Sustained transgene expression in rat kidney with naked plasmid DNA and PCR-amplified DNA fragments

Recently, we developed a kidney-targeted gene transfer technique, in which naked DNA was injected into the renal vein while the renal vein and artery were clamped. Kidney-targeted DNA transfer with only the renal vein clamped is an important modification that may permit less invasive catheter-based gene transfer in future clinical applications. The preparation of PCR-amplified DNA fragments is less time-consuming than that of naked plasmid DNA. We examined rat erythropoietin (Epo) plasmid, pCAGGS-Epo, or PCR-amplified DNA fragment, fCAGGS-Epo, transfer into the rat kidney with only the renal vein clamped. The Epo level peaked at week 3 and then was sustained for 24 weeks, which resulted in significant erythropoiesis. This modified technique, allowing long-term expression of both PCR-amplified DNA fragments and naked plasmid DNA, could potentially be used for catheter-based gene transfer in humans, and could help determine the physiological functions of putative genes.     

Amphidiniopsis uroensis sp. nov. and Amphidiniopsis pectinaria sp. nov. (Dinophyceae): Two new benthic dinoflage llates from Japan

Two new armoured, heterotrophic sand‐dwelling marine dinoflagellates, Amphidiniopsis uroensis Toriumi, Yoshimatsu et Dodge sp. nov. and Amphidiniopsis pectinaria Toriumi, Yoshimatsu et Dodge sp. nov. were collected from Japanese sandy beaches, and their morphological features observed by light microscopy and scanning electron microscopy (SEM). The cell size of A. uroensis is 28–31 μm in length and 23–28 μm in width. The plate formula is Po 3′, 3a, 6″, 3c, 4s (+1 acc.), 5″′, 2″″. The thecal surface is ornamented with small processes, pores and spines, however, the surface of plate 2a is smooth. The epitheca possesses a narrow ridge that is extended along on the suture between 1′ and 3′. Plate 1″ connects with the right sulcal (Sd) and right sulcal accessory (Sda) plates, so the cingulum is incomplete. A nucleus is situated in the central part of the cell. There are a few small spines at the antapex. There are no stigma or chloroplasts. Amphidiniopsis pectinaria cells are 33–40 urn in length and 29–35 μm in width. The plate formula is Po 4′, 3a, 7″, 3c, 4s (+1 acc.), 5″′, 2″″. Plate 1″ connects directly with Sd and Sda plates, so the cingulum is incomplete. The thecal surface is ornamented with small processes, spines and pores. The epitheca is provided with a narrow ridge that is extended along on the suture between plates 1′, 4′ and 7″. The ornamentation on the antapical plates is unique. It is arranged in 10 straight rows on the hypotheca; each row has a strong spine at its posterior end. In addition, there is a long spine at the antapex. There are no stigma or chloroplasts. A nucleus is located in the central part of the cell.     

Analysis of mating type determination by transplantation of O macronuclear karyoplasm in Paramecium tetraurelia

The odd (O) or even (E) mating type in Paramecium tetraurelia is determined during the first cell cycle after new macronuclear development. The present paper demonstrates that mating type E is irreversibly determined at the end of the first cell cycle. Direct evidence comes from transplanting O macronuclear karyoplasm containing O-determining factor into E autogamous cells during a new postzygotic macronuclear development. Transplantation of O macronuclear karyoplasm into E autogamous cells at 7–8 hr after the origin of the macronucleus from a product of the synkaryon produces nearly 100% O mating type among the exautogamous cell lines but almost none 10–11 hr after the origin of the macronucleus (around the end of the first cell cycle). The macronuclear anlagen at the stage at which mating type E seems to be fixed contains about 20 times as much DNA as the vegetative G1 micronucleus. The O-determining factor shifting E cells toward O mating type by transplanting O macronuclear karyoplasm is also produced by the newly developed macronucleus in an effective concentration at 10–11 hr after the sensitive period and produced at full levels by the third cell cycle. The level of O factor in the macronucleus then gradually declines with subsequent repeated rounds of DNA synthesis and is finally lost by the eighth cell cycle.     

Free sialooligosaccharides found in the unfertilized eggs of a freshwater trout, Plecoglossus altivelis. A large storage pool of complex-type bi-, tri-, and tetraantennary sialooligosaccharides

Unfertilized eggs of Plecoglossus altivelis (a kind of freshwater trout, "Ayu" in Japanese) were found to contain a relatively large pool of three sialooligosaccharides. These free oligosaccharides were accumulated in the cytoplasm up to concentrations of about 35 ng per egg, which correspond to about 107 micrograms/g. Their structures were determined by a combination of chemical (composition and methylation analysis and Smith degradation) and instrumental (fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and 400-MHz1H NMR) analyses. The structures established represent a typical type of bi-, tri-, and tetraantennary sialooligosaccharides all of which end with di-N-acetylchitobiose structure (-GlcNAc beta 1-4GlcNAc) and alpha -2,3-linked NeuAc at reducing and nonreducing termini, respectively. These data show that these free sialooligosaccharides, which were originally protein-linked components, must be released during oogenesis. Although a specific functional requirement for their release is not known, the occurrence of such free sialooligosaccharides in normal animal cells or tissues has not been previously reported.     

Different metabolic fate of two carbons of glycolate in its conversion to serine in Euglena gracilis z

In our preceding work (A. Yokota, Y. Nakano, and S. Kitaoka, 1978, Agric. Biol. Chem. 42, 121-129), extensive decarboxylation of glycolate carboxyl carbon during its metabolism in Euglena gracilis suggested occurrence of a metabolic pathway of glycolate different from that of higher C3 plants. In the present report, we establish the Euglena glycolate pathway from characteristics of the decarboxylation of the carboxyl carbon and from the metabolic fate of hydroxymethyl carbon of glycolate. The ratio of the decarboxylation of the carboxyl carbon of glycolate to the total metabolized carbon increased with increasing metabolic rate in an asymptotic fashion. Thus, the ratio was 20% at the metabolic rate of 0.05 nmol of glycolate/10(6) cells/min, but it was over 60% at the rate of more than 0.35 nmol/10(6) cells/min after 2 min of incubation. Metabolic products were also changed depending on the rate of metabolism of glycolate; glycine was the main product at the low rate of glycolate metabolism and the contribution of glycine was reversed by the increased contribution of evolved CO2 at the high rates. At the metabolic rate of 1.5 nmol of glycolate/10(6) cells/min, the rate of the decarboxylation was 1.0 nmol of CO2/10(6) cells/min, which could not be explained by the extremely low activity of glycine synthase in Euglena. Experiments with [2-14C]glycolate showed that exogenously added formate and methionine caused accumulation of radioactive formate. Based on these results, we have proposed that the glycolate metabolism of E. gracilis consists of glycine and formate pathways and that the relative contribution of both pathways to the glycolate metabolism depends on the metabolic rate of glycolate.     

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca²⁺/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused Ca²⁺ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca²⁺-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca²⁺ challenge-induced injury in a dose-dependent manner (10⁻¹⁰–10⁻⁸ M ). Cyclosporin A at 1 µ M mimicked the effect of FK506. Rapamycin (1 µ M ) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 n M ), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca²⁺ challenge-induced increase in cytosolic Ca²⁺ level nor Na⁺-Ca²⁺ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca²⁺ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.     

Biosynthesis of polymyxin E III. Total synthesis of polymyxin E by a cell-free enzyme system

Polymyxin E, an antimicrobial branched cyclic decapeptide, was synthesized by an enzyme fraction partially purified from crude extracts of the producing organism, $\text{Aerobacillus}\text{polyaerogenes}$. For the synthesis, three constituent amino acids (L-2,4-diaminobutyric acid, L-leucine, and L-threonine), ATP, Mg²⁺ and an acylating system consisting of octanoyl CoA and an ammonium sulfate fraction of cell extracts are required.     

In vitro stimulation by retinol of porcine pancreatic esterase activity toward esters of short-chain fatty acids.

1. Retinol exerted a remarkable stimulating effect (approx. 260% increase), essentially similar to that (300%) of phytol, on the so-called esterase activity displayed by crude pancreatic lipase [EC 3.1.1.3] toward true solutions of esters, but none of the typical lipase activity toward emulsions of water-insoluble esters. 2. Comparison of the stimulatory effects of retinol derivatives on the esterase activity revealed that retinyl acetate was the most active, being sustantially similar in effect to retinol; retinal was fairly active, while retinoic acid, retinyl palmitate, and beta-ionone were far less active. 3. With various isoprenoid compounds, the efficiency of stimulation increased with the carbon chain length, attaining a maximum at 15 to 20 carbon atoms. Above this chain length the efficiency decreased rapidly. 4. Comparison of the effects of retinol and phytol on the esterase activity of various other lipolytic enzymes indicated that this kind of activator may be relatively specific to porcine pancreatic esterase activity.