High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixure of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10⁴ and 10⁵. HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Siblings with renal tubular acidosis and nerve deafness. The first family in Japan

Summary Two siblings with renal tubular acidosis (RTA) and nerve deafness were examined. It was found by ammonium chloride and bicarbonate loading tests that the 6-year-old brother had a hybrid type of RTA and his 4-year-old sister, a distal type of RTA. Enzyme activity and amount of enzyme protein of carbonic anhydrase isoenzyme I and II in red blood cells, measured using an immunoadsorbent method, were normal in both cases. Although this indicated that the RTAs of these patients are not generated by the carbonic anhydrase deficiency, an investigation with renal tissue is necessary to arrive at a final conclusion.     

Light and electron microscopy of the ducts and their subepithelial tissue in the rat ventral prostate

Summary The ducts of the rat ventral prostate have been studied by light and electron microscopy for elucidation of their role in prostatic function. The epithelium of the main duct consists of simple columnar cells and polymorphic basal cells. The columnar cells show no indication of secretory activity. The basal cells contain bundles of filaments of 5–6 nm thickness and numerous pinocytotic vesicles. The ducts are surrounded by layers of circular smooth muscle cells interspersed with nerve axons. On ultrastructural grounds the ducts do not appear to secrete material into the seminal fluid, but apparently the muscular coat actively helps drain the gland during ejaculation.     

Antitumor activity of Nocardia cell wall skeleton preparations in transplantable tumors in syngeneic mice and patients with malignant pleurisy

Summary The antitumor activity of the cell wall skeleton preparations of four species of Nocardia, N. brasiliensis strain 146, N. coeliaca strain 122, N. polychromogenes strain 6, and N. rubra, which showed potent adjuvant activity on the induction of cell-mediated cytotoxicity in allogeneic mice, was examined with the aid of EL-4 leukemia, melanoma B16, and MH-134 hepatoma in syngeneic mice. Preliminary clinical trials were performed and the results suggest that the cell wall skeleton of N. rubra, upon intrapleural injection, may be useful as an immunotherapeutic agent for patients with malignant pleurisy. The chemical properties of these cell wall skeleton preparations are described.     

Studies on delayed hypersensitivity to protein antigen. Induction of delayed hypersensitivity by chemically modified antigen.

it was shown in our previous paper that mice primed with chemically modified bacterial alpha-amylase (BaA), which was neither cross-reactive with anti-BaA antibody nor able to induce a humoral anti-BaA response, developed enhanced responses to a subsequent challenge with native BaA and that the magnitude of the immunological memory was closely related to the priming dose of modified BaA. This paper describes the experimental conditions for induction of delayed hypersensitivity (DH) by modified BaA in relation to the development of immunological memory for antibody response to native BaA. Mice primed with either an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of modified BaA in complete Freunds adjuvant (CFA) developed enhanced anti-BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to a subsequent challenge with BaA. In contrast, when mice were immunized with an s.c. injection of the modified BaA only, a significant level of DH to native BaA could be induced, as measured by the footpad reaction (FPR). The highest degree of DH was observed in mice given 50 micrograms of modified BaA. DH was detectable within 5 days and persisted for 25 days after immunization. In the reciprocal combination of native BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to DH was examined. The primary anti-BaA responses induced by an i.p. injection of large doses of BaA was markedly higher than those induced by an s.c. injection, while DH was exhibited only in mice given s.c. injection of BaA in CFA. With respect to DH to native BaA induced by the modified BaA, it was shown that C3H/He mice were high and C57BL/6 mice were low responders.     

Role of luteinizing hormone-releasing factor (LH-RF) and LH on maintenance of early gestation in rats.

The role of luteinizing hormone (LH) and LH-releasing hormone (LH-RH) in the maintenance of early pregnancy in rats was studied. Serum levels of progesterone (P) and LH were measured daily in untreated pregnant rats from Day 4 through parturition. Serum levels of P and LH were determined on Days 11 and 15 of pregnancy in animals treated with antisera to LH (LH-A/S) and to LH-RH (LH-RH-A/S) on Days 8-10. Serum levels of P peaked on Days 7 and 16 in untreated animals, after which they declined sharply just before delivery. Serum LH fluctuated between 30-160 ng/ml during pregnancy but did not exhibit any distinctive peaks. Treatment with .2 ml LH-A/S on Days 8-10 reduced serum P to virtually undetectable levels on Day 11, and only a slight recovery was evident on Day 15. Lower doses of LH-A/S had no effect. Administration of 1.3 ml LH-RH-A/S had no effect on serum levels of P or LH, and did not impede fetal development. The results indicate that LH is essential to the luteotropic complex of early pregnancy in the rat, and also that LH-RH-A/S can maintain to some extent basal levels of P and LH during early pregnancy.     

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10⁴). PII-D (mol. wt 2 × 10⁵) and PII-T (mol. wt 8 × 10⁵) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K_m's and relative values of V_m's for several substrates, suggesting that they had the same common active site.PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction.Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V_m's.A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.     

G6PD Ube, a glucose-6-phosphate dehydrogenase variant found in four unrelated Japanese families.

A total of 6,120 Japanese males were screened for glucose-6-phosphate dehydrogenase deficiency (G6PD). Five cases with the deficiency were discovered. Two of them and an additional two cases have the same variant, G6PD Ube, characterized by moderate enzyme deficiency, fast moving enzyme activity on electrophoresis, high Ki Nadph, utilization of substrate analogues, kinetics, pH optima, and stability. This variant was distinguished for G6PD A- and from other Oriental variants by biochemical parameters. Differences in the frequency and type of the variants between southern Asia and Japan, suggest that the Japanese who have been isolated on islands where malaria is not endemic, may have developed their own variant traits.