Oxygen uptake rate of immobilized growing hybridoma cells

Summary The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.     

Effects of antipyretics and anticancer drug on human cells at high temperature condition

The effects of antifebriles and anticancer drug on human vascular endothelial cells (HVE) and several cultured human cells were investigated. The HVE were isolated from umbilical cord veins by enzyme treatment and cultured successively in aerated synthetic medium, RPMI-1640, with 20% preclostrum new born calf serum. The presence of factor VIII antigen in the HVE was determined by enzyme-labeled antibody method. Cell count and protein amount were examined at regular intervals. At 3 hour-expose, sulpyrine was more toxic to the cultured cells than aspirin at 37 degrees C. The cytotoxicity of sulpyrine was markedly enhanced at 40 degrees C than at 37 degrees C. However, there was no enhancement in the cytotoxicity of aspirin at 40 degrees C. Cultured HVE and normal human fetal lung (HAIN-55) cells at 37 degrees C were sensitive to sulpyrine, and their sensitivity of the cells to the drug were markedly enhanced when they were incubated at 41 degrees C. In contrast, sensitivity of malignant human cells (HeLa cells) to sulpyrine was not found at 37 degrees C, however sensitivity of the cells to the drug was manifested at 41 degrees C of incubation. There was no effect of 5-fluorouracil (FU) on the growth of HVE and HAIN-55 cells at 41 degrees C, while HeLa cells showed high susceptibility to FU at the same temperature. The results showed the possibility that normal human cells may be sensitive to antifebrile drugs but not to anticancer drug at ordinary and high temperature, whereas malignant human cell may be susceptible to both antifebrile drugs and anticancer drug at high temperature.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Enclosure experiment on the control mechanism of planktonic bacterial standing stock

In order to understand the control mechanisms of a large, stable bacterial standing stock, enclosure experiments were conducted in a eutrophic lake, where both bacterial productivity and grazing pressure were very high. Total bacterial number in the different enclosures ranged from 1.2 to 2.7×10⁷ cells mL⁻¹ throughout the experiment. The average bacterial cell production rate estimated from a grazer eliminating experiment was 6.3×10⁵ cells mL⁻¹ h⁻¹. Difference in the bacterial cell production rate between shaded and unshaded enclosures was not apparent. Bacteria showed a reduction in standing stock of only about 25–30% even after the supply of light was cut to 1%. Bacteria in the shaded enclosures then recovered their production rate in the first 12 days of perturbation. Grazing pressure in the shaded enclosures was not less than that for the control. Thus, it was considered a control mechanism of bacterial stable standing stock that the bacteria shifted their organic substrate from extracellular dissolved organic carbon freshly released from phytoplankton to that already stocked in the water column, though it is not known whether the dominant bacteria were the same.     

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.     

Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Neural coding of speech sound in the telencephalic auditory area of the mynah bird

Summary The responses of neurons in field L in the auditory neostriatum of the mynah bird, Gracula religiosa, were recorded during presentation of intact or manipulated mimic voices. A typical mimic voice konnichiwa elicited responses in most of the neurons. Neurons in the input layer (L2) of field L showed many peaks on peristimulus time histograms while those in other layers (L1 and L3) exhibited only one or two peaks. Several neurons in L1 and L3 responded only to the affricative consonant /t/ in the intact mimic voices. They did not respond to the affricative consonant in the isolated segment or to the one in the playbacked voice in reverse. Forty-five percent of the neurons (33/ 73) decreased in firing rates at the affricative consonant in the isolated segment compared with in the intact voice. Some of these neurons, in which neither the affricative consonant in the isolated segment nor bursts of noise alone elicited responses, exhibited clear phasic responses to /t/ in the case when bursts of noise with particular central frequencies preceded the affricative consonant. The responsiveness of these neurons appears to receive temporal facilitation. These results suggest that these neurons code the temporal relationship of speech sound.Abbreviations HVc hyperstriatum ventrale, pars caudale - TFN temporally facilitated neuron - TSN temporally suppressed neuron