[Transcriptome profiling of differentiated lenses through RNA sequencing].

Objective: To gain insight into the transcriptional landscape including mRNA, long non-coding RNA (lncRNA), and circular RNA (circRNA) of the differentiated lens. Methods: Experiment research. The total RNAs of the differentiated lenses were extracted and purified. Total RNAs of 16-week, 23-week, and 25-week differentiated lenses were then sequenced using Illumina HiSeq 2500, and analyzed using bioinformatics tools. The top expressed and differentially expressed mRNAs and lncRNAs were screened. The expressions of overlap genes among the 16-week, 23-week, and 25-week lenses were analyzed by Venn diagram. The expression tendency of lens-specific genes was obtained and verified with real-time polymerase chain reaction. Results: A total of 67 518 311 mapped reads were obtained from differentiated lenses at 16 weeks, 99 440 160 at 23 weeks, and 67 262 320 at 25 weeks. The gene overlap expression analysis showed 740 of the top 1 000 highly expressed mRNAs, 170 of the top 300 highly expressed lncRNAs, and 69 of the top 100 highly expressed circRNAs overlapping expressed in lenses at 16, 23, and 25 weeks, respectively. Lens specific gene expression analysis revealed that the expression of crystallin (CRY) AA, CRYGA, CRYGB, CRYGC, CRYGD, CRYGEP, and CRYGS was upregulated, while the expression of gap junction (GJ) A3 and GJA8 was downregulated with the differentiation of lenses. Conclusion: The lens transcriptome profile shows that more than half of the high expressed mRNA, lncRNA and circRNA at different differentiation stages are overlapping expressed, and all of them have high expression of lens specific protein genes, such as CRY, GJ etc. (Chin J Ophthalmol, 2020, 56: 356-363).

First Report of Bacterial Head Rot of Broccoli Caused by Pseudomonas fluorescens in China.

During warm and humid periods in the winters from 2005 to 2008, head rot symptoms on broccoli (cv. Sijilv) (Brassica oleracea L. var italica Planch) were observed in commercial fields in Ningbo, Zhejiang Province, China. In agreement with the report of Cui and Harling (1), water-soaked lesions developed on the buds and then progressed into a brown-black soft rot. Longitudinal sections of the symptomatic inflorescences showed brown discoloration and rotting of the internal tissues. Broccoli production is hampered by the disease, with disease incidence ranging from 65 to 81%. Bacteria were isolated by streaking on nutrient agar (3) and individual colonies formed after 2 to 3 days of incubation at 28°C. Fifteen of thirty isolates induced hypersensitive reactions (HR) on tobacco leaves (Nicotiana tabacum cv. Samsun) within 48 h. All the HR-positive strains were fluorescent on King's medium B and the colonies were smooth, convex, entire, and round. Classical bacteriological tests indicated that the fluorescent strains were gram negative, obligate aerobes, arginine dihydrolase positive, and oxidase positive. Also, the fluorescent strains were positive for the production of levan from sucrose. Five representative strains were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc., Newark, DE) with the aerobic bacterial library (TSBA50). The five strains were identified as Pseudomonas fluorescens with Biolog and FAME similarity indexes of 0.61 to 0.68 and 0.52 to 0.58, respectively. The 16S rRNA gene sequence of broccoli strain PFB-01 (GenBank Accession No. GQ352649) was determined according to Li et al. (2). A subsequent GenBank search showed that this sequence had 98% nucleotide identity with the type strain of P. fluorescens (ATCC 17386T, GenBank Accession No. AF094726). Koch's postulates were completed by the inoculation of broccoli heads (cv. Sijilv) with cell suspensions (107 CFU/ml) of the above five strains by spraying on the surface of subcorymbs. Each treatment had five replicates. All strains induced head rot symptoms similar to those observed in natural infections. No symptoms were noted on the control plants inoculated with sterile water. Bacteria were successfully reisolated from symptomatic heads and confirmed by the cellular fatty acid composition. To our knowledge, this is the first report in China that P. fluorescens is the causal pathogen of bacterial head rot of broccoli. References: (1) X. Cui and R. Harling. Phytopathology 96:408, 2006. (2) B. Li et al. J. Phytopathol. 154:711, 2006. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

Isoquinoline alkaloids from Thalictrum delavayi.

Two new protoberberine alkaloids, 2,3,9,10-dimethylenedioxy-8-oxoprotoberberine (1) and 2,3,9,10-dimethylenedioxy-1,8-dihydroxyprotoberberine (2), together with nine known isoquinoline-type alkaloids were isolated from the roots of Thalictrum delavayi. Their structures were elucidated by spectral methods. Among these compounds, pseudoprotopine showed competitive inhibition activity by DA receptor binding assay (D1) in vitro. The competitive inhibitions were 87.5% (10(-4) M) and 15.6% (10(-6) M), respectively.

Synthesis and antitumour activity of novel diterpenequinone salvicine and the analogs.

A novel diterpenequinone named salvicine (4), structurally modified derivative of a natural product, and a series of the novel analogs have been prepared. Most of the analogs were found to be potently active against tumor cell lines in vitro. Further study on 4 in vivo demonstrated that it possessed a significant antineoplastic activity against murine S-180 Sarcoma and Lewis lung cancer, and human lung adenocarcinoma xenografts A-549 and LAX-83. The preclinical studies of 4 are now under way.

Inhibition of p56(lck) tyrosine kinase by isothiazolones.

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.

Hemiparkinsonism in monkeys following unilateral common carotid artery infusion of MPTP. A study of behavior, biochemistry and histology.

After local surgical exposure, we administrated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) directly into the right common carotid artery of 5 rhesus monkeys. All the monkeys manifested akinesia, rigidity and postural tremor of the contralateral limbs, and spontaneous circling toward the MPTP treated side. These disturbances began to appear 3-4 days after injection, peaking at one month, and continued until the day of sacrifice. After treatment with madopar and apomorphine, marked improvements of the motor impairments appeared and a striking reversal of the direction of rotation away from the MPTP-treated side occurred in a dose-dependent manner. The ipsilateral neurotoxicity was confirmed biochemically by 99% reduction in the caudate-putamen dopamine levels and histologically by selective cell loss in the substantia nigra of the MPTP-treated side. It is concluded that this primate model of hemiparkinsonism is easy to reproduce and life is maintained with good health otherwise. So it may be more feasible for behavioral and pharmacological studies of Parkinson's disease.

[Changes in monoamine neurotransmitters in CSF during inhibition of defensive hypertensive reactions by inputs of deep peroneal nerve].

Our previous work has indicated that excitation of hypothalamic defence area (HDA) could lead to an increase in the release of central monoamine neurotransmitters, pressor and other defence responses. The present experiments showed that stimulation of deep peroneal nerve with a current of low frequency and low intensity could inhibit the pressor, abolish the increase of noradrenaline and its metabolite MHPG and attenuate that of 5-HIAA (a 5-HT metabolite), dopamine and its metabolites DOPAC and HVA in CSF. The data suggest that inputs of deep peroneal nerve may inhibit the increased release of central monoamine neurotransmitters, and then defence reactions; and inhibition of NA release in some nuclei of the brain is an important mechanism of inhibition of defence pressor by inputs of deep peroneal nerve.

Partial protection from the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by Pro-Leu-Gly-NH2(PLG; MIF-1).

The administration of MPTP to man and monkey has been shown to cause a neurotoxic effect on the nigrostriatal dopamine system. MPTP was injected in C57-BL black mice, 36 mg per kg for 7 days, which resulted in permanent reduction of dopamine and serotonin levels in the striatum. In the mice pretreated with PLG, although the striatal dopamine level was also reduced, mean dopamine and serotonin levels were significantly higher than in mice given MPTP alone. It is concluded that PLG could protect at least partially the neurotoxic effect of MPTP.

Determination of serum catecholamine metabolites in neuroblastoma by high performance liquid chromatography with electrochemical detection.

A method for determining serum catecholamine metabolites such as vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenyl glycol (MHPG) and homovanillic acid (HVA) in neuroblastoma by using high performance liquid chromatography and electrochemical detector is described. The separation of catecholamine metabolites was performed on a reverse phase column with an eluting system containing citric acid-potassium hydrogen phosphate buffer and methanol as the organic modifier. The experimental results showed that VMA and HVA levels in the serum of neuroblastoma patients were 15-30 times higher than that of the normal control group. The same phenomenon also occurred in patients with stage II neuroblastoma. Serum VMA, MHPG and HVA levels reduced to normal in patients suffering from neuroblastoma after surgery. Serum catecholamine metabolites analysed by using HPLC/ECD is more simple, sensitive and reliable than that by usual urine assay and might be used for the diagnosis of neuroblastoma even in early stage.