Crosslinking of membranes: the effect of dimethylsuberimidate, a bifunctional alkylating agent, on mitochondrial electron transport and ATPase.

Isolated mitochondrial inner membranes were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effect on electron transport and ATPase activity was determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that treatment resulted in the polymerization of membrane polypeptides. Electron transport and ATPase activity was strongly inhibited by this reagent. However, at comparable levels of amidination, a monofunctional imidate, ethylacetimidate, was less inhibitory than the bifunctional reagent. Furthermore, dimethylsuberate, a hydrolysis product of dimethylsuberimidate, caused minimal inhibition of activity. Dimethylsuberimidate treatment resulted in a decrease in the apparent K_m of the ATPase, while the monoimidate did not alter the K_m. The results obtained suggest that inhibition by dimethylsuberimidate may be due, in part, to the molecular crosslinking of inner membrane components.     

Multiple sites of vanadate and peroxovanadate action in Xenopus oocytes

In Xenopus laevis oocytes, the insulin mimics, vanadate and peroxovandates (PV), stimulated the uptake of ³H-2-deoxyglucose and incorporation of ³⁵S-methionine into protein. For both hexose transport and protein synthesis, peroxovandates (produced by reacting vandate and H₂O₂) were at least as potent as vandate. Microinjection of peroxovandates into the oocytes stimulated 2-deoxyglucose uptake. However, methionine incorporation was not stimulated by microinjection of peroxovanadate or vanadate solutions. Consistent with these results and with the possibility that vandate and peroxovandates enter the cell on a phosphate transporter, raising the medium phosphate concentration from 1 mM to 10 mM blocked vanadate-stimulated hexose transport and partially reduced peroxovanadates stimulation of hexose transport. Increased medium phosphate did not reduce stimulation of protein synthesis by either effector. Taken together, these data indicate that vanadate/peroxovanadates act at both intracellular and extracellular sites. Action at the former stimulates hexose uptake and action at the latter, protein synthesis. © 1995 Wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

What's new: To boldly glow…. Applications of laser scanning confocal microscopy in developmental biology

The laser scanning confocal microscope (LSCM)

1 LSCM: laser scanning confocal microscope; FISH: fluorescence in situ hybridisation; DiO₆: 3,3′-dihexyloxacarbocyanine iodide; NBD-ceramide: 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-caproyl)sphingosine; DiO: 3,3′-dioctadecyloxacarbocyanine perchlorate; DiI: 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate; CCD: charge-coupled device; DIC: differential interference contrast; FURA2: (-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy)-2-)2′-amino-5′-methylphenoxy)-ethane-N,N,N′,N′-tetraacetic acid, sodium salt);BCECF: 2′,7′-bis-(carboxyethyl)-5-(and-6-)-carboxyfluorescein;fluo-3: 1-(2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-2-(2′amino-5′-methylphenoxy)-ethane-N,N,N′,N′,-tetraacetic acid, ammonium salt; DAPI: 4′,6-diamidino-2-phenylindole, dihydrochloride; PET: positron emission tomogrophy; CT: computer-assisted tomogrophy; CiD: cubitus interruptus dominus; MRC: Medical Research Council; TOTO-1: benzothiazolium-4-quinolinium dimer; YOYO-1: benzoxazolium-4-quinolinium dimer; ex.: excitation wavelength; em.: emission wavelength.

is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.     

Sense organs on the antennal flagellum of mayflies (Ephemeroptera)

A few very small sensory pegs with the characteristics of chemoreceptors are present in both males and females on the antennal flagellum of the mayflies, Stenacron interpunctatum (Say)

1 Stenacron interpunctatum (Say) is Stenonema interpunctatum (Say) of earlier literature.

and Cloeon sp. They are situated near the proximal end of the flagellum on its ventro-lateral surface and have escaped the attention of earlier investigators.     

Enantioselectivity of esterases in human brain

Subcellular fractions of three human brain specimens were found to contain esterase activities which hydrolyzed racemic oxazepam 3-acetate (rac-OXA). All three human brain preparations were highly selective toward the S-enantiomer of rac-OXA. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Highly stereoselective acid-catalyzed homonucleophilic substitution reactions

Enantiomeric 3-O-methyltemazepam and 3-Oethyltemazepam were highly stereoselectively substituted by the 3-methoxy group of methanol in acidic anhydrous methanol and by the 3-ethoxy group of ethanol in acidic anhydrous ethanol, respectively. The stereoselectivity of the homonucleophilic substitution reactions was determined by circular dichroism spectropolarimetry and gas chromatography-mass spectrometry. In anhydrous solutions containing 0.5 M D₂SO₄ at 50°C, for example, the stereoselectivity was ∼63:1 for enantiomeric 3-O-methyltemazepam in CD₃OD and ∼94:1 for enantiomeric 3-O-ethyltemazepam in C₂D₅OD. The high stereoselectivity at C3 position was primarily due to the presence of a methyl group at N1 position. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Taurine regulates insulin release from pancreatic beta cell lines

Background

Pancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.

Methods

Pancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.

Results

In both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.

Conclusions

Taurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

The transformation of τ-particles into T4 heads. I. Evidence for the conservative mode of this transformation.

Asynchroneous T4 phage head maturation includes the step of P23 cleavage: P23 of head-related τ-particles is cleaved into P23

1 This paper is part of the thesis of R. K. L. Bijlenga. It is number X of the series: “Studies on the morphopoieses of the head of phage T-even.”

of capsids with a conservative mode of transformation as evidenced by “heavy” labeling in temperature shift-down experiments with mutant 24 (tsL90). Assuming a subunit pool, data indicate in situ cleavage on individual precursor particles. The interpretation becomes less interesting when assuming a compartmentation of the membrane surface; this hypothesis is not ruled out.     

Morphological development of normal and phyllody expressing Rosa hybrida cv. Motrea flowers

A large number of soybean (Glycine max L. Merr.)flowers and young pods abscise rather than develop into mature pods. Flower andpod drop or abortion accounts for the majority of total reproductive abscissionand influences potential soybean yield. The objectives of this study were todetermine the patterns of flower, pod and seed development under treatmentswiththe growth regulators, 2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP) and6-benzylaminopurine (BAP), applied at the early reproductive stages, and toexamine the association of reproductive abscission with growth characteristicsand agronomic traits, including seed yield and seed weight. Small seeded [cvPungsan (11.1±0.4 g100-seed^–1)] and large seeded [cv Manlee(21.0±0.5 g 100-seed^–1)]genotypes were separately planted in the greenhouse and field, and treated witheither 2,4-DP or BAP. 2,4-DP (a synthetic auxin) and BAP (a syntheticcytokinin)were each applied at three concentrations (i.e. high, intermediate or low):0.12mM, 0.08 mM, 0.04 mM, and 1.5mM, 1 mM, 0.5 mM respectively. High andlow concentrations were employed for greenhouse experiments to examine thenumber of flowers per plant in pots. With the exception of low BAP (0.5mM) treatment in Pungsan, all treatments increased total podnumberwith various numbers of seeds per pod. Low 2,4-DP (0.04 mM) inbothgenotypes or BAP (0.5 mM) in Manlee significantly reduced flowerabortion and delayed abscission of pods in both genotypes, resulting inincreased pod setting rates. Under field conditions using intermediateconcentrations, 1 mM BAP significantly increased 100-seed weightto22.3 g at R1 in Manlee and 11.9 g at R3 in Pungsan.BAP (1 mM) at R3 in Pungsan significantly improved seed yield(40.1g plant^–1). Maturity was not significantlyaffected by either application in Manlee, but was significantly affected by BAPin Pungsan. In Pungsan, 2,4-DP increased pod number, plant height and nodenumber, but decreased 100-seed weight in Pungsan treated at R1, causing nosignificant change of seed yield. This study suggested that exogenousregulatorssignificantly influenced reproductive and growth characteristics, andconsequently seed yield, but increase of pod number was not always beneficialfor seed yield.     

Light-dependent inactivation of photosynthetic oxygen evolution during NaCl treatment of photosystem II particles: The role of the 24-kDa protein

Photosystem (PS) II particles prepared from spinach thylakoids with Triton X-100 were treated with 1.5 M NaCl either in the light or dark. Under both conditions, the 24-kDa and 18-kDa proteins were released from the particles, but rebound to them when the NaCl concentration was reduced to 34 mM by dilution. Oxygen evolution measured after the dilution was inactivated following NaCl treatment in the light, but not following treatment in the dark. The inactivation in the light was suppressed when 5 mM CaCl₂ was added during or after the NaCl treatment. Based on these observations, a scheme is proposed for the mechanism of light-dependent inactivation of oxygen evolution during NaCl treatment of PS II particles and for the function of the 24-kDa protein in regulating the conformation of a supposed Ca²⁺-binding intrinsic protein.Abbreviations Chl chlorophyll - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 4-morpholineethanesulphonic acid - PS photosystem - SDS sodium dodecylsulphate     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

Structure and synthesis of a lipid-containing bacteriophage. Chemical modifications of bacteriophage PM2 and the resulting alterations in acyl-chain motion in the PM2 membrane.

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidlethanolamine residues, were selectively cross-linked in the presence of 0.1-0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 10(6)-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagen was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-p-phenylenedimaleimide, however.     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.     

Kanzi: The ape at the brink of the human mind. By Sue Savage-Rumbaugh and Roger Lewin. New York: John Wiley & Sons. 1994. 299 pp. ISBN 0-471-58591-2 $24.95 (cloth)

The archaeological evidence of ancient cranial surgery is limited to cases of trepanation and cauterization. I report here on the only known case of cranial surgery in direct association with the osseous image of a nontrauma-induced soft tissue lesion (sinus pericranii). This case, from Alameda County, California (Late Middle Period, ca. 300–500 AD), is the earliest and only definitive evidence of invasive surgery from prehistoric North America.

1 Throughout this work, all reference to North American evidence excludes cases from south of the border between the United States and Mexico.

Because this individual presents the only bony evidence of cranial surgery other than trepanation or cauterization, it contributes substantially to our extremely limited understanding of medical practices in preliterate societies. © 1995 Wiley-Liss, Inc.     

Expression of OsAMT1 (1.1-1.3) in rice varieties differing in nitrogen accumulation

OsAMT is a high-affinity ammonium transporter responsible for NH ₄ ⁺ uptake by rice plants. To investigate the expression patterns of OsAMT in different genotypes in relation to nitrogen accumulation, we measured the expression of OsAMT1.1, OsAMT1.2, and OsAMT1.3 using Real-Time PCR (RT-PCR) in GD (higher N accumulation) and NG (lower N accumulation) seedlings of the Oryza sativa L. cultivar treated with 0.1 mM NH₄NO₃ and 2 mM NH₄NO₃. We found that the expression level of OsAMT1.1 was significantly higher than those of OsAMT1.2 and OsAMT1.3 in the roots treated with 0.1 mM NH₄NO₃, suggesting that OsAMT1.1 contributed the most to N accumulation among the three genes. In GD root, OsAMT1.1 had significantly higher expression levels when it was up-regulated by 0.1 mM NH₄NO₃ than when down-regulated by 2 mM NH₄NO₃. OsAMT1.1 was mainly found in GD roots treated with 0.1 mM NH₄NO₃. We conclude that the OsAMT1.1 in GD roots, which was significantly up-regulated by low N and down-regulated by high N, was the dominating factor in determining the higher N acquisition in GD than in NG at 0.1 mM NH₄NO₃.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. II. Selective action of chlorfluazuron on wheat germ agglutinin binding and cuticle ultrastructure

The action of the chitin synthesis inhibitor, chlorfluazuron, was investigated in Spodoptera frugiperda wing imaginal discs cultured in vitro. Electron microscopy and cytochemical labeling with a lectin, wheat germ agglutinin (WGA), were used to monitor morphogenesis, as well as the presence and localization of chitin and non-polymerized N-acetyl-D-glucosamine (GlcNAc). Chlorfluazuron (CFA) selectively inhibited 20- hydroxyecdysone–stimulated chitin synthesis and procuticle deposition in imaginal discs, without otherwise affecting their morphogenesis. Tracheole migration, evagination, exocytosis, and endocytosis in the epithelial cells, and the presence of non-polymerized GlcNAc in the extracellular matrix, were observed in both CFA-treated and control wing discs. On the other hand, CFA prevented the appearance of WGA-labeled chitin in newly formed procuticle, while the deposition of proteinaceous cuticulin and epicuticle was unaffected. A brief treatment with CFA resulted in WGA labeling of non-polymerized GlcNAc, but not chitin in the procuticle region. The lack of chitin in CFA treated wing discs was correlated with the appearance of an amorphous, non-lamellar procuticle region. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Correlation between glycerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths,Manduca sexta (L.)

Analysis by TLC and HPLC revealed that the triacylglycerols comprise the most abundant lipid class in the sex pheromone glands of Manduca sexta females. Also, conjugated olefinic acyl analogs of the major pheromone aldehydes occur principally in the triacylglycerols. The amount of triacylglycerols with conjugated diene acyl moieties significantly decreased when the period of pheromone production was extended by 7 h beyond the normal period of pheromone production by 3 injections of pheromone biosynthesis activating neuropeptide (PBAN) at 3 h intervals. This decrease indicates that the triacylglycerols stored in the gland may serve as major sources of pheromone precursors in the biosynthesis of the sex pheromone aldehydes. Furthermore, analysis of pheromone aldehydes and triacylglycerols in the gland from moths treated with PBAN showed that the proportions of the triacylglycerols with conjugated diene moieties were closely correlated with the proportions of aldehydes found in the same gland. This correlation suggests that the proportions of fatty acids bound to certain triacylglycerols regulates the proportions of aldehydes in biosynthesis of the pheromone blend in M. sexta. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Dynamic organization of mitochondrial membranes: A crosslinking study with dimethylsuberimidate

Rat liver mitochondria were treated with dimethylsuberimidate, a bifunctional alkylating agent, and the effects were evaluated kinetically. Concurrently with the modification of amino groups, mitochondrial proteins were crosslinked and the organelles lost their osmotic response. When the dimethylsuberimidate reaction was performed in the presence of succinate, more primary amino groups were available when compared with a sucrose medium. Concomitantly, osmotic stabilization and crosslinking of mitochondrial proteins were accelerated. The activity of aspartate aminotransferase was also studied in crosslinked mitochondria. The enzyme activity was only slightly modified when mitochondria were amidinated in a sucrose medium and solubilized thereafter with Triton X-100 or cetyltrimethylammonium bromide. In contrast, in the presence of succinate, 60% of activity was lost after solubilization with Triton X-100, but not after solubilization with cetyltrimethylammonium bromide. This finding was correlated with the changes in intramitochondrial localization of the enzyme (A. Waksman and A. Rendon, 1974,Biochimie54, 907–924). When carbonylcyanide-p-trifluoromethoxyphenylhydrazone was added in both cases (sucrose or sucrose plus succinate), the rates of osmotic stabilization, amidination reaction, crosslinking of proteins, and aspartate aminotransferase activity were similar to those observed in a sucrose medium alone. The present results suggest that organizational changes of the mitochondrial membranes induced by succinate, including intramitochondrial protein movement, are prevented by carbonylcyanide-p-trifluoromethoxyphenylhydrazone.