NO and cGMP facilitate adenosine production in rat hearts via activation of ecto-5′-nucleotidase

 We examined whether nitric oxide (NO), a possible cardioprotective substance, can increase the production of interstitial adenosine in the ventricular myocardium. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5′-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5′-monophosphate (AMP) at a rate of 1.0 μl min^–1. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography. Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5′-nucleotidase, and the level of adenosine reflected the activity of ecto-5′-nucleotidase in the tissue. S-Nitroso-N-acetylpenicillamine (SNAP, 0.3–3 mM), an NO donor, increased the dialysate adenosine measured in the presence of AMP (100 μM) in a concentration-dependent manner. However, in the presence of an NO-oxidizing agent, 2-(4-carboxyphenyl-4,4,5,5-tetramethylimidazoline)-1-oxyl 3-oxide (carboxy-PTIO, 1 mM), the effect of SNAP was abolished. Another NO donor, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409, 1 mM) also increased adenosine production. 8-Bromo-cGMP (0.1–3 mM), a membrane-permeable cGMP analogue and a potent activator of cGMP-dependent protein kinase, increased the level of AMP-primed dialysate adenosine in a concentration-dependent manner. These results suggest that NO facilitates the production of interstitial adenosine in rat hearts in situ, via cGMP-mediated activation of ecto-5′-nucleotidase. Received: 20 February 1998 / Received after revision and accepted: 9 July 1998     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>: cytochemical localization of a NTPDase1 and an ecto-5′-nucleotidase and effects of adenine nucleotides on cellular viability

Nucleoside triphosphate diphosphohydrolase 1 (NTPDase1), which hydrolyzes extracellular ATP and ADP, and ecto-5-nucleotidase, which hydrolyzes AMP, are characterized for Trichomonas vaginalis. Ultrastructural cytochemical microscopy showed NTPDase1 and ecto-5-nucleotidase activities on the surface of the parasites. High levels of extracellular adenine nucleotides and adenosine did not exert cytolytic effects in intact cells of T. vaginalis. Our results suggest that these enzymes are relevant for the survival of the parasite during exposure to extracellular nucleotides. Since the ecto-localization of these enzymes is essential for the maintenance of adenosine extracellular levels, this nucleoside could be important for the purine salvage pathway in the parasite.     

Susceptibility in vitro of clinically metronidazole-resistant Trichomonas vaginalis to nitazoxanide, toyocamycin, and 2-fluoro-2′-deoxyadenosine

This study investigates the susceptibility of a clinically metronidazole (Mz)-resistant isolate of Trichomonas vaginalis to alternative anti-trichomonal compounds. The microaerobic minimal inhibitory concentration (MIC) of the 5-nitroimidazole (NI) drug, Mz, against a typical Mz-susceptible isolate of T. vaginalis is around 3.2 μM Mz while the clinically, highly Mz-resistant isolate has an MIC of 50–100 μM. This isolate was cross-resistant to other members of the 5-NI family of compounds including tinidazole and other experimental compounds and maintained resistance under anaerobic conditions. In addition, this isolate was cross-resistant to the 5-nitrothiazole compound nitazoxanide and the 5-nitrofuran derivative, furazolidone. Adenosine analogues toyocamycin and 2-fluoro-2′-deoxyadenosine with no nitro group were also less effective against the clinically Mz-resistant isolate than a Mz-susceptible one. Three other isolates which were determined to be Mz-resistant soon after isolation lost resistance in the long term. One other isolate has maintained some level of permanent Mz resistance (MIC of 25 μM). A multi-drug resistance mechanism may be involved in these clinically Mz-resistant isolates.     

Allergen-induced airway hyperresponsiveness is absent in ecto-5′-nucleotidase (CD73)-deficient mice

Adenosine is formed from extracellular purines by ecto-5′-nucleotidase (CD73) and is an essential player in allergic airway inflammation. The contribution of adenosine and other purines to electrolyte transport and mucociliary clearance was studied in airways of allergen challenged mice. No signs for allergen-induced inflammation were found in CD73−/− mice, and adenosine monophosphate (AMP) was unable to elicit airway Cl⁻ secretion in these animals. Tracheas of ovalbumin (OVA)-treated BALB/c and CD73+/+ mice were hyperresponsive towards methacholine when assessed by Penh and direct optical measurement of contraction. In addition Cl⁻ secretion activated by ATP and ADP was enhanced. These changes were not observed in CD73−/− mice. Expression of CFTR or CLCA was unchanged upon OVA treatment of CD73 mice, suggesting enhanced Cl⁻ secretion due to upregulated purinergic pathways. Mucociliary clearance was determined by measuring particle transport in excised mouse tracheas and was strongly enhanced in OVA-challenged CD73+/+ mice, but remained unchanged in CD73−/− mice. While mucociliary clearance is activated by allergen exposure independent of functional ecto-5′-nucleotidase, airway inflammation is largely dependent on CD73. Thus, ecto-5′-nucleotidase may provide a novel target for therapeutic intervention, probably by local application of ecto-5′-nucleotidase inhibitors through inhalation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19±3 pmol) and adenine nucleotides (101±7 pmol). Experiments performed in the presence of tubocurarine (5 M) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5-nucleotidase.Brief accounts of some of the results in this study have been published previously (refs. [6, 7]).     

Inhibition of ecto-ATPase activity by curcumin in hepatocellular carcinoma HepG2 cells

Effects of curcumin, a major constituent of turmeric, on ecto-nucleotidases have not been clarified. Here, we investigated whether curcumin affects ecto-nucleotidase activities in human hepatocellular carcinoma HepG2 cells. In the cells, high levels of Mg²⁺-dependent activity of ecto-nucleotidases were observed in the presence of 1 mM adenosine triphosphate (ATP). The activity was inhibited by ecto-ATPase inhibitors such as suramin, ZnCl₂ and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid. On the other hand, the activity was significantly decreased at alkaline pH (pH 9) and was not inhibited by levamisole, an inhibitor of alkaline phosphatase. In the presence of ATP, curcumin inhibited the activity in a concentration-dependent manner (IC₅₀ = 6.2 μM). In contrast, curcumin had no effects on ecto-nucleotidase activity in the presence of ADP (1 mM) or AMP (1 mM). The K _m value for ATP hydrolysis of curcumin-sensitive ecto-ATPase was similar to the value of NTPDase2, an isoform of ecto-nucleoside triphosphate diphosphohydrolase. These results suggest that curcumin is a potent inhibitor of ecto-ATPase and may affect extracellular ATP-dependent responses.     

Effect of menstrual cycle phase on sprinting performance

This study examined the effects of menstrual cycle phase (MCP) upon sprinting and recovery as well as upon metabolic responses to such exercise. Eight females performed a repeated 30-s sprint on a non-motorised treadmill interspersed with a 2-min rest in three phases of the MCP, follicular (low 17β-estradiol and progesterone), just prior to ovulation (midcycle trial, highest 17β-estradiol concentration and low progesterone) and in the luteal phase (high 17β-estradiol and high progesterone). MCP was verified later by radioimmunoassay of 17β-estradiol and progesterone. Peak power output (PPO) and mean power output (MPO) were unaltered (P > 0.05) due to MCP [PPO for sprint 1: 463 (18) W vs. 443 (15) W vs. 449 (18) W; PPO for sprint 2: 395 (17) W vs. 359 (16) W vs. 397 (17) W; MPO for sprint 1: 302 (15) W vs. 298 (13) W vs. 298 (14) W; MPO for sprint 2: 252 (10) W vs. 248 (10) W vs. 259 (12) W for follicular, midcycle and luteal trial, mean (SEM), respectively]. Similarly, percentage recovery of PPO and MPO (the PPO or MPO during sprint 2 expressed as a percentage of the PPO or MPO during sprint 1) was also unchanged (P > 0.05). Blood lactate, blood pH and plasma ammonia after sprinting and estimated plasma volume were also unaltered by MCP (P > 0.05). These findings suggest that hormonal fluctuations due to MCP do not interfere with maximal intensity whole body sprinting and the metabolic responses to such exercise.     

Cell microvesicles during experimental endotoxemia

The dynamics of microvesicle formation in arterial blood in generalized Schwartzman phenomenon was studied. Successive (with 24-h interval) intravenous injections of endotoxin to rabbits in a dose of 1 mg/kg and 3 mg/kg caused an increase in the content of microvesicles in the blood, some of them containing ecto-5′-nucleotidase. Biphasic changes in arterial blood clotting time and erythrocyte hemolysis were observed. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 517–520, November, 2006     

Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes

 The possibility of the protein kinase C (PKC) pathway being a mechanism underlying the desensitization of carbachol- (CCh-)activated nonselective cationic current (I _CCh) was investigated in a study of guinea-pig gastric myocytes. Using the conventional whole-cell patch-clamp technique with symmetrical CsCl-rich solution in pipette and bath, I _CCh was induced by bath application of 50 μM CCh. With 0.5 mM EGTA [ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] in the pipette solution (0.5 mM [EGTA]_i), I _CCh decayed spontaneously (desensitization of I _CCh) to around 20% within 10 min. Desensitization of I _CCh was significantly attenuated with 2 mM [EGTA]_i. At a concentration of 20 μM OAG (1-oleoyl-2-acetyl-sn-glycerol), a PKC activator, inhibited I _CCh at 0.5 mM [EGTA]_i but far less at 2 mM [EGTA]_i (18% and 81% of control, respectively). The same cationic current induced by intracellular guanosine-5′-O-(3-thiotriphosphate) (GTP[γ-S]) was not inhibited by OAG with 0.5 mM [EGTA]_i. The pretreatment of gastric myocytes with PKC inhibitors, either 1 μM chelerythrine or 1 μM peptide inhibitor, attenuated the desensitization of I _CCh. [Ca²⁺]_i was also measured by single cell microfluorometry using fura-2. Under CCh stimulation with 2 mM [EGTA]_i, [Ca²⁺]_i did not increase above 100 nM while it increased to around 260 nM with 0.5 mM [EGTA]_i. These results suggest that the desensitization of I _CCh is partly due to the Ca²⁺-dependent PKC pathway in guinea-pig gastric myocytes. Received: 27 August 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

Effects of epinephrine and 17β-estradiol sulfate on transmembrane potentials of guinea pig cardiomyocytes

Changes in the transmembrane potentials of guinea pig cardiomyocytes caused by epinephrine and 17β-estradiol sulfate are studied. It is shown that 17β-estradiol attenuates the effect of epinephrine on these cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 611–613, December, 1996     

beta-Sitosterol modulation of monocyte-endothelial cell interaction: a comparison to female hormones

ObjectiveTo investigate the effect of β-sitosterol, 17β-estradiol and progesterone on oxidized LDL (oxLDL)-stimulated human umbilical venous endothelial cell (HUVEC) expression of intercellular adhesion molecule-1 (ICAM-1), THP-1 monocyte chemotactic activity, migration and adhesion of THP-1 cells co-cultured with HUVECs.MethodsICAM-1 expression was determined by immunofluorescence in HUVEC monolayers treated with LDL or oxLDL and 17β-estradiol, progesterone or β-sitosterol. Monocyte chemotactic activity was performed in Transwell chambers by culturing HUVECs with different stimuli and steroids, THP-1 cells labeled with [³H] thymidine were added to the upper chamber and the radioactivity was measured. Migration assays were performed using Transwell chambers but monocytes were labeled with BCECF-AM and THP-1 cells adhered to HUVECs were visualized by fluorescence microscopy. MCP-1 was quantified by ELISA.ResultsICAM-1 expression was inhibited by β-sitosterol alone, when combined with 17β-estradiol or progesterone, or with both hormones. It was shown that 7.5 μM β-sitosterol decreased migration and adhesion of THP-1 cells to HUVECs cultured in the presence of oxLDL. This effect was also observed in HUVEC cultures in the presence of β-sitosterol, the 17β-estradiol and progesterone mixture, and in the presence of the two hormones. It was shown that 7.5 μM β-sitosterol significantly inhibited chemotaxis of [³H] thymidine labeled THP-1 cells in oxLDL-stimulated HUVEC cultures. MCP-1 concentrations in the supernatants of oxLDL-stimulated HUVEC cultures were inhibited by 7.5 μM β-sitosterol as well as by progesterone and the mixture of the two female hormones.     

Genome organisation and sequence comparison suggest intraspecies incongruence in M RNA of Watermelon bud necrosis virus

Watermelon bud necrosis virus (WBNV), a member of the genus Tospovirus, family Bunyaviridae is an important viral pathogen in watermelon cultivation in India. The complete genome sequence properties of WBNV are not available. In the present study, the complete M RNA sequence and the genome organisation of a WBNV isolate infecting watermelon in Delhi (WBNV-wDel) were determined. The M RNA was 4,794 nucleotides (nt) long and potentially coded for a movement protein (NSm) of 34.22 kDa (307 amino acids) on the viral sense strand and a Gn/Gc glycoprotein precursor of 127.15 kDa (1,121 amino acids) on the complementary strand. The two open reading frames were separated by an intergenic region of 402 nt. The 5′ and 3′ untranslated regions were 55 and 47 nt long, respectively, containing complementary termini typical of tospoviruses. WBNV-wDel was most closely related (79.1% identity) to Groundnut bud necrosis virus, an important tospovirus that occurs in several crops in India, and was different (63.3–75.2% identity) from the other cucurbit-infecting tospoviruses known to occur in Taiwan and Japan. Sequence analysis of NSm and Gn/Gc revealed phylogenetic incongruence between WBNV-wDel and another isolate originating from central India (WBNV-Wm-Som isolate). The Wm-Som isolate showed evolutionary divergence from the wDel isolate in the Gn/Gc protein (74.6% identity) potentially due to recombination with the other tospoviruses that are known to occur in India. This is the first report of a comparison of complete sequences of M RNA of WBNV.     

Hypoxic inhibition of K+ currents in isolated rat type I carotid body cells: evidence against the involvement of cyclic nucleotides

 Whole-cell patch-clamp recordings were used to evaluate the effects of the cyclic nucleotides adenosine 3’,5’-cyclic monophosphate (cAMP) and guanosine 3’,5’-cyclic monophosphate (cGMP) on ionic currents in type I carotid body cells isolated from rat pups, and to investigate whether cyclic nucleotides are involved in K⁺ current inhibition by hypoxia. In the presence of 500 μM isobutylmethylxanthine, currents were not significantly modified by 8-bromo-cAMP (2 mM), dibutyryl-cAMP (5 mM) or 8-bromo-cGMP (2 mM). Currents were also unaffected by the phosphodiesterase (PDE)-resistant protein kinase A activators Sp-cyclic adenosine-3′,5′-monophosphorothioate (Sp-cAMPS) and Sp-8-bromoadenosine-3′,5′-monophosphorothioate (Sp-8-bromo-cAMPS) (50 μM), or by β-phenyl-1,N ²-ethenoguanosine-3′,5′-cyclic monophosphate (PET-cGMP) (100 μM) or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP; 500 μM). Ca²⁺ channel currents were also unaffected by Sp-8-Br-cAMPS, PET-cGMP and SNAP at the same concentrations. In the absence of cyclic nucleotide analogues, hypoxia (P_O2 17–23 mmHg) reversibly inhibited K⁺ currents. This degree of hypoxic inhibition was not significantly altered by the PDE-resistant protein kinase A inhibitors Rp-cyclic adenosine-3′,5′-monophosphorothioate (Rp-cAMPS) (50 μM) or Rp-8-bromoadenosine-3′,5′-monophosphorothioate (Rp-8-bromo-cAMPS) (200 μM). Similarly, PET-cGMP (100 μM) and SNAP (500 μM) did not alter the degree of inhibition caused by hypoxia. At the same concentrations used in type I cell experiments, Sp-8-bromo-cAMPS, PET-cGMP and SNAP completely relaxed isolated guinea-pig basilar arteries preconstricted with 20 mM K⁺-containing solutions. Our results indicate that cyclic nucleotides alone are not an important factor in the regulation by O₂ tension of K⁺ currents in rat type I carotid body cells. Received: 12 June 1996 / Accepted: 14 August 1996     

The key steroidogenic enzyme 3β-hydroxysteroid dehydrogenase in <Emphasis Type="Italic">Taenia solium</Emphasis> and <Emphasis Type="Italic">Taenia crassiceps</Emphasis> (WFU)

Larval and adult stages of Taenia solium and Taenia crassiceps WFU strain were analyzed by histochemical and biochemical methods to determine the existence of steroid pathways. The presence of the key enzyme 3β-hydroxisteroid-dehydrogenase (3β-HSD) was examined in frozen sections of cysticerci obtained from mice and segments of tapeworms obtained from the intestine of hamsters. 3β-HSD activity was detected by nitroblue-tetrazolium products after incubation with dehydroepiandrosterone, androstendiol, or pregnenolone. Tapeworm tissues exhibited 3β-HSD activity in the subtegumentary areas of the neck and immature proglottids following incubation with androstendiol, as well as surrounding the testes in mature proglottids. T. solium cysticerci exhibited 3β-HSD activity in the subtegumentary tissues. The synthesis of steroid hormones involving the activity of 3β-HSD was studied in cysticerci or tapeworms incubated in the presence of tritiated steroid precursors. The culture media were analyzed by thin layer chromatography and showed synthesis of androstendiol, testosterone, and 17β-estradiol by cysticerci, androstendiol, and 17β-estradiol by tapeworms. The results strongly suggest the activity of 3β-HSD in taeniid parasites that have at least a part of the enzymatic chain required for androgen and estrogen synthesis and that the enzymes are present in the larval stage and from the early strobilar stages to the mature proglottids.     

Complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus,a member of a new species in the genus <Emphasis Type="Italic">Kobuvirus</Emphasis>, family <Emphasis Type="Italic">Picornaviridae</Emphasis>

Kobuvirus is a new genus in the family Picornaviridae. Two species are currently known: Aichi virus (human kobuvirus) and Bovine kobuvirus (U-1). In this study, the complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus (Kobuvirus/swine/S-1-HUN/2007/Hungary, EU787450) were determined. The structure of the S-1-HUN genome, VPg–5′UTR–leader protein–structural proteins (VP0, VP3, VP1)–non-structural proteins (2A–2C, 3A–3D)–3′UTR–poly(A) tail, was found to be typical of picornavirus. The 8210-nucleotide (nt)-long RNA genome contains a large open reading frame (7467 nt) encoding a potential polyprotein precursor of 2488 amino acids (aa) that has 57/56% and 63/64% nt/aa identity with Aichi virus and U-1, respectively. The 5′UTR contains a hepacivirus/pestivirus-like internal ribosomal entry site (IRES type IV group-B-like) with conserved pseudoknot, II and IIIa–f domains. A tandem repeat (a 30-amino-acid-long motif) was detected in 2B. Thirty-nine (65%) of the 60 fecal samples from pigs under the age of 6 months at the tested farm were positive (the incidence was 90% under the age of 3 weeks). Porcine kobuvirus belongs to a potential new species—the third—in the genus Kobuvirus. Nucleotide sequence data reported are available in the GenBank database under accession number EU787450.     

Evidence that acute taurine treatment alters extracellular AMP hydrolysis and adenosine deaminase activity in zebrafish brain membranes

Taurine is one of the most abundant free amino acids in excitable tissues. In the brain, extracellular taurine may act as an inhibitory neurotransmitter, neuromodulator, and neuroprotector. Nucleotides are ubiquitous signaling molecules that play crucial roles for brain function. The inactivation of nucleotide-mediated signaling is controlled by ectonucleotidases, which include the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5′-nucleotidase. These enzymes hydrolyze ATP/GTP to adenosine/guanosine, which exert a modulatory role controlling several neurotransmitter systems. The nucleoside adenosine can be inactivated in extracellular or intracellular milieu by adenosine deaminase (ADA). In this report, we tested whether acute taurine treatment at supra-physiological concentrations alters NTPDase, ecto-5′-nucleotidase, and ADA activities in zebrafish brain. Fish were treated with 42, 150, and 400 mg L⁻¹ taurine for 1 h, the brains were dissected and the enzyme assays were performed. Although the NTPDase activities were not altered, 150 and 400 mg L⁻¹ taurine increased AMP hydrolysis (128 and 153%, respectively) in zebrafish brain membranes and significantly decreased ecto-ADA activity (29 and 38%, respectively). In vitro assays demonstrated that taurine did not change AMP hydrolysis, whereas it promoted a significant decrease in ecto-ADA activity at 150 and 400 mg L⁻¹ (24 and 26%, respectively). Altogether, our data provide the first evidence that taurine exposure modulates the ecto-enzymes responsible for controlling extracellular adenosine levels in zebrafish brain. These findings could be relevant to evaluate potential beneficial effects promoted by acute taurine treatment in the central nervous system (CNS) of this species.     

Effects of 17β-estradiol and its isomer 17α-estradiol on learning in rats with chronic cholinergic deficiency in the brain

It was shown for the first time that estrogens 17β- and 17α-estradiols compensate impaired cognitive functions in rats with partial chronic deprivation of cholinergic functions in the central nervous system induced by intracerebral administration of selective cholinergic neurotoxin AF64A. 17β-Estradiol produced strong dose-dependent changes in the weights of hormone-sensitive endocrine glands, while 17α-estradiol did not affect the weight of the gonads and slightly influenced (in high concentration) the weights of the adrenal glands and thymus. The positive effects of exogenous 17β- and 17α-estradiols on cognitive functions are due to their antioxidant properties, rather than due to specific action on hormone-sensitive endocrine glands. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 5, pp. 525–527, May, 2000     

Rotavirus-neutralizing antibodies inhibit virus binding to integrins α2β1 and α4β1

Summary Rotavirus outer capsid proteins VP5^*, VP8^* and VP7 elicit neutralizing, protective antibodies. The α2β1 integrin is a cellular receptor for rotavirus that is bound by VP5^*. Some rotaviruses also recognize the α4β1 integrin. In this study, the effects of antibodies to rotavirus on virus binding to recombinant α2β1 and α4β1 expressed on K562 cells were determined. All neutralizing monoclonal antibodies to VP5^* tested (YO-2C2, 2G4, 1A10) and two to VP7 (RV-3:2, RV-4:2) inhibited rotavirus binding to α2β1. Rotavirus binding to α4β1 was reduced by 2G4 and neutralizing antibody F45:2, directed to VP7. However, a neutralizing antibody to VP8^* (RV-5:2) and one to VP7 (RV-3:1) did not affect rotavirus binding to these integrins. Virus-cell binding was unaffected by non-neutralizing antibody RVA to the rotavirus inner capsid protein VP6. The attachment of human rotavirus strain Wa to these integrins was inhibited by infection sera with neutralizing activity collected from two children hospitalised with severe rotavirus gastroenteritis. A negative reference serum did not affect rotavirus-cell attachment. As the binding of rotaviruses to α2β1 and α4β1 is inhibited by neutralizing antibodies to VP5^* and VP7, and serum from children with rotavirus disease, rotavirus recognition of these integrins may be important for host infection.     

Activity of doripenem against extended-spectrum β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates

The in vitro activity of doripenem was evaluated against a recent collection of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates (201 ESBL-producing Enterobacteriaceae [153 Escherichia coli and 48 Klebsiella pneumoniae] and 201 P. aeruginosa). Comparator agents included amikacin, tobramycin, ciprofloxacin, cefepime, cefotaxime, ceftazidime piperacillin-tazobactam, imipenem, and meropenem. Both doripenem and meropenem inhibited 100% of the ESBL-producing Enterobacteriaceae at ≤0.5 μg/mL. For these isolates, the MIC₉₀ of doripenem (0.12 μg/mL) was 4-fold lower than that of imipenem (0.5 μg/mL). Against P. aeruginosa, the MIC₉₀ of doripenem and meropenem was 2 μg/mL, 4-fold lower than that of imipenem (8 μg/mL). At an MIC of ≤2 μg/mL, doripenem, meropenem, and imipenem inhibited 90.5%, 89.6%, and 82.1% of P. aeruginosa isolates, respectively. Doripenem maintained activity against imipenem-nonsusceptible isolates of P. aeruginosa; at an MIC of ≤4 μg/mL, it inhibited 15 of the 25 isolates with MICs for imipenem of >4 μg/mL. Doripenem is active against ESBL-producing Enterobacteriaceae and P. aeruginosa isolates. Its activity is similar to that of meropenem and slightly better than that of imipenem. The results of this study suggest that doripenem could be an alternative therapeutic agent for infections caused by these organisms.     

Remodeling of Hyperplastic Pituitaries in Hypothyroid us-Subunit Knockout Mice After Thyroxine and 1713-Estradiol Treatment: Role of Apoptosis

Hyperplasia of pituitary thyrotrophs is often associated with hypothyroidism. In this study, the effects of thyroxine and 17β-estradiol on thyrotroph hyperplasia was analyzed using a hypothyroid mouse model resulting from targeted disruption of the glycoprotein hormone α-subunit (αSU) gene, which leads to lack of functional thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) and underdevelopment of the thyroid and gonads. Thyroxine replacement for 2 mo resulted in a decrease in the relative percent of thyrotrophs and an increase of lactotrophs and somatotrophs numbers to normal values. A twofold increase in the relative percent of gonadotrophs was observed compared to wild-type mouse pitutary. Treatment for 2 mo with 17β-estradiol led to an increase in lactotroph numbers to normal levels, but had no influence on thyrotroph hyperplasia. Rearrangement of the hyperplastic pituitary phenotype after hormonal replacement proceeded without any evidence of pituitary cell necrosis. A slight increase in apoptotic cell death was observed in hormone-treated pituitaries, and this was localized to TSH cells by double-labeling experiments. Chronic thyroxine treatment resulted in increased expression of Bcl-2 protein in hypertrophied pituitary cells, whereas 17β-estradiol increased expression of Bad protein in prolactin cells. These results suggest that apoptotic cell death is involved in reversal of thyrotroph hyperplasia in the presence of thyroid hormone. Thyroxine and 17β-estradiol may influence cell death in this model by regulating expression of the Bcl-2 protein family in a cell-type specific manner.