Cell type-specific localization of sphingosine kinase 1a in human tissues.

Cell type-specific localization of sphingosine kinase 1a (SPHK1a) in tissues was analyzed with a rabbit polyclonal antibody against the 16 C-terminal amino acids derived from the recently reported mouse cDNA sequence of SPHK1a. This antibody (anti-SPHK1a antibody) can react specifically with SPHK1a of mouse, rat, and human tissues. Utilizing its crossreactivity to human SPHK1a, the cell-specific localization of SPHK1a in human tissues was histochemically examined. Strong positive staining for SPHK1a was observed in the white matter in the cerebrum and cerebellum, the red nucleus and cerebral peduncle in the midbrain, the uriniferous tubules in the kidney, the endothelial cells in vessels of various organs, and in megakaryocytes and platelets. The lining cells of sinusoids in the liver and splenic cords in the spleen showed moderate staining. Columnar epithelia in the intestine and Leydig's cells in the testis showed weak staining patterns. In addition, TPA-treated HEL cells, a human leukemia cell line, showed a megakaryocytic phenotype accompanied with increases in immunostaining of both SPHK1a and SPHK enzyme activity, suggesting that SPHK1a may be a novel marker of megakaryocytic differentiation and that this antibody is also useful for in vitro study of differentiation models.(J Histochem Cytochem 49:845-855, 2001)     

Subthalamic Nucleus Deep Brain Stimulation Modulate Catecholamine Levels with Significant Relations to Clinical Outcome after Surgery in Patients with Parkinson’s Disease

Aims

Although subthalamic nucleus deep brain stimulation (STN-DBS) is effective in patients with advanced Parkinson’s disease (PD), its physiological mechanisms remain unclear. Because STN-DBS is effective in patients with PD whose motor symptoms are dramatically alleviated by L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, the higher preoperative catecholamine levels might be related to the better clinical outcome after surgery. We aimed to examine the correlation between the preoperative catecholamine levels and postoperative clinical outcome after subthalamic nucleus deep brain stimulation. The effectiveness of STN-DBS in the patient who responded well to dopaminergic medication suggest the causal link between the dopaminergic system and STN-DBS. We also examined how catecholamine levels were modulated after subthalamic stimulation.

Methods

In total 25 patients with PD were enrolled (Mean age 66.2 ± 6.7 years, mean disease duration 11.6 ± 3.7 years). Mean levodopa equivalent doses were 1032 ± 34.6 mg before surgery. Cerebrospinal fluid and plasma catecholamine levels were measured an hour after oral administration of antiparkinsonian drugs before surgery. The mean Unified Parkinson’s Disease Rating Scale scores (UPDRS) and the Parkinson’s disease Questionnaire-39 (PDQ-39) were obtained before and after surgery. Of the 25 patients, postoperative cerebrospinal fluid and plasma were collected an hour after oral administration of antiparkinsonian drugs during on stimulation at follow up in 11 patients.

Results

Mean levodopa equivalent doses significantly decreased after surgery with improvement in motor functions and quality of life. The preoperative catecholamine levels had basically negative correlations with postoperative motor scores and quality of life, suggesting that higher preoperative catecholamine levels were related to better outcome after STN-DBS. The preoperative plasma levels of L-DOPA had significantly negative correlations with postoperative UPDRS- III score in off phase three months after STN-DBS. The preoperative cerebrospinal fluid (CSF) 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxytryptamine (5-HT) levels had significantly negative correlations with postoperative UPDRS- III score in off phase one year after STN-DBS and the preoperative CSF homovanilic acid (HVA) levels had significant negative correlations with postoperative UPDRS- III score in on phase three months after STN-DBS. In PDQ-39 SI (summary index), preoperative plasma dopamine (DA) level had significantly negative correlations with postoperative PDQ-39 SI one year after STN-DBS suggesting that higher preoperative plasma DA level resulted in better quality of life (QOL) one year after STN-DBS. The stepwise multiple linear regression study revealed that higher preoperative plasma HVA levels had negative influence on the postoperative motor symptoms (i.e., increase in the score of UPDRS), whereas higher preoperative CSF L-DOPA levels had positive influence on the postoperative motor symptoms and QOL (decrease in the score of UPDRS and PDQ-39 SI) The catecholamine levels were not significantly reduced postoperatively in 11 patients despite the significant reduction in levodopa equivalent doses. Unexpectedly, CSF HVA levels significantly increased from 0.00089±0.0003 ng/μl to 0.002±0.0008 ng/μl after STN-DBS.

Conclusion

The preoperative catecholamine levels might affect the postoperative motor symptoms and quality of life. The catecholamine levels were not significantly reduced postoperatively despite the significant reduction in levodopa equivalent doses.     

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.     

Naturally occurring antibodies in humans can neutralize a variety of influenza virus strains, including H3, H1, H2, and H5

Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the V(H)1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use V(H)1-69. The possible epitope recognized by these clones is discussed.     

Significant species-dependence of P700 redox potential as verified by spectroelectrochemistry: comparison of spinach and Theromosynechococcus elongatus

The redox potentials of P700, the primary electron donor of photosystem (PS) I, of spinach and Thermosynechococcus elongatus were determined by means of spectroelectrochemistry with an error range of +/-2-3 mV, to find that the redox potential of P700 in T. elongatus is lower by ca. 50 mV as compared with spinach. The shift in the P700 redox potential of PS I core particles prepared by harsh detergent treatments remained to within 10 mV for both organisms. These results show that the 50 mV difference in the P700 redox potential between the two organisms is not a detergent-induced artifact but reflects an intrinsic property of each PS I.     

Floral-induced and constitutive defense against florivory: a comparison of chemical traits in 12 herb species

Little is known about how plants protect flowers—their reproductive organs—against florivory. Additionally, the induced floral defense system has been examined in only a few species. We tested the inducibility of putative floral defenses and investigated the relationship between natural florivory and the floral defenses of 12 naturally growing plant species. The relationships between florivory and four chemical traits (nitrogen, phosphorus, total phenolics, and condensed tannins) were investigated in 12 plant species. We also studied whether flowers induce changes in chemical defenses in response to artificial damage in 10 plant species. A higher concentration of floral nitrogen was associated with a decreasing frequency of florivore attacks. Among the four traits of the 10 plant species studied, no trait changed in response to the artificial damage. We suggest that induced defense systems may not be advantageous for flowers, although it is also possible that these species simply do not use induced defense in any of their plant parts.