Plant Regeneration from Isolated Microspore of Indica Rice

Isolated microspores of rice (Oryza saliva L.) cultivars, IR36and IR43, belonging to the recalcitrant indica subspecies werecultured. Two types of microspores were observed after isolationfrom the fresh anthers and from pre-cultured anthers—onetype consisted of vacuolated, larger-sized grains, while theother was composed of microspores of smaller sizes with densecytoplasm. Within few days in culture, all the smaller sizedgrains were dead, and only the large grains were viable andproduced pollen embryos. After 30 days from culture, microcalliwere transferred to semisolid modified Murashige and Skoog mediumcontaining 1 mg/liter each of kinetin and naphthaleneaceticacid and kept under continuous light at 25?C. IR36 showed onlycell division while IR43 gave 32 green plants from these experiments. (Received January 18, 1990; Accepted July 4, 1990)     

Mother-infant interactions of wild-born,individually-caged cynomolgus monkeys (Macaca fascicularis) during the first 14 weeks of infant life

This study documents age-related changes in the interactions of wild-born cynomolgus macaque mothers and their infants living in individual cages during the first 14 weeks of infant life. Body contact between mother and infant, maternal holding, and infant sucking were found to decrease, and the mothers showed an increased frequency of aggression toward their infants with age. These results were broadly similar to those reported for mother-infant interactions in other macaques living in social groups. Nevertheless, a clear difference between the present cynomolgus macaques and other macaques in social groups was apparent. The cynomolgus macaque mothers tended to permit their infants to move about freely without displaying maternal protectiveness such as restraint or retrieval, unlike other macaque mothers in social groups. Such maternal behaviors might derive from the experience of living in individual cages for many years and the relative safety of living in individual cages. The lack of maternal restraint and retrieval could be responsible for the observed sex differences in behavior: male infants moved more actively, and broke, and made contact with their mothers more frequently than did female infants. Moreover, mothers of female infants held and groomed them more frequently and were less aggressive toward them.     

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures.

Two primary cell targets for human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4+ T lymphocytes and monocyte-derived macrophages (MDM). HIV-1 encodes envelope glycoproteins which mediate virus entry into these cells. We have utilized infected and radiolabelled primary peripheral blood mononuclear cell (PBMC) and MDM cultures to examine the biochemical and antigenic properties of the HIV-1 envelope produced in these two cell types. The gp120 produced in MDM migrates as a broad, diffuse band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels compared with that of the more homogeneous gp120 released from PBMCs. Glycosidase analyses indicated that the diffuse appearance of the MDM gp120 is due to the presence of asparagine-linked carbohydrates containing lactosaminoglycans, a modification not observed with the gp120 produced in PBMCs. Neutralization experiments, using isogeneic PBMC and MDM-derived macrophage-tropic HIV-1 isolates, indicate that 8- to 10-fold more neutralizing antibody, directed against the viral envelope, is required to block virus produced from MDM. These results demonstrate that HIV-1 released from infected PBMC and MDM cultures differs in its biochemical and antigenic properties.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Characterization of an oxygen-dependent inducible promoter system, the nar promoter, and Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to see whether the nar promoter cloned onto pBR322 can be used as an inducible promoter. To increase the expression level, the nar promoter was expressed in E. coli where active nitrate reductase cannot be expressed from the nar operon on the chromosome. A plasmid with the lacZ gene expressing beta-galactosidase instead of the structural genes of the nar operon was used to simplify an assay of induction of the nar promoter. The following effects were investigated to find optimal conditions: methods of inducing the nar promoter, optimal nitrate and molybdate concentrations maximally inducing the nar promoter, the amount of expressed beta-galactosidase, and induction ratio (specific beta-galactosidase activity after maximal induction/specific beta-galactosidase activity before induction.)The following results were obtained from the experiments: induction of the nar promoter was optimal when E. coli was grown in the presence of 1% nitrate at the beginning of culture; expression of beta-galactosidase was not affected by molybdate; the induction ratio was maximal, approximately 300, when the overnight culture was grown in the flask for 2.5 h (OD(600) is congruent to 1.3) before being transferred to the fermentor; the amount of beta-galactosidase per cell and per medium volume was maximal when E. coli was grown under aerobic conditions to OD(600) = 1.7; then the nar promoter was induced under microaerobic conditions made by lowering dissolved oxygen level (DO) to 1-2%. After approximately 6 h of induction, OD(600) became 3.2 and specific beta-galactosidase activity became 36,000 Miller units, equivalent to 35% of total cellular proteins, which was confirmed from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (c) 1996 John Wiley & Sons, Inc.     

Growth kinetics of Lactobacillus acidophilus under ohmic heating

Lactobacillus acidophilus OSU133 was inoculated into MRS broth in a fermenter vessel and incubated at 30, 35, or 45 degrees C with agitation. Incubation temperatures were attained by conventional or ohmic heating. An electrical current at low (15 V) or high (40 V) voltage was used to heat the culture directly during fermentations under ohmic heating. The growth parameters (lag period, minimum generation time, and maximum growth) and changes in pH were determined during fermentation. Metabolic activities (consumption of glucose and production of lactic acid and bacteriocin) were determined during fermentation at 35 degrees C under both heating methods. Lag period for L. acidophilus was affected appreciably by the method of heating, but the magnitude of these changes depended on the fermentation temperature. When fermentation was done at 30 degrees C, lag period decreased by 94% under low-voltage ohmic, compared with conventional, heating methods. Ohmic heating did not change the generation time significantly and caused slight, but significant (p < 0.01) decrease in maximum growth. Therefore, the electric current enhances the early stages, but it inhibits the late stages of growth. Ohmic, compared with conventional, heating resulted in higher final pH and lower bacteriocin activity in the fermented medium. However, ohmic heating at 35 degrees C had minimal effect on glucose utilization and lactic acid production by L. acidophilus. Results show that measurement of the electric current when ohmic heating is done at a constant voltage may be used in monitoring such fermentations. In conclusion, ohmic heating is potentially useful in certain applications related to fermented foods. (c) 1996 John Wiley & Sons, Inc.