Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

Construction of integrated system for selection of fused plant protoplasts

Summary An integrated system has been constructed to instantly identify and efficiently sort the heterokaryons formed by plant protoplast fusion. The system is composed of the following functions: a) a transport system, b) an electro-manipulator, c) a cell harvester, d) a flow cytometer/cell sorter, and e) a control device. The conditions for an efficient and reproducible enrichment of the heterokaryons have been investigated by this system using the fluorescein isothiocyanate stained protoplasts preparing from Glycyrrhiza glabra cell cultures and unstained protoplasts of Abrus precatorius cell cultures which contain a large quantity of chlorophyll.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2, 4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscissic acid - FITC fluorescein isothiocyanate This paper is part 96 in the series Studies on Plant Tissue Cultures. For part 95 see Orinara Y., Noguchi T. and Furuya T. (1993) submitted for publication.     

Changes in Levels of Heme a, Protoheme and Protochlorophyll(ide) in Submerged Rice Seedlings after Exposure to Air

Rice (Oryza sativa L. cv. Yamabiko) seedlings germinated underwater for 5 days contained small amounts of heme a and protohemebut no protochlorophyll(ide) [Pchl(ide)]. Levels of hemes andPchl(ide) increased rapidly upon transfer to air. When expressedin terms of fresh weight of tissue, hemes reached the levelsin aerobic controls after 24 h of contact with air, but Pchl(ide)did not. A comparison of the increases during 24-h adaptationto air in levels of heme a and Pchl(ide), which are specificto mitochondria and plastids, respectively, suggested that thedevelopment of mitochondria preceded that of plastids. The rateof synthesis of 5-aminolevulinic acid (ALA) was low in submergedseedlings, as compared to the rate in aerobic controls, butit increased during air adaptation. The sum of the amounts ofheme a, protoheme and Pchl(ide) increased in parallel with theamount of porphyrins, equivalent to the amount of ALA synthesizedduring the experimental period. When submerged seedlings thathad been pretreated with levulinic acid were exposed to air,no Pchl(ide) was formed. In contrast, Pchl(ide) accumulatedunder water when submerged seedlings were fed with ALA. Theseresults indicate that the synthesis of ALA, the limiting stepin the synthesis of Pchl(ide), is repressed under hypoxic conditions. ¹ Present address: KRI International, Inc., Kyoto Research Park17, Chudoji Minami-machi, Shimogyo-ku, Kyoto, 600 Japan. ² Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

Rapid identification of Vibrio anguillarum by Colony hybridization

A 562 base pair fragment of DNA from a serotype A strain of Vibrio anguillarum was cloned into pUC9 and used as a hybridization probe for the rapid identification of Vibrio anguillarum by colony hybridization. The probe was tested on nine different fish pathogens, 15 Vibrio isolates, 2 organisms closely related to Vibrio, and 9 serotypes of V. anguillarum. The probe hybridized only with the DNA of V. anguillarum serotypes A and H. The sequence of the 562 nucleotides have been determined. This probe allows rapid, reliable, and specific detection of V. anguillarum in freshwater ayu, Plecoglossus altivelis.     

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.     

Simple and rapid quantitative assay of C-labelled urea in human serum using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for ¹³C-labelled urea ([¹³C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [¹³C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [¹³C]urea from intrinsic urea present at high concentration in serum. In addition, a ¹³C nuclear magnetic resonance spectroscopic quantitative method for [¹³C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [¹³C]urea in human serum and urine is also presented.     

Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Halotolerant Green Alga Dunaliella tertiolecta

A Ca²⁺-dependent protein kinase (CDPK) that has been partiallypurified and characterized previously [Yuasa and Muto (1992)Arch. Biochem. Biophys. 296: 175] was further purified to about20,000-fold from the soluble fraction of Dunaliella tertiolecta.The enzyme preparation contained 60- and 52-kDa polypeptidesboth of which phosphorylated casein as a substrate. Both polypeptidesshowed a Ca²⁺-dependent increase in mobility during SDS-PAGEand ⁴⁵Ca²⁺-binding activity after SDS-PAGE and electroblottingonto a nitrocellulose membrane, suggesting that both the 60-and 52-kDa CDPKs directly bind Ca²⁺. The protein kinase inhibitors,K-252a and staurosporine, inhibited the CDPK competitively withrespect to ATP. An antibody raised against the 60-kDa CDPK crossreactedwith both the 60- and 52-kDa polypeptides. Both molecular specieswere autophosphorylated in the presence of Ca²⁺, and a highlyphosphorylated 80-kDa band appeared in addition to these phosphorylatedbands at 60 and 52 kDa in SDS-PAGE. However, the specific activityof CDPK was not changed by prior autophosphorylation when theautophosphorylated enzyme was assayed as a mixture of thesephosphorylated molecular species. Only the 60-kDa polypeptidewas immunodetected in subcellular fractions of Dunaliella cells.The 52-kDa polypeptide increased during storage of the enzyme.These results suggest that the 52-kDa polypeptide is a proteolyticartifact produced during purification. Immunoreactive bandsof 60-kDa were detected in extracts of several green algae butnot in extracts of higher plants or a brown alga. ¹This research was partly supported by Grants-in-Aid from theMinistry of Education, Science and Culture, Japan (No. 06454013and 06304023) and Research Fellowship of the Japan Society forthe Promotion of Science for Young Sciencists. ²Research Fellow (PD) of the Japan Society for the Promotionof Science.     

Effects of shear flow on photosynthesis in a dilute suspension of microalgae

This study investigates the effects of shear stress on photosynthesis in dilute suspensions of Spirulina platensis and Chlorella by measuring the oxygen production rate using a coaxial, double-rotating-cylinder apparatus that generates Couette shear flow. Our device enables up to 0.6 Pa shear stress to be applied, which has the hydrodynamic effect of generating the algal motion and acutely augmenting the oxygen production rate of Spirulina, primarily because the surface area of algae exposed to illumination is increased. However, there is shear-flow limitation on any increase in oxygen production, and the shear stress at maximum oxygen production rate tends to decrease with increasing temperature. The comparative study with Chlorella showed the reverse relationship between oxygen production and shear stress, and the cause of this difference is discussed in terms of several factors such as size, shape, hydrodynamic stress capacity and others.