Complex asparagine-linked oligosaccharides in Mgat1-null embryos

To investigate the developmental role of complex N-linked oligosaccharides,we previously inactivated the mouse Mgat1 gene which encodesUDP-N-acetylglucosamine:     

Alterations of O-glycan biosynthesis in human colon cancer tissues

Human colon cancer is associated with antigenic and structuralchanges in mucin-type carbohydrate chains (O-glycans). To elucidatethe control of the biosynthesis of these O-glycans in coloncancer, we have studied glycosyltransferase and sulphotransferaseactivities involved in the assembly of elongated O-glycan structures.We analysed homogenates prepared from cancer tissue, adjacentnormal and distal normal tissue from 20 patients. Several transferaseactivities showed pronounced changes in cancer tissue. The changescorrelate with previous findings of a loss of O-glycans in cancermucins, but did not always correlate with levels of Tn, sialyl-Tn,T and Le^x antigens in homogenates or with the differentiationstatus and Duke's stages of the cancer tissue or the patient'sblood type, sex and age. UDP-GlcNAc: Gal NAc-R ß3-N-acetylglucosaminyltransferase(where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetyl-D-galactosamine)synthesizing O-glycan core 3, GlcNAcß1-3GalNAc-, CMP-sialicacid: GalNAc-peptide     

Variation in Concentration of Ribulose-1, 5-bisphosphate Carboxylase in Leaves of Barley, Wheat and Hybrids of Crested Wheatgrasses

Amounts of the enzyme ribulose-1,5-bisphosphate carboxylasewere estimated in seedling leaves of barley (Hordewn vulgareL.) and flag leaves of wheat (Triticum aestitum L.) by radialimmuno diffusion. A fourfold variation among barley varietiesfor amount of RuBPCase at the seedling stage was observed (c.3.5–15mg g^–1 fr. wt). Range in variation for amountof flag leaf RuBPCase among wheat varieties was 6-09-9.39 mgRuBPCase g^–1 fr. wt. F₁ hybrids from interspecific andintergeneric crosses of crested wheatgrasses (Agropyron andElymus spp.) and their amphidiploid analogues were comparedfor amount of RuBPCase in the most recent fully expanded leavesharvested before seed set. Amount of enzyme varied from 3.4to 77.6 mg g^–1 fr. wt among the hybrids. No effect chromosomenumber on enzyme concentration was observed among 13 hybridsand their amphidiploid counterparts. Key words: RuBPCase, wheatgrasses     

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3 ^+/+ and Mgat3 ^–/– mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3 ^–/– mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3 ^–/– mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3 ^–/– mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc. Published in 2003.     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Photoadaptation in Antarctic phytopfankton: variations in growth rate, chemical composition and P versus I curves

The response of phytoplankton to variations in the light regimewas studied during the VULCAN and ACDA cruises in the Antarctic.Unenriched batch cultures of 12–19 days' duration reachedchl concentrations of 10–50 µg^–1 and exhibitedexponential growth rates, with the maximal rate being 0.41 doubl,day^–1. Ice edge algae exhibited maximum growth rates atphoton flux densities (PFD) of 30–100 µE m^–2S^–1and the growth rate was reduced by about 30% at 500–1000µE m^–2S^–1 The chl/C ratio ranged between 0.004and 0.018, with the lowest ratios at PFDs above 500 µEm^–2S^–1 chl/C ratios were also below maximum at PFDsbelow 40–50 µE m^–2S^–1 The C:N:P ratioswere close to the Redfield ratios; the Si/C ratio averaged 0.16(atoms), and the ATP/C ratio averaged from 0.0024 to 0.0050in different culture senes. When thawed after having been frozenfor 10 days, shade-adapted cultures were in a much better conditionthan sun-adapted ones. P versus I data showed that the maximumassimilation number varied from 0.75 to 4.4 µg C (µgchl)^–1h^–1. It varied inversely with the chl/C ratio;therefore the maximum carbon turnover rate varied little betweensamples (0.024/0.035 h^–1). Low biomass communities exhibitedrelatively high values for (the initial slope of P versus Icurves), low values for 1_sat (160–330 µE m^–2S^–1),and they were susceptible to photoinhibition. In contrast, communitiesdominated by Odontella weissflogii exhibited low values for, a high value for I_sat (560 µE m^–2S^–1 andthey tolerated high PFDs. The photo-adaptational status of thephytoplankton in natural water samples is discussed relativeto the profile of water column stability and mixing processes.     

Effect of Salinity on Growth, Nodulation and Nitrogen Yield of Chickpea (Cicer arietinum L.)

Chickpea cultivar ILC 482 was inoculated with salt-tolerantRhizobium strain Ch191 in solution culture with different saltconcentrations added either immediately with inoculation or5 d later. The inhibitory effect of salinity on nodulation ofchickpea occurred at 40 dS m^–1 (34.2 mol m^–3 NaCl)and nodulation was completely inhibited at 7 dS m^–1 (61.6mol m^–3 NaCl); the plants died at 8 dS m^–1 (71.8mol m^–3 NaCl). Chickpea cultivar ILC 482 inoculated with Rhizobium strain Ch191^spcstrwas grown in two pot experiments and irrigated with saline water.Salinity (NaCl equivalent to 1–4 dS m^–1) significantlydecreased shoot and root dry weight, total nodule number perplant, nodule weight and average nodule weight. The resultsindicate that Rhizobium strain Ch191 forms an infective andeffective symbiosis with chickpea under saline and non-salineconditions; this legume was more salt-sensitive compared tothe rhizobia, the roots were more sensitive than the shoots,and N₂ fixation was more sensitive to salinity than plant growth. Key words: Cicer arietinum, nodulation, N₂ fixation, Rhizobium, salinity     

Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus

The protein encoded by the envelope gene of Friend spleen focus-formingvirus is responsible for the acute leukaemogenicity of thisvirus. In order to correlate glycosylation and intracellularprocessing of this protein with viral pathogenicity, envelopegene products of pathogenic and apathogenic glycosylation mutantswere expressed in Rat-1 cells and metabolically labelled with[6-³H] glucosamine. Following immunoprecipitation, primary andsecondary gene products (gp55, gp65) were separated by preparativepolyacrylamide gel electrophoresis. Oligosaccharides were releasedfrom tryptic glycopeptides by treatment with endo-ß-N-acetylglucosaminidaseH (gp55), peptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagineamidase F (gp65) or by reductive ß-elimination. Resultingglycans were characterized by cochromatography with authenticoligosaccharide standards using different HPLC systems and digestionwith exoglycosidases. The results revealed that the primaryenvelope gene products of pathogenic glycosylation mutants were,in part, further processed in Rat-1 cells similar to wild-typeglycoprotein, resulting in polypeptides carrying complex-typeN-glycans as well as partially sialylated O-linked oligosaccharides.In contrast, corresponding glycoproteins encoded by apathogenicmutants were found to remain at the level of the primary translationproduct exclusively comprising high-mannose-type N-glycans.Hence, intracellular maturation of the envelope gene productsin this model cell line seems to correlate with the in vivopathogenicityof the glycosylation mutants studied. carbohydrate structure glycoprotein murine leukaemia virus oligosaccharide processing SFFV     

Xylem fluxes of fixed N through nodules of the legume Acacia littorea and haustoria of an associated N-dependent root hemiparasite Olax phyllanthi

Nodulated 1-1.5-year-old plants of Acacia littorea grown inminus nitrogen culture were each partnered with a single seedlingof the root hemiparasite Olax phyllanthi. Partitioning of fixedN between plant organs of the host and parasite was studiedfor the period 4–8 months after introducing the parasite.N fluxes through nodules of Acacia and xylem-tapping haustoriaof Olax were compared using measured xylem flows of fixed Nand anatomical information for the two organs. N₂ fixation duringthe study interval (635 µg N g FW nodules^–1 d^–1)corresponded to a xylem loading flux of 0.20 µg N mm^–2d^–1 across the secretory membranes of the pencycle parenchymaof the nodule vascular strands. A much higher flux of N (4891µg mm^–2 d^–1) exited through xylem at the junctionof nodule and root. The corresponding flux of N from host xylemacross absorptive membranes of the endophyte parenchyma of Olaxhaustorium was 1.15 µg N mm^–1 d^–1, six timesthe loading flux in nodules. The exit flux from haustorium toparasite rootlet was 20.0 pg N mm^–1 d^–1, 200-foldless than that passing through xylem elements of the nodule.Fluxes of individual amino compounds in xylem of nodule andhaustorium were assessed on a molar and N basis. N flux valuesare related to data for transpiration and partitioning of Cand N of the association recorded in a companion paper. Key words: Olax phyllanthi, host-parasite relationships, N flux, Acacia, N₂ fixation     

A study of feeding in predacious ciliates using prey ciliates labeled with fluorescent microspheres

Feeding in predacious estuarine ciliates was investigated ina series of laboratory experiments using a new method of preylabeling which facilitates microscopic indentification of ingestedprey items. Ingestion rates of Mesodinium pulex, Euplotes vannusand E.woodruffi were estimated using the appearance, insidethe predator, of bacteriovorous ciliates (Metanophrys sp., Cyclidiumsp.and Pleuronema sp ) labeled with fluorescent microspheres. Preyremain motile and have presumably unaltered surface characteristics.Ingestion rates of log-growth phase predators increased withprey density. Mesodinium pulex ingested 0 15–0.32 cellsh^–1 over a prey concentration of 60–2300 ml^–1.Maximum ingestion rates of E. woodruffi and E. vannus were 4.5and 3.4 cells h^–1 respectively, estimated at prey abundancesof 75 and 172 cells ml^–1 respectively. Comparisons offeeding rates on prey of different sizes, and the effects ofstarvation, indicated that ingestion is likely limited by differentfactors in ‘raptorial’ (M pulex) and ‘filterfeeding’ (Euplotes spp.) predators.     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Purification and Characterization of Soluble and Membrane-Associated DNA Polymerases from a Virus PBCV-1 Infected Green Alga Chlorella NC64A

DNA polymerases were purified several hundred-fold from the10 000 x g soluble (polymerase I) and particulate (polymeraseIII) fractions prepared from virus PBCV-1 infected ChlorellaNC64A extracts. Both DNA polymerases exhibited optimal activitywith activated calf thymus DNA at pH 8.5. DNA polymerase I required3.0 mol m^–3 MgSO₄ and 150 to 250 mol m^–3 KCl foroptimum activity whereas, DNA polymerase III required 2.0 molm^–3 MgSO₄ and 150 mol m^–3 KCl. Both enzymes wereinhibited by pyrophosphate, actinomycin D, ethidium bromide,dideoxythymidine triphosphate, and N-ethylmaleimide but wererelatively insensitive to aphidicolin. DNA polymerase I differedfrom DNA polymerase III in its response to cations (particularlyNH₄Cl), elution from a DEAE cellulose column, and molecularweight. Key words: Algal virus, DNA polymerase, Chlorella     

Beta N-acetylglucosaminyltransferase V (Mgat5) deficiency reduces the depression-like phenotype in mice

The central nervous system (CNS) is rich in glycoconjugates, located on cell surface and in extracellular matrix. The products of Golgi UDP-GlcNAc: N -acetylglucosaminyltransferases (encoded by Mgat1, Mgat2, Mgat4 and Mgat5) act sequentially to generate the GlcNAc-branched complex-type N -glycans on glycoprotein receptors. While elimination of all the branched N -glycans in Mgat1^−/− mouse embryos is lethal at neural tube fold stage, decreased branching is associated with late developmental defects similar to type 2 of congenital disorders of glycosylation, with developmental and psychomotor abnormalities. To study the role of complex-type N -glycans in brain function, we tested Mgat5^−/− mice in a battery of neurological and behavioral tests. Despite the absence of tri- and tetra-antennary products, Mgat5^−/− mice were not different from their wild-type littermates in physical and neurological assessments, anxiety level, startle reactivity and sensorimotor gating. However, they displayed a robust decrease in the immobility time in the forced swim test and the tail suspension test independent of locomotor activity, interpreted as a change in depression-like behavior. This effect was accentuated after chronic mild stress. Comparable increase in plasma corticosterone of Mgat5^+/+ and Mgat5^−/− mice in response to acute stress shows an intact function of the hypothalamus–pituitary–adrenal axis. A change in social interactions was also observed. Our results indicate that Mgat5 modification of complex-type N -glycans on CNS glycoproteins is involved in the regulation of depression-like behavior.     

Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl^– removal, and energy depletion activate Ca²⁺-permeable cation channels with Ca²⁺-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1^–/– mice) than in their wild-type littermates (AE1^+/+ mice) despite increased percentages of reticulocytes (AE1^–/–: 49%, AE1^+/+: 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1^–/–erythrocytes/reticulocytes (10%) than in AE1^+/+ erythrocytes (1%). Osmotic shock (addition of 400 mM sucrose), Cl^– removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1^–/– erythrocytes/reticulocytes than in AE1^+/+ erythrocytes. The increase of annexin binding following exposure to the Ca²⁺ ionophore ionomycin (1 µM) was, however, similar in AE1^–/– and in AE1^+/+ erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca²⁺ permeability in AE1^–/– erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1^–/– mice than in AE1^+/+ mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca²⁺ entry and subsequent scrambling of cell membrane phospholipids. annexin; cell volume; osmolarity; phosphatidylserine; energy depletion     

Micropropagation of Pissardi Plum

An efficient medium for multiplication of Prunus cerasifera,J. F. Ehrh. cv. ‘Atropurpurea’, Pissardi plum, consistedof Linsmaier-Skoog basal medium (LS) supplemented with 3% sucrose,10mg 1^–1 N⁴-(²-isopentenyl) adenine (2iP), and 162 mg1^–1 phloroglucinol (Phl). Phl in the medium significantlyenhanced growth (P < 0.1 %) over cultures maintained on LSmedium with 10 mg 1^–1 2iP and no Phl. Plantlets were rootedon a half-strength Murashige-Skoog (MS) medium supplementedwith 0.2 mg 1^–1 indole-3-butyric acid (IBA). Prunus cerasifera, Pissardi plum, micropropagation, phloroglucinol, N⁶-(²-isopentenyl) adenine     

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction

The biosynthesis of complex asparagine (N)-linked oligosaccharidesin vertebrates proceeds with the linkage of N-acetylglucosamine(GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:ß-D-mannosideß1–4 N-acetylglucosaminyltransferase III (GlcNAc-TIII,EC2.4.1.144) catalyzes the addition of GlcNAc to the mannosethat is itself ß1–4 linked to underlying N-acetylglucosamine.GlcNAc-TIII thereby produces what is known as a ‘bisecting’GlcNAc linkage which is found on various hybrid and complexN-glycans. GlcNAc-TIII can also play a regulatory role in N-glycanbiosynthesis as addition of the bisecting GlcNAc eliminatesthe potential for     

Light, temperature and nitrogen as interacting factors affecting diel vertical migrations of dinoflagellates in culture

Diel vertical migrations of the marine dinoflagellates Gonyaulaxpolyedra Stein and Ceratium furca (Ehr.) Clap, et Lachm. werefollowed in a laboratory tube (2.02 m x 0.25 m) under a 12:12hlight:dark cycle. The effects of temperature stratification,two levels of surface irradiance and nitrogen depletion on patternsof vertical migrations were examined. At temperatures between22–26°C with small temperature gradients, both speciesmigrated at a rate of 0.7 –1.0 m h^–1. Steeper thermoclines(ca. 0.8°C 0.1 m^–1) with temperatures below ca. 20°Ccaused a marked decrease in swimming speed which resulted inaccumulations of cells in these thermocline regions. Under conditionsof nutrient sufficiency both algae migrated into the surfacelayers at irradiance values of over 1000 µE m^–2s^–1. Increasing nitrogen depletion caused the downwardmigration of both algae to commence progressively earlier inthe day and before the end of the light period. The earlierdownward migrations enabled a more complete descent throughthe thermocline. Nitrogen depleted cells of Gonyaulax continuedto undertake vertical migrations but avoided high irradiancesthus forming subsurface maxima at irradiance levels close to150 µE m^–2 s^–1. Ceratium cells which exhaustedboth inorganic nitrogen and phosphorus ceased to migrate accompaniedby a large change in cellular fluorescence.     

Genetic assessment of the importance of galectin-3 in cancer initiation, progression, and dissemination in mice

The galectin family of β-galactoside binding lectins isinvolved in normal and pathological processes. Altered expressionof galectin-3 has been described in many cancers, and studiesof cancer cell lines have implicated this lectin in variousaspects of the tumorigenic cascade. The goal of this reportwas to directly assess the importance of galectin-3 in tumorbiology by introducing the galectin-3 null mutation (galectin-3^–/–)into mouse lines genetically programmed to develop cancers.We used two mouse models of human intestinal cancer, the Apc^Minand Apc^1638N lines, to study tumor initiation and tumor progression.We also crossed the galectin-3^–/– mice with PyMTtransgenic animals, a model in which primary mammary gland tumorsgive rise to lung metastases at high frequency. Unexpectedly,we show that the absence of galectin-3 does not affect the evolutionof the disease in any of these three situations.     

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

We previously showed that plasma membrane Ca²⁺-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca²⁺ ([Ca²⁺]_i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1^+/–, Pmca4^+/–, Pmca4^–/–, and Pmca1^+/–Pmca4^–/– mice. There were no differences in basal [Ca²⁺]_i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca²⁺]_i (130–180%) in smooth muscle from Pmca1^+/– and Pmca1^+/–Pmca4^–/– bladders than those in WT or Pmca4^–/–. The responses to carbachol (CCh: 10 µM) were also greater in Pmca1^+/– (120–150%) than in WT bladders. In contrast, the responses in Pmca4^–/– and Pmca1^+/–Pmca4^–/– bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca²⁺]_i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4^–/– (130–190%) and Pmca1^+/–Pmca4^–/– (120–250%) bladders, but not in Pmca1^+/– bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca²⁺ clearance, while PMCA4 is essential for the [Ca²⁺]_i increase and contractile response to the CCh receptor-mediated signal transduction pathway. PMCA; bladder smooth muscle; gene-altered mice