L-histidine decarboxylase as a probe in studies on histamine

Because the Falck-Hillarp formaldehyde fluorescence method, which was superbly applied to identify catecholaminergic and serotonergic neurons, is not applicable to histamine, the first author (T.W.) developed an antibody to L-histidine decarboxylase (HDC) for identification of the histaminergic neuron system in the brain. The anti-HDC antibody was of great use for mapping the location and distribution of this histaminergic neuron system. (S)-alpha-fluoromethylhistidine, a specific and potent irreversible inhibitor of HDC, was also very useful in studies on functions of the neuron system. The activity of HDC is increased by various agents, treatments, and physiological conditions. We found new compounds that increased HDC activity (i.e., tetradecanoylphobol acetate (TPA), other tumor promoters, and staphylococcal enterotoxin A); and using mast cell-deficient mutant (W/W(v)) mice, we obtained evidence that this increase occurred in macrophages. To further characterize the mechanism of increases in HDC activity, the second author (H.O.) cloned human HDC cDNA and a human HDC gene. In studies on the regulation mechanism of the HDC gene, which is expressed only in limited types of cells such as mast cells, enterochromaffin-like cells in the stomach, cells in the tuberomammillary nucleus of the brain, and macrophages, CpG islands in the promoter region of the HDC gene were found to be demethylated in cells expressing the gene, whereas they are methylated in other cells that do not express the HDC gene. In collaboration with many other researchers, we developed HDC knockout mice. The resulting research is producing a lot of interesting findings in our laboratory as well as in others. In summary, HDC has been and will be useful in studies on functions of histamine.     

Metabolic profiling of normal and hypertensive rat kidney tissues by hrMAS-NMR spectroscopy

Intact kidney tissue samples of normal and spontaneously hypertensive rats (SHRs) were analyzed by hrMAS-NMR spectroscopy and principal component analysis (PCA). Radial components (cortex, outer stripe of the outer medulla, inner stripe of the outer medulla, and papilla) were sampled from various regions across the kidney from multiple animals in order to establish inter- and intra-animal variability. The effects of temperature were also measured. Papilla was differentiated from the other tissue types, and this variation by tissue type was greater than the effect of temperature on the samples (spectra were compared from samples at 2 and 30 °C). This study also revealed long term stability issues of tissue storage at -80 °C. The PCA showed that the greatest differentiation between normal rats and SHRs was found in the cortex and the regions in the NMR spectra that were correlated with this variation were identified.Abbreviations hrMAS High-resolution magic angle spinning - NMR Nuclear magnetic resonance - PCA Principal component analysis - CSA Chemical shift anisotropy - DD Dipolar coupling - SHR Spontaneously hypertensive rat     

A microwave-assisted sequential extraction of water and dilute acid soluble arsenic species from marine plant and animal tissues

This paper describes the use of dilute nitric acid for the extraction and quantification of arsenic species. A number of extractants (e.g. water, 1.5 M orthophosphoric acid, methanol-water and dilute nitric acid) were tested for the extraction of arsenic from marine biological samples, such as plants that have proved difficult to quantitatively extract. Dilute 2% (v/v) nitric acid was found to give the highest recoveries of arsenic overall and was chosen for further optimisation. The optimal extraction conditions for arsenic were 2% (v/v) HNO₃, 6 min⁻¹, 90 °C. Arsenic species were found to be stable under the optimised conditions with the exception of the arsenoriboses which degraded to a product eluting at the same retention time as glycerol arsenoribose. Good agreement was found between the 2% (v/v) HNO₃ extraction and the methanol-water extraction for the certified reference material DORM-2 (AB 17.1 and 16.2 μg g⁻¹, respectively, and TETRA 0.27 and 0.25 μg g⁻¹, respectively), which were in close agreement with the certified concentrations of AB 16.4 ± 1.1 μg g⁻¹ and TETRA 0.248 ± 0.054 μg g⁻¹.To preserve the integrity of arsenic species, a sequential extraction technique was developed where the previously methanol-water extracted pellet was further extracted with 2% (v/v) HNO₃ under the optimised conditions. Increases in arsenic recoveries between 13% and 36% were found and speciation of this faction revealed that only inorganic and simple methylated species were extracted.     

Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.     

Low volume microwave digestion for the determination of selenium in marine biological tissues by graphite furnace atomic absorption spectroscopy

A microwave digestion method for the determination of marine biological tissues has been developed to allow determination of selenium in small sample sizes (< 0.1 g). The benefits of this technique include maintaining concentrates in extracts without the subsequent over dilution encountered when using larger vessels, increased sample throughput and reduced loss of volatile material. Freeze dried biological material (< 0.1 g) and nitric acid (1 ml) were placed into 7 ml screw top Teflon vessels which are completely sealed on capping. Two 7 ml vials were placed into larger 120 ml vessels fitted with a Teflon spacer and 10 ml of distilled water. The effects of microwave power and time, and sample mass on selenium recovery from three marine standard reference materials (NIST SRM 1566a Oyster Tissue, NRCC DORM-1 Dogfish Muscle and NRCC TORT-1 Lobster Hepatopancreas) were examined. The optimum conditions: 600 W, 2 min; 0 W, 2 min; 450 W, 45 min, allowed quantitative recoveries of selenium from these and three other standard reference materials (NRCC DOLT-1 Dogfish liver, NIST RM-50 Albacore tuna and IAEA MA-A-2 fish flesh). Studies on sample mass showed that the analysis of sample masses from 0.025 to 0.1 g gave selenium concentrations within the certified range. Six species of selenium: selenite, selenate, selenomethionine, selenocysteine, selenocystamine, and trimethyl selenonium were added to oyster, dogfish, and lobster tissues. Recoveries were near quantitative for all species (94–105%) except trimethyl selenonium (90–101%).     

Virtual Screening of Alkaloid and Terpenoid Inhibitors of SMT Expressed in Naegleria sp.

The pathogenic form of thermophilic Naegleria sp. i.e., Naegleria fowleri, also known as brain eating amoeba, causes primary amoebic encephalitis (PAM) with a >97% fatality rate. To date, there are no specific drugs identified to treat this disease specifically. The present antimicrobial combinatorial chemotherapy is hard on many patients, especially children. Interestingly, Naegleria fowleri has complex lipid biosynthesis pathways like other protists and also has a strong preference to utilize absorbed host lipids for generating energy. The ergosterol biosynthesis pathway provides a unique drug target opportunity, as some of the key enzymes involved in this pathway are absent in humans. Sterol 24-C Methyltransferase (SMT) is one such enzyme that is not found in humans. To select novel inhibitors for this enzyme, alkaloids and terpenoids inhibitors were screened and tested against two isozymes of SMT identified in N. gruberi (non-pathogenic) as well as its homolog found in yeast, i.e., ERG6. Five natural product derived inhibitors i.e., Cyclopamine, Chelerythrine, Berberine, Tanshinone 2A, and Catharanthine have been identified as potential drug candidates based on multiple criteria including binding affinity, ADME scores, absorption, and, most importantly, its ability to cross the blood brain barrier. This study provides multiple leads for future drug exploration against Naegleria fowleri.     

良性前列腺增生组织对钛宝石激光的吸收和散射特性

研究了经尿道等离子体双极电切除术(PKRP)切除和经尿道前列腺电切术(TURP)切除的前列腺增生(BPH)组织对640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860和880 nm波长的钛宝石激光的光学特性及其差异,实验采用双积分球测量系统以及反向倍增法获取BPH组织的吸收和散射特性参数。结果表明：PKRP和TURP切除的BPH组织对这1３个不同波长的激光的吸收系数和约化散射系数都是随着激光波长增大而明显减小的,PKRP切除的BPH组织对这同一波长的激光的吸收系数和约化散射系数都明显较TURP切除的BPH组织对相应的波长的激光的吸收系数和约化散射系数要小,PKRP和TURP切除的BPH组织的吸收系数以及约化散射系数的最大值都在640 nm,其值分别为(0.885±0.022)和(0.955±0.024)mm-1以及(1.564±0.039)和(1.658±0.042)mm-1,其差异分别为7.91%以及6.01%,其最小值都在880 nm,其值分别为(0.443±0.011)和(0.455±0.011)mm-1以及(1.117±0.028)和(1.197±0.030)mm-1,其差异分别为2.71%以及7.16%。PKRP和TURP切除的BPH组织对660 nm的吸收系数的差异最大,其值为8.95%,其差异最小在860 nm,其值为1.75%,PKRP和TURP切除的BPH组织对800 nm的约化散射系数的差异最大,其值为9.13%,其差异最小在640 nm,其值为6.01%。     

热凝固致良性前列腺增生组织在590～1 064 nm的吸收和散射特性的变化

研究了自然的和热凝固的良性前列腺增生(BPH)组织在590～1 064 nm光谱范围的光学特性及其差异,实验采用带积分球附件的分光光度计以及反向倍增法获取组织样品的吸收和散射特性参数。结果表明：热凝固导致BPH组织在590～1 064 nm光谱范围的吸收系数明显地减小的,自然的和热凝固的BPH组织的吸收系数都有一个峰值在990 nm处,其值分别为0.438和0.416 mm-1,自然的和热凝固的BPH组织的吸收系数的最大差异在1 064 nm,其值为86.79%,其最小差异在920 nm,其值为4.74%。热凝固导致BPH组织在600～1 064 nm光谱范围的约化散射系数明显地增大,而在590 nm处,热凝固导致BPH组织的约化散射系数却是明显地减小,自然的和热凝固的BPH组织的约化散射系数都有一个峰值在970 nm处,其值分别为1.090和1.449 mm-1,其另一个峰值都在1 050 nm处,其值分别为1.116和1.627 mm-1,自然的和热凝固的BPH组织的约化散射系数的最大差异在1 060 nm,其值为47.73%,其最小差异在600 nm,其值为4.86%。