipoxvirus (APV) antibodies by enzyme-linked immunosorbent assay (ELISA), and blood cells from 55 birds were also tested for Plasmodium spp. by PCR. Forty-five birds (69.2%) tested seropositive to APV. Song thrushes (Turdus philomelos) presented the highest seroprevalence at 100% (4/4), followed by Eurasian blackbirds (Turdus merula) (96.86%, 31/32), chaffinches (Fringilla coelebs) (54.55%, 6/11), starlings (Sturnus vulgaris) (25%, 3/12), greenfinches (Carduelis chloris) (25%, 1/4), and European goldfinches (Carduelis carduelis) (0%, 0/2). Plasmodium spp. DNA was detected in 15/55 birds (27.3%), including 11 Eurasian blackbirds, one song thrush, and three starlings. Eight Eurasian blackbird isolates (73%) grouped within the subgenus Novyella. Two Eurasian blackbird isolates and the song thrush isolate clustered within a different group with previously reported lineages LINN1 and AFTRU5. In addition, all three starling isolates clustered within the well-characterized lineage Plasmodium (Huffia) elongatum GRW06. All Plasmodium-positive Eurasian blackbirds and the song thrush were seropositive to APV, whereas only 67% of Plasmodium-positive starlings showed evidence of previous exposure to APV. A significant relationship between birds seropositive to APV and birds infected by Plasmodium spp. was observed (chi2 = 5.69, df = 1, P = 0.0086). To the authors' knowledge this is the first report describing the seroprevalence of APV and its association with Plasmodium spp. infection in introduced bird species in New Zealand.

Avipoxvirus infections in brown kiwi (Apteryx mantelli).

CASE HISTORY: Nodular lesions were found on the skin of two immature brown kiwi (Apteryx mantelli) less than 6 months of age living freely on Ponui Island off the North Island of New Zealand. The lesions were observed during routine external examination undertaken as a part of the management of other research projects, one in 2006 and the other in 2011. Apart from the skin lesions, both birds showed no signs of illness and the lesions resolved spontaneously over a 2-month period. PATHOLOGICAL FINDINGS: The first case showed several 3-mm diameter firm, brown nodules located on the skin below the hock of both legs. The second case had a single multinodular mass that measured 7×20 mm, on the base of the bill. A portion of the mass and scab samples were collected for diagnosis. Histological examination of the nodules revealed severe ballooning degeneration of keratinocytes and epithelial hyperplasia. Round eosinophilic structures resembling avipoxvirus (APV) intracytoplasmic inclusion bodies (Bollinger bodies) were observed in the layers of keratinocytes. In deeper layers of the epidermis, there was evidence of secondary bacterial growth and inflammation. DIAGNOSIS: DNA was extracted from tissue samples and subjected to PCR analysis. Avipoxvirus 4b core protein gene was detected in both samples by PCR. Bootstrap analysis of APV 4b core protein gene revealed that APV isolates from two kiwi comprised two different subclades. One isolate displayed 100% sequence homology to subclade B1, and the other presented 100% sequence homology to subclade A3. CLINICAL RELEVANCE: This study confirmed that kiwi are susceptible to APV infection and that at least two different strains of APV are present in the population examined. Since there is no information on the origin, virulence, or prevalence of APV in kiwi, a seroprevalence study would be useful to elucidate the degree of exposure and immune response to the disease. This would allow a more informed approach to risk management of the disease in wild and captive populations.

Concurrent avian malaria and avipox virus infection in translocated South Island saddlebacks (Philesturnus carunculatus carunculatus).

CASE HISTORY: Outbreaks of mortality in South Island saddlebacks (Philesturnus carunculatus carunculatus) that had been translocated to two offshore islands in the Marlborough Sounds of New Zealand were investigated during the summers of 2002 and 2007. Both outbreaks were associated with a severe decrease in numbers of saddlebacks of up to 60% of approximately 200 birds. CLINICAL AND PATHOLOGICAL FINDINGS: Many of the surviving birds were in poor condition, and had skin lesions on the legs and head. Necropsy showed pale liver and lungs, and a swollen spleen. Histopathology revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes and splenic histiocytes. The skin lesions consisted of epithelial proliferations containing numerous Bollinger bodies typical of avipox virus (APV) infection. Two different APV were isolated, using PCR, from two different birds exhibiting skin lesions. Each isolate had 100% sequence homology with APV members from either Clade A or Clade B. In addition, PCR analysis revealed that the Plasmodium elongatum present in infected birds belonged to a strain that was endemic in the population of North Island saddlebacks (Philesturnus carunculatus rufusater). DIAGNOSIS: Concurrent infections with Plasmodium spp. haemoparasites and APV were identified as the likely cause of death in the birds examined. CONCLUSIONS AND CLINICAL RELEVANCE: Although the Plasmodium spp. identified is thought to be endemic to saddlebacks in New Zealand, the affected birds were likely to be immunocompromised by concurrent APV infection or through lack of genetic diversity. Both the introduced mosquito Culex quinquefasicatus and the native mosquito Culex pervigilans are likely vectors for both these diseases, and the provision of water supplies less favourable to mosquito-breeding is recommended.

The prevalence of psittacine beak and feather disease virus infection in native parrots in New Zealand.

AIM: To determine the prevalence of psittacine beak and feather disease virus (BFDV) infection in native parrots in New Zealand. METHODS: One hundred and sixty-nine wild native parrots and 143 captive native parrots throughout New Zealand were examined for the presence of BFDV, from June 2003 to January 2005. Feathers of each bird, and blood samples from 15 birds, were collected and submitted for PCR assay to detect BFDV. RESULTS: All of the samples from wild native parrots were negative for BFDV by PCR assays. Similarly, of the 143 PCR tests from captive native parrots 139 (97%) were negative for BFDV. However, a pair of red-crowned parakeets and two Antipodes Island parakeets from different captive facilities were found to be infected with BFDV. The infected birds showed no clinical signs suggestive of psittacine beak and feather disease (PBFD), although the second Antipodes Island parakeet was found dead, and had pathological changes consistent with acute septicaemia. CONCLUSIONS: The results indicate a very low prevalence of BFDV among free-living native parrots although captive birds, in particular native parakeets, are susceptible to BFDV infection, and the Antipodes Island parakeets may be susceptible to PBFD.

The prevalence of beak and feather disease virus infection in wild populations of parrots and cockatoos in New Zealand.

AIM: To determine the prevalence of beak and feather disease virus (BFDV) infection in exotic parrots and cockatoos in the wild in New Zealand. METHODS: Eastern rosellas (Platycercus eximius, n=162) were caught from Te Puke, Wellington and Dunedin, using mistnets, between April 2004 and February 2006, and sulphur crested cockatoos (Cacatua galerita, n=255) were captured for pet-trading from November 2001 to September 2004. Feathers from both species were tested for BFDV, using an established polymerase chain reaction (PCR) test. Post-mortem examinations were conducted on some of the eastern rosellas, and selected tissues from 24 birds positive for BFDV were examined using routine histological methods for the presence of characteristic inclusion bodies. RESULTS: Of the eastern rosellas, 24/162 (14.8%) were positive for BFDV, and the 95% confidence interval (CI) for true prevalence was estimated as 8.6-20.4%, which varied between regions. Eastern rosellas that were positive for BFDV showed no clinical or histological signs of disease or inclusion bodies. Of the sulphur-crested cockatoos, 70/255 (28%) were positive for BFDV, and the 95% CI for true prevalence was calculated as 22-33%. CONCLUSIONS: The surprisingly high prevalence of BFDV in wild eastern rosellas and sulphur-crested cockatoos has serious implications for the conservation of native parrots and the export of wild-trapped parrots and cockatoos from New Zealand. Serological studies for BFDV in wild exotic parrots, and molecular studies of virus genotype, are recommended to further characterise the origin and epidemiology of the disease in populations of wild exotic parrots and cockatoos in New Zealand.

Preparation and biological evaluation of 188Re-ethylenediamine-N,N,N',N'-tetrakis(methylene phosphonic acid) as a potential agent for bone pain palliation.

This study was undertaken in order to prepare 188Re labelled ethylenediamine-N,N,N',N'-tetrakis(methylene phosphonic acid) (EDTMP), and to determine its potential as a therapeutic radiopharmaceutical for the palliation of metastatic bone pain. The effects of pH, incubation methods, and concentrations of stannous chloride, EDTMP, and ammonium perrhenate as a carrier on radiochemical yield and stability were evaluated. Biodistribution studies were performed in male Wistar rats after intravenous injection of 188Re-EDTMP and compared with those of hydroxyethylidene diphosphonate (HEDP). Greater than 95% radiochemical yield of 188Re-EDTMP was obtained under the optimal conditions (0.1 mmol x ml(-1) of EDTMP, 0.5 mg x ml(-1) of stannous chloride, and pH 1.0). Heating the reaction mixture (boiling water for 15 min, and microwave heating for 15 s) and the addition of ammonium perrhenate increased the radiochemical stability (>90% at 3 h, and >80% at 48 h). The biodistribution of 188Re-EDTMP showed high bony uptake and rapid clearance from other organs, and high bone-to-soft tissue ratios, which are similar to 188Re-HEDP. In conclusion, 188Re-EDTMP was prepared with high radiochemical yield and stability, and showed favourable biological characteristics. Microwave heating was a convenient and rapid method for the preparation of 188Re-EDTMP. It is considered that 188Re-EDTMP is a potential therapeutic agent for bone metastasis.

BAPTA/AM, an intracellular calcium chelator, induces delayed necrosis by lipoxygenase-mediated free radicals in mouse cortical cultures.

1. Disruption of calcium homeostasis during neurodegenerative diseases is known to trigger apoptotic or necrotic death in neuronal cells. Recently, the authors reported that intracellular calcium restriction by NMDA receptor antagonists induces apoptosis in cortical cultures. To evaluate whether further restriction of intracellular free calcium can induce apoptosis or necrosis, we examined the neurotoxic characterization of BAPTA/AM, a permeable free calcium chelator, in mouse cortical cultures. 2. Exposure of mixed (glia and neuron) cortical cultures (DIV 13-16) to 3-10 microM BAPTA/AM (non-toxic concentration for glial cells) for 24-48 hr resulted in delayed and necrotic neuronal death. The necrotic findings included swelling and loss of mitochondria and endoplasmic reticulum (ER) with neuronal membrane rupture 24 hr after treatment with BAPTA/AM. Simultaneously, we observed a few TUNEL-positive cells in the neuronal subpopulation of the same cultures. 3. The neurotoxicity evoked by BAPTA/AM (10 microM) was significantly attenuated by the addition of 0.5 microM cycloheximide (a protein synthesis inhibitor), 10 microM actinomycin D (an RNA transcription inhibitor), a high extracellular potassium concentration (total 15 mM KCl), 100 microM t-ACPD (a metabotrophic agonist), 100 microM alpha-tocopherol (a free radical scavenger), 100 microM deferoxamine (a ferric ion chelator), 100 microM L-NAME (a nitric oxide synthase (NOS) inhibitor), 50 microM DNQX (a non-NMDA receptor blocker), and 3-30 microM esculetin (a lipoxygenase inhibitor). However, 0.3-3 mM ASA (a cyclooxygenase inhibitor), 100 ng/ml nerve growth factor (NGF), 10 microM MK-801 (a NMDA receptor antagonist), 20 microM zVAD-fmk (caspase inhibitor) and 50 U/ml catalase failed to inhibit the injury. 4. However, NGF and catalase blocked the neurotoxicity induced by BAPTA/AM in young neuronal cells (DIV 6). BAPTA/AM (10 microM) did not alter the expression of inducible nitric oxide synthase (iNOS) on glial cells. 5. These results suggest that the feature of neuronal death induced by BAPTA/AM exhibits predominantly delayed necrosis mediated by lipoxygenase-dependent free radicals.

Serotonin receptor and transporter ligands - current status.

The serotonin (5-HT) receptor system has 14 different subtypes classified by pharmacology and function. Many ligands are widely used for therapeutic and diagnostic purposes in some severe human diseases. Most of the ligands that are specific for each 5-HT receptor have distinctive chemical structures with regard to pharmacophore elements including 4-arylpiperazine, benzimidazole, benzamide, chroman, aminopyridazine, tetralin, and polycycles. However, their affinity and selectivity for 5-HT, dopamine and alpha1 receptors depend on their substituents and linker spacers. 5-HT transporter inhibitors have also been developed as potential antidepressants. In contrast to classical tricyclic compounds, newly developed secondary amine derivatives such as paroxetine and tetralin show high binding affinity and selectivity. Radioisotope-labeled ligands have also been developed, including [carbonyl-(11)C]WAY 100635 for 5-HT1A receptor, [(11C) or (18)F]ketanserine derivatives for 5-HT(2) receptor, [(125)I]DAIZAC for 5-HT(3) receptor, and [123I]IDAM for 5-HT transporter, and these are accumulated in brain regions that are rich in the respective receptors. This review summarizes the recent development of 5-HT receptor- and transporter-specific ligands and their pharmacological properties on the basis of their chemical structures.

Simple and reliable preparation of pentavalent 99Tcm-dimercaptosuccinic acid at alkaline pH without oxygen bubbling.

We investigated a simple and reliable method for the preparation of 99Tcm-dimercaptosuccinic acid (99Tcm(V)-DMSA) without the addition of oxygen. The effect of pH, amount of reducing agent, and oxygen addition in the synthesis of 99Tcm(V)-DMSA were evaluated. At pH 9, we obtained a radiochemical yield of 95% +/- 1.2% within 10 min and a high stability until 7 h, with 92% +/- 1.5% radiochemical purity. However, at a pH lower than 9, the radiochemical yield was below 90% within 10 min, and a longer reaction time was needed to obtain a radiochemical yield above 90%. The addition of oxygen did not have an additional effect on the radiochemical yield or its stability at pH 9, whereas it increased the radiochemical yield of 99Tcm-(V)-DMSA at pH 7. It was noted that the smaller the amount of reducing agent used, the higher was the radiochemical yield obtained at pH 7. However, at pH 9, the radiochemical yield was not dependent on the amount of reducing agent. In conclusion, the synthesis of 99Tcm(V)-DMSA was more dependent on the pH of the reaction mixture than on the amount of reducing agent or the addition of oxygen. The adjustment of pH 9 was the easiest and most effective method for the synthesis of 99Tcm(V)-DMSA using a commercial kit for 99Tcm(III)-DMSA.

Molecular characterization of the NeIF2Bbeta gene encoding a putative eIF2B beta-subunit in Nicotiana tabacum.

The NeIF2Bbeta cDNA encoding beta-subunit of the translation initiation factor 2B (eIF2B-beta) was identified from Nicotiana tabacum through protein interaction with PRK1, a reproductive-organ-specific receptor-like kinase (Park et al., 2000). The eIF2B is a guanine nucleotide-exchange protein that consists of five subunits, which function in the regulation of translation in eukaryotic cells. The NeIF2Bbeta that shows a high homology in the amino acid sequence with other beta-subunits also exhibits sequence similarity to a and delta subunits of eIF2B from yeast and animals. The NeIF2Bbeta gene was expressed in all of the tissues examined, but the mRNA level was higher in reproductive tissues than in vegetative tissues. During anther development, the NeIF2Bbeta mRNA was detected in all stages with a slightly higher level in the earliest stage. The NeIF2Bbeta-GFP fusion protein was mainly localized in the cytosol.

Automation of the synthesis of highly concentrated 188Re-MAG3 for intracoronary radiation therapy.

We have developed an efficient method and an automated synthetic system for the preparation of highly concentrated 188Re-MAG3. Routine production of 188Re-MAG3 for use in intracoronary radiation therapy was performed by compressed air driven semi-automated shielded system. 188Re-MAG3 was prepared with a commercial kit and reducing agents, purified and concentrated by C18 Sep-Pak cartridges to desired radioactivity and volume. Using this automated system, reproducible radiolabeling yields of 80-85% were obtained.

Synthesis of substituted-(l)-tryptophanols from an enantiomerically pure aziridine-2-methanol.

Enantiomerically pure (l)-tryptophanol (5) was synthesized from 4(R)-iodomethyl-2-oxazolidinone (2) and indolylmagnesium bromide in three steps (52% overall yield). Using this procedure, we also prepared various tryptophanols with substituent(s) on the indole ring. Furthermore, optically active 4(R)-iodomethyl-2-oxazolidinone was readily prepared from an enantiomerically pure aziridine-2(S)-methanol in high yield. [reaction: see text]