Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Application of 39K NMR Spectroscopy to Potassium Transport in Mung Bean Root Tips

The intracellular K⁺ concentration and its change in mung bean[Vigna mungo (L.) Hepper] root tips were investigated non-invasivelywith ³⁹K nuclear magnetic resonance spectroscopy using a membraneimpermeable shift reagent, dysprosium (III) tripolyphosphate[Dy(PPP_i)^7–₂]. The K⁺ resonance was shifted to highermagnetic field in proportion to the concentration of the shiftreagent. In addition to a reference capillary peak for measuringthe K⁺ concentration, two well-resolved peaks (intra- and extracellularK⁺ resonances) were observed in the 39K NMR spectra of mungbean root tips. The intracellular K⁺ concentration was determinedto be 41 mM, which was similar to the value obtained by flamephotometry. When 20 mM KCl was added to the external medium,the intensity of the intracellular K⁺ resonance gradually increasedand the net K⁺ uptake rate was calculated to be 4.1 micromolesper gram fresh weight per hour. After removal of KCl from theperfusion medium, the intracellular K⁺ concentration considerablydecreased. With ³¹P NMR method, 2.5 mM Dy(PPP_j)^7–1₂ and20 mM KCl had little effect on the ATP level in the cells. Wehave indicated that the ³⁹K NMR method can be used to determinethe K⁺ levels and net fluxes of the K⁺ transport in perfusedroot tips successively. (Received April 6, 1988; Accepted September 29, 1988)     

Expression of intermediate filaments and other special markers by testicular germ cell tumors. With reference to embryogenesis.

Distribution of intermediate filament proteins (IFs) and several special markers was studied in 39 testicular germ cell tumors and 8 embryos and foetuses. The similarity and difference between development of germ cell tumor and embryogenesis were immunohistochemically investigated. Seminoma and embryonal carcinoma, as tumoral counterparts of undifferentiated germ cells, were characterized by little IF expression. This study revealed that the maturing and differentiating process in germ cell tumor is different from normal embryonal development and the tumor cells showed leaping maturing steps in tumorigenesis. Immunostaining for IFs helped to discover the further differentiation occurring in embryonal carcinoma and to demonstrate heterogeneous elements in non-seminoma germ cell tumors, which sometimes might not be apparent by light microscopical observation of H&E staining section. According to the findings, two patterns in mixed germ cell tumors are suggested; i.e., combined and diffuse types. The mechanism of tumorigenesis of the two types is supposed to be different. Clinically, the prognosis of most patients with testicular germ cell tumor is fairly good because of the improved chemotherapies that are dependent on histological diagnosis.     

Significant antitumor effect of a synthetic lipid A analogue,DT-5461, on murine syngeneic tumor models

Summary The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

Testicular development in Chinese Meishan boars

The developmental process of the testis and age-related changes in the morphology of rete testicular spermatozoa were investigated in Meishan boars at 1 to 364 days of age. Testicular weight and the diameter of seminiferous tubules increased rapidly until 150 to 180 days of age. Leptotene stage spermatocytes, round spermatids and spermatozoa were first found in the section of seminiferous tubules at 30 to 45, 60 and 75 days of age, respectively. However, after 105 to 120 days of age, most rete testicular spermatozoa were morphologically normal. These results indicate that Meishan boars reach puberty as early as 75 days of age, though the testes acquire the ability to produce morphologically normal spermatozoa at about 120 days.     

Synthesis of polyamide supports for use in peptide synthesis and as peptide-resin conjugates for antibody production.

We have synthesized beaded, hydrophilic cross-linked, aminoalkyl polydimethylacrylamide supports upon which peptides have been assembled using standard Boc or Fmoc chemistry in automated equipment. The resins were prepared by the free radical-initiated co-polymerization of N,N-dimethylacrylamide, N,N'-bisacrylyl-1,3-diaminopropane, and a functional monomer which were contained in a reverse-phase, detergent-emulsified suspension. The functional monomers used were N-(2-(methylsulfonyl)ethyloxycarbonyl)-allyl-amine (MSC-allylamine), N-acrylyl-1,6-diaminohexane hydrochloride or N-methacrylyl-1,3-diamino-propane hydrochloride. The MSC protecting group was removed by treatment of the resin with methanolic base during workup. After coupling of N-alpha-t-butyloxycarbonyl-alanine (Boc-alanine), amino acid analyses gave resin loading capacities between 0.15 mmol/g and 1.4 mmol/g, depending on the concentration and composition of the functional monomer. The resulting polymers were highly swollen by polar solvents including aqueous buffers. Peptides were synthesized on these supports after attaching the first amino acid directly or through a cleavable ester linker. When the carboxyl-terminal amino acid was coupled as the 4-oxymethylbenzoic acid derivative, the peptide could be deprotected and remain attached to the hydrophilic polymer since the peptide-benzyl ester bond was stable to HF deprotection at 0 degrees in the presence of 10% anisole and 1% ethanedithiol. The resulting peptidyl-resin could be swollen in aqueous buffers and injected into animals for the production of antibodies.     

Cervicovaginal and endometrial cytology in ovarian cancer

The clinical significance of cytologic examination was studied in 114 patients with ovarian cancer who had received preoperative cytologic examinations. The overall positive rate of the cytologic examinations was 26.3% (30 of 114): 22 (19.3%) of the 114 cases had positive cervicovaginal smears while 13 of 31 endometrial aspiration smears (41.9%) were positive. The positive rate was not related to the volume of ascites but rather to its presence or absence. Thus, if ascites was observed, the positive rate was about 2.1 times higher than if it was absent. In two of four cases of ovarian cancer with no endometrial invasion but a positive cytologic examination of ascitic fluid, fallopian tube specimens contained cancer cells; this suggests that ovarian cancer cells may reach the cervix and/or vagina by passing through the fallopian tube, particularly if ascites is present. Since cytologic examination, especially of endometrial aspiration smears, shows a high positive rate if ovarian cancer cells are observed in the abdominal cavity, cytology should be used as an important ancillary method for the assessment of ovarian cancer.