Aging of chick embryo fibroblasts in vitro : III. Polyploid cell accumulation

The accumulation of polyploid cells during the lifespan of chick embryo fibroblasts was examined. The nuclear area of each stained nucleus in the chick cells is virtually proportional to its relative DNA content. Changes in mean nuclear area of the cells with advancing aging were observed. The mean nuclear area increases at the latest culture stage when growth rate notably declines, and their increase follows an increase in multinuclear cells. In the senescent cells, there are the ploidy classes of 2C, 4C, 8C, 16C, 32C and 64C, being defined as 2ⁿC. The mechanism of age-dependent polyploidization is also discussed.     

Control of renal bicarbonate transport

Single rabbit renal tubules were perfused in vitro to elucidate the factors that control bicarbonate transport. One factor studied was the preexisting acid-base status of the rabbits. Cortical collecting ducts from acidotic rabbits (given ammonium chloride) transported bicarbonate from lumen to bath. Collecting ducts from alkalotic rabbits (given sodium bicarbonate) transported bicarbonate in the opposite direction. Thus, bicarbonate transport by collecting ducts in vitro was conditioned by the preexisting state of the rabbit in vivo. In contrast, bicarbonate transport by proximal straight tubules and cortical thick ascending limbs was not affected by ammonium chloride or sodium bicarbonate given to the rabbits. Parathyroid hormone, the second factor studied, strongly inhibited bicarbonate absorption by proximal straight tubules.     

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast,Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Peroxisomal NADP-linked isocitrate dehydrogenase (Ps-NADP-IDH) was purified for the first time from Candida tropicalis cells grown on n-alkane as a carbon source, which was effective in proliferation of peroxisomes. The properties of Ps-NADP-IDH were compared with those of mitochondrial NAD-linked isocitrate dehydrogenase (Mt-NAD-IDH) purified from the cells grown on acetate, in which peroxisomes did not proliferate. Ps-NADP-IDH was a homodimer of identical subunits (45 kDa), while Mt-NAD-IDH was suggested to be a heterooctamer composed of two types of subunits with different molecular masses (41 and 38 kDa). Kinetic studies revealed that Ps-NADP-IDH gave Michaelis-Menten saturation curves against isocitrate and NADP concentrations, whereas Mt-NAD-IDH was an allosteric enzyme regulated by ATP, AMP, and citrate. Inhibition by 2-oxoglutarate, a precursor of glutamate, was observed only for Ps-NADP-IDH. Both enzymes were inhibited by concomitant addition of oxalacetate and glyoxylate. The function of Ps-NADP-IDH seems to be completely discriminated from that of Mt-NAD-IDH as reflected by their distinct subcellular localizations. Furthermore, the properties of Ps-NADP-IDH were also compared with those of other mitochondrial and cytosolic IDHs from sources reported previously.     

Inhibitio of Na transport by prostacyclin (PGI2) in rabbit cortical collecting tubule

The effects of prostacyclin (PGI₂) on transepithelial potential difference (PD) and sodium transport were examined in rabbit cortical collecting tubules (CCT) perfused in vitro. Addition of PGI₂ (10^?6M) to the bathing medium, which was bubbled with 95% O₂ – 5% CO₂, caused a reversible decrease in PD averaging 49±9.4 (SE)%. Maximal effect was evident between 5–10 min. After addition of PGI₂ and PD returned spontaneously towards control values within 30 min., corresponding to the rapid degradation of PGI₂. In a more alkaline bathing solution achieved by bubbling with 100% O₂, in which the degradation of PGI₂ is known to be delayed markedly, the decrease in PD by PGI₂ was continuous and dose-dependent, with half-maximal and maximal effects achieved at 10^?7 M and 10^?5 M, respectively. Neither 10^?8 M PGI₂ nor vehicle alone exerted significant effects on PD. 6-keto-PGF_1α (10^?5M), believed to be the major metabolite of PGI₂, had no effect on PD. Lumen-to-bath flux of Na decreased with PGI₂ from 9.0 to 5.6 pEq/cm/sec (n=4, p<0.005), although bath-to-lumen flux did not change significantly. In summary, PGI₂ caused a dose-dependent decrease in PD of rabbit CCT and inhibited Na absorption in this segment in vitro. These results suggest that PGI₂ may play an important role in regulating Na transport in CCT.     

Establishment of cell surface engineering and its development

Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique.     

Application of colour laser microscope for observing living biological specimens

Pollen-mother cells from Lilium longiflorum, human metaphase chromosomes and human spermatozoa, were observed under a colour laser microscope. Helical mitochondrial strands were clearly evident in the middle piece of human spermatozoa. The technique enables living specimens to be conveniently and effectively examined.     

Comparison of two forms of catalytic antibody displayed on yeast-cell surface

Two forms of the Fab fragment of the catalytic antibody 6D9 were individually displayed on yeast-cell surface in fusion to the C-terminal half of -agglutinin: one was 6D9 Fab1, in which the light chain of the Fab (Lc) fragment is displayed on cell surface and the heavy chain of the Fab (Fd) fragment is secreted and linked to the Lc fragment with a disulfide bond; the other was 6D9 Fab2, in which the Fd fragment is displayed on cell surface and the Lc fragment is secreted and linked to the Fd fragment with a disulfide bond. Analysis by flow cytometry indicated that some 6D9 Fab2 fragments were unable to construct an appropriate conformation, and that most of the 6D9 Fab1 fragments displayed on yeast-cell surface exhibited higher binding affinity, stability, and catalytic activity. Conformation of the surface-displayed hetero-dimeric Fab fragment mainly depended on the intermolecular disulfide bond between the Lc and Fd fragments. The conformation of 6D9 Fab1 was more stable than that of Fab2. In the reducing environment of solution containing 25 nM DTT, the function of 6D9 Fab2 was almost completely lost. The successful display of 6D9 Fab1 on yeast-cell surface provides a novel approach to the engineering of catalytic antibodies.     

The E. coli K-12 chromosome flanked by two IS10 sequences transposes

Summary Transposon are commonly found among prokaryotes and usually range up to 20 kilobases. In this study, we were interested to determine whether a larger DNA segment could transpose. We observed that the E. coli K-12 chromosome, 4,000 kilobases in size, when flanked by two IS10 sequences, could transpose to pACYC177 at a frequency of 10^-8 per cell per generation. We suggest that this transposition event occurs independently of the size and without duplication of the entire DNA sequence flanked by the IS10 elements.     

Effects of Hydrostatic Pressure on Photosynthetic Systems. I. Preferential Destruction of the O2-Evolving Center

The effects of hydrostatic pressure treatments on the photosyntheticactivities of isolated thylakoids and PSII membranes were studied,and the following results were obtained. (1) The O₂-evolvingactivity was selectively inhibited by treatment at 200 MPa andabove, while the electron transport in the PSII reaction center,as measured by the photoreduction of 2,6-dichlorophenolindophenol(DCIP) with 1,5-diphenylcarbazide (DPC), was markedly enhanced.(2) The activity of PSI, as measured by the photoreduction ofmethylviologen with reduced DCIP, was not much affected evenafter treatment at 500 MPa, whereas this electron transportbecame uncoupled from phosphorylation at 200 MPa and above.(3) In pressure-treated PSII membranes, the EPR signal of Y⁺_zbecame photoinducible with the concomitant appearance of anEPR signal for Mn²⁺. (4) The 33-kDa extrinsic protein was retainedin inhibited PSII membranes, but the 17- and 23-kDa extrinsicproteins were lost. (5) Cross-linking of PSII proteins by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) suppressed the pressure-induced inactivation of the evolutionof O₂. (6) In pressure-treated PSII membranes, higher concentrationsof DCMU were required to inhibit the photoreduction of DCIP.Features of the pressure-induced inhibition of various reactionsin photosynthetic membranes are discussed on the basis of theseresults. ¹On leave from Advanced Research Laboratory, Hitachi Ltd., Hatoyama,Saitama, 350-03 Japan.