Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKTI1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild-type in short-term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athktl/sos3 shoots are Na+-hypersensitive in long-term growth assays. Thus wild-type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Effect of maternal epidural analgesia on fetal intrapartum oxygen saturation

The use of maternal epidural analgesia in labor may be associated with nonreassuring fetal heart rate (FHR) patterns. Fetal oxygen saturation (FSpO(2)) monitoring may improve assessment of fetal well-being during this time. Mean FSpO(2) values were compared over seven 5-minute epochs: 5 minutes prior to an epidural event (combined insertion of epidural/top-up epidural analgesia and infusion pump bolus), to 30 minutes following the event, including possible effects of maternal position and FHR pattern on FSpO(2) values. Mean FSpO(2) values were significantly different between the 5 minutes prior (49.5%) versus 16-20 minutes (44.3%, p

Noninvasive measurement of cerebral bioimpedance for detection of cerebral edema in the neonatal piglet

The association of sustained cerebral edema with poor neurological outcome following hypoxia-ischaemia in the neonate suggests that measurement of cerebral edema may allow early prediction of outcome in these infants. Direct measurements of cerebral impedance have been widely used in animal studies to monitor cerebral edema, but such invasive measurements are not possible in the human neonate. This study investigated the ability of noninvasive cerebral impedance measurements to detect cerebral edema following hypoxia-ischaemia. One-day-old piglets were anaesthetized, intubated and ventilated. Hypoxia was induced by reducing the inspired oxygen concentration to 4-6% O-2. Noninvasive cerebral bioimpedance was measured using gel electrodes attached to the scalp. Cerebral bioimpedance was also measured directly by insertion of two silver-silver chloride electrodes subdurally. Noninvasive and invasive measurements were made before, during and after hypoxia. Whole body impedance was measured to assess overall fluid movements. Intracranial pressure was measured continuously via a catheter inserted subdurally, as an index of cerebral edema. There was good agreement between noninvasive and invasive measurements of cerebral impedance although externally obtained responses were attenuated. Noninvasive measurements were also well correlated with intracranial pressure. Whole body impedance changes did not account for increases in noninvasively measured cerebral impedance. Results suggest that noninvasive cerebral impedance measurements do reflect intracranial events, and are able to detect cerebral edema following hypoxia-ischaemia in the neonate. (C) 2002 Elsevier Science B.V. All rights reserved.

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of. which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA, variant, may be functional. (C) 2002 Elsevier Science B.V. All rights reserved.

Lanczos subspace time-independent wave packet calculations of S(D-1)+H-2 reactive scattering

In this paper. we present the results of quantum dynamical simulations of the S (D-1) + H-2 insertion reaction on a newly developed potential energy surface (J. Chem. Phys. 2001, 114, 320). State-to-state reaction probabilities. product state distributions, and initial-state resolved cumulative reaction probabilities from a given incoming reactant channel are obtained from a time-independent wave packet analysis, performed within a single Lanczos subspace. Integral reaction cross sections are then estimated by J-shifting method and compared with the results from molecular beam experiment and QCT calculations.

Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (< 10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Gaileria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25KFP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasl::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604,2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket or Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

Expression of MUC1 splice variants in benign and malignant ovarian tumours

MUC1 is expressed on the surface of ovarian cancer cells. Nine different splice variants of MUC1 have been described, but no study has reported on the expression of MUC1 iso-forms in human ovarian cancer. Our study compares patterns of expression of MUC1 splice variants of malignant and benign ovarian tumours. Ovarian tissue samples were taken from patients with benign ovarian tumours (n = 34) and from patients who had surgery for primary (n = 47) or recurrent (n = 8) ovarian cancer. RT-PCR for MUC1 splice variants A, B, C, D, X, Y, Z, REP and SEC was performed and their expression compared to clinical and histopathologic parameters. Variants A, D, X, Y and Z were more frequently expressed in malignant than in benign tumours. All primary ovarian cancer cases were positive for variant REP but negative for variant SEC. No significant association of the expression of MUC1 splice variants with the response to chemotherapy or patient survival could be demonstrated. Expression of MUC1 splice variants A, D, X, Y, Z and REP is associated with the presence of malignancy, whereas expression of MUC1/SEC is associated with the absence of malignancy. (C) 2002 Wiley-Liss, Inc.

Conservation of the cardiostimulant effects of (-)-Norepinephrine across Ser49Gly and Gly389Arg Beta1-adrenergic receptor polymorphisms in human right atrium in vitro

OBJECTIVES The goal of this study was to determine whether the cardiostimulant effects of the endogenous beta(1)-adrenergic receptor (AR) agonist, (-)-norepinephrine are modified by polymorphic (Serine49Glycine [Ser49Gly], Glycine389Arginine [Gly389Arg]) variants of beta(1)-ARs in the nonfailing adult human heart. BACKGROUND Human heart beta(1)-ARs perform a crucial role in mediating the cardiostimulant effects of (-)-norepinephrine. An understanding of the significance of Ser49Gly and Gly389Arg polymorphisms in the human heart is beginning to emerge, but not as yet in adult patients who have coronary artery disease (CAD). METHODS The potency and maximal effects of (-)-norepinephrine at beta(1)-ARs (in the presence of beta(2)-AR blockade with 50 nM ICI 118,551 [erythro-DL-1(7-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol]) for changes in contractile force and shortening of contractile cycle duration were determined in human right atrium in vitro from 87 patients undergoing coronary artery bypass grafting who were taking beta-blockers before surgery. A smaller sample of patients (n = 20) not taking beta-blockers was also investigated. Genotyping for two beta(1)-AR polymorphisms (Ser49Gly and Gly389Arg) was determined from a sample of blood taken at the time of surgery. RESULTS (-)-Norepinephrine caused concentration-dependent increases in contractile force and reductions in time to reach peak force and time to reach 50% relaxation. There were no differences in the potency or maximal effects of (-)-norepinephrine in the right atrium from patients with different Ser49Gly and Gly389Arg polymorphisms. CONCLUSIONS The cardiostimulant effects of (-)-norepinephrine at beta(1)-ARs were conserved across Ser49Gly and Gly389Arg polymorphisms in the right atrium of nonfailing hearts from patients with CAD managed with or without beta-blockers. (C) 2002 by the American College of Cardiology Foundation.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Malaria vectors on Buka and Bougainville islands, Papua New Guinea

Anophelines were sampled from 82 locations oil Buka and Bougainville islands in Papua New Guinea by larval collections, carbon dioxide-baited Mosquito traps, and human biting catches. Anopheles farauti s.s. was collected in larval Surveys but infrequently in mosquito traps on both islands; on Buka Island this species was readily collected in human biting catches. Anopheles faraunti 2 was commonly collected in larval surveys on both islands however. it was not collected in either mosquito traps or human biting catches. Anopheles punctulatus was found only on Buka Island, where it was commonly collected as larvae, but rarely in human biting catches and mosquito traps. Anopheles lungae was collected Lis larvae from only I site on Bougainville. Anopheles farauti s.s. led consistently throughout the night (1900-0600 h): small peaks at midnight and dawn were not statistically significant. Of 1,156 An. farauti s.s. specimens examined by enzyme-linked immunosorbent assay for malaria sporozoites. 20 were found to be positive: 12 were positive for Plasmodium falciparum and 8 were positive for P. vivax (247 variant = 5: 210 variant = 3). Anopheles farauti s.s. seems to be the major malaria vector on these islands, whereas An. punctulatus may play a minor role on Buka Island. Anophele farauti 2 is unlikely to be involved in malaria transmission on Buka or Bougainville islands.

The 2000 Ogura Lecture: Olfactory neural cells: An untapped diagnostic and therapeutic resource

Objective. This is an over-view of the cellular biology of upper nasal mucosal cells that have special characteristics that enable them to be used to diagnose and study congenital neurological diseases and to aid neural repair. Study Design: After mapping the distribution of neural cells in the upper nose, the authors' investigations moved to the use of olfactory neurones to diagnose neurological diseases of development, especially schizophrenia. Olfactory-ensheating glial cells (OEGs) from the cranial cavity promote axonal penetration of the central nervous system and aid spinal cord repair in rodents. The authors sought to isolate these cells from the more accessible upper nasal cavity in rats and in humans and prove they could likewise promote neural regeneration, making these cells suitable for human spinal repair investigations. Methods: The schizophrenia-diagnosis aspect of the study entailed the biopsy of the olfactory areas of 10 schizophrenic patients and 10 control subjects. The tissue samples were sliced and grown in culture medium. The ease of cell attachment to fibronectin (artificial epithelial basement membrane), as well as the mitotic and apoptotic indices, was studied in the presence and absence of dopamine in those cell cultures. The neural repair part of the study entailed a harvesting and insertion of first rat olfactory lamina propria rich in OEGs between cut ends of the spinal cords and then later the microinjection of an OEG-rich suspension into rat spinal cords previously transected by open laminectomy. Further studies were done in which OEG insertion was performed up to 1 month after rat cord transection and also in monkeys. Results: Schizophrenic patients' olfactory tissues do not easily attach to basement membrane compared with control subjects, adding evidence to the theory that cell wall anomalies are part of the schizophrenic lesion of neurones. Schizophrenic patient cell cultures had higher mitotic and apoptotic indices compared with control subjects. The addition of dopamine altered these indices enough to allow accurate differentiation of schizophrenics from control patients, leading to, possibly for the first time, an early objective diagnosis of schizophrenia and possible assessment of preventive strategies. OEGs from the nose were shown to be as effective as those from the olfactory bulb in promoting axonal growth across transected spinal cords even when added I month after injury in the rat. These otherwise paraplegic rats grew motor and proprioceptive and fine touch fibers with corresponding behavioral improvement. Conclusions. The tissues of the olfactory mucosa are readily available to the otolaryngologist. Being surface cells, they must regenerate (called neurogenesis). Biopsy of this area and amplification of cells in culture gives the scientist a window to the developing brain, including early diagnosis of schizophrenia. The Holy Grail of neurological disease is the cure of traumatic paraplegia and OEGs from the nose promote that repair. The otolaryngologist may become the necessary partner of the neurophysiologist and spinal surgeon to take the laboratory potential of paraplegic cure into the day-to-day realm of clinical reality.

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PCH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20% with glucose loading. Cot-relation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not Suppressed by oral glucose loading in non-diabetic mothers.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Short report: Molecular genetic characterization of an unusually severe case of hydatid disease in Alaska caused by the cervid strain of Echinococcus granulosusi

Distinct Echinococcus granulosus life cycle patterns have been described in North America: domestic and sylvatic. Gene sequences of the sylvatic E. granulosus indicate that it represents a separate variant. Case-based data have suggested that the course of sylvatic disease is less severe than that of domestic disease. which led to the recommendation to treat cystic echinococcosis patients in the Arctic by careful medical management rather than by aggressive surgery. We recently reported the first two documented E. granalosus human cases in Alaska with accompanying severe sequelae. Here we describe the results of molecular genetic analysis of the cyst material of one of the subjects that supported identification of the parasite as the sylvatic (cervid) strain and not the domestic (common sheep strain), which was initially thought to be implicated in these unusually severe Alaskan cases.