Natural developmental variations in leaf and plant senescence in Arabidopsis thaliana.

Leaf senescence is a developmentally regulated process that contributes to nutrient redistribution during reproductive growth and finally leads to tissue death. Manipulating leaf senescence through breeding or genetic engineering may help to improve important agronomic traits, such as crop yield and the storage life of harvested organs. Here, we studied natural variations in the regulation of plant senescence among 16 Arabidopsis thaliana accessions. Chlorophyll content and the proportion of yellow leaves were used as indicator parameters to determine leaf and plant senescence respectively. Our study indicated significant genotype effects on the onset and development of senescence. We selected three late- and five early-senescence accessions for further physiological studies. The relationship between leaf and plant senescence was accession-dependent. There was a significant correlation between plant senescence and the total number of leaves, siliques and plant bolting age. We monitored expression of two senescence marker genes, SAG12 and WRKY53, to evaluate progression of senescence. Our data revealed that chlorophyll content does not fully reflect leaf age, because even fully green leaves had already commenced senescence at the molecular level. Integrating senescence parameters, such as the proportion of senescent leaves, at the whole plant level provided a better indication of the molecular status of the plant than single leaf senescence parameters.

Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis.

The major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)-based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col-0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single-tube/single-capillary format. In addition, for 110 framework markers matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col-0 and C24. Subsets of 10-62 of the established markers discriminate between various combinations of the accessions Col-0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col-0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) x Col-0 cross.

Involvement of dopamine D(2) receptors in complex maze learning and acetylcholine release in ventral hippocampus of rats.

In the current study we focus on the involvement of dopamine D(2) receptors in the ventral hippocampus in memory performance and acetylcholine release. Using the aversively motivated 14-unit T-maze (Stone maze) the injection of raclopride, a D(2) receptor antagonist, into the ventral hippocampus (8 microg/kg) was found to impair memory performance. Co-injection of quinpirole, a D(2) receptor agonist (8 microg/kg), overcame the impairment in performance. Microdialysis study revealed that quinpirole infusion (10-500 microM) into the ventral hippocampus stimulated acetylcholine release in a dose-dependent manner, and systemic injection of quinpirole (0.5 mg/kg, i.p.) also stimulated acetylcholine release in the ventral hippocampus. Infusion of eticlopride, another D(2) receptor antagonist, into the ventral hippocampus suppressed acetylcholine release in the hippocampus induced by systemic injection of quinpirole. Taken together, we suggest that D(2) receptors in the ventral hippocampus are involved in memory performance, possibly through the regulation of acetylcholine.

Multi-trait QTL mapping in barley using multivariate regression.

Many studies of QTL locations record several different traits on the same population, but most analyses look at this information on a trait-by-trait basis. In this paper we show how the regression approach to QTL mapping of Haley & Knott (1992) may be extended to a multi-trait analysis via multivariate regression, easily programmed in statistical packages. A procedure for identifying QTL locations using forward selection and bootstrapping is proposed. The method is applied to examine the locations for QTLs for six yield characters (the number of fertile stems, the grain number of the main stem, the main stem grain weight, the single plant yield, the plot yield and the thousand grain weight) in a doubled haploid population of spring barley. Several chromosomal locations with effects on more than one trait are found. The method is also suitable for examining a single trait measured in different years or environments, and is used here to examine data on heading date, a highly heritable trait, and plot yield, a trait with moderate heritability and showing QTL-environment interactions.

Expression of tandem invertase genes associated with sexual and vegetative growth cycles in potato.

The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the beta-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.

Linkage analysis in tetraploid potato and association of markers with quantitative resistance to late blight (Phytophthora infestans).

We have constructed a partial linkage map in tetraploid potato which integrates simplex, duplex and double-simplex AFLP markers. The map consists of 231 maternal and 106 paternal markers with total map lengths of 990.9 cM and 484.6 cM. The longer of the two cumulative map lengths represents approximately 25% coverage of the genome. In tetraploids, much of the polymorphism between parental clones is masked by 'dosage' which significantly reduces the number of individual markers that can be scored in a population. Consequently, the major advantage of using AFLPs--their high multiplex ratio--is reduced to the point where the use of alternative multi-allelic marker types would be significantly more efficient. The segregation data and map information have been used in a QTL analysis of late blight resistance, and a multi-allelic locus at the proximal end of chromosome VIII has been identified which contributes significantly to the expression of resistance. No late blight resistance genes or QTLs have previously been mapped to this location.

Isolation, characterisation and mapping of simple sequence repeat loci in potato.

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1-5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.

Phenserine, a novel acetylcholinesterase inhibitor, attenuates impaired learning of rats in a 14-unit T-maze induced by blockade of the N-methyl-D-aspartate receptor.

The present study evaluated the interaction of the glutamatergic and acetylcholinergic systems in memory formation, with an overall emphasis on developing multi-system approaches for treating age-related cognitive decline and Alzheimer' s disease. Specifically, we used a 14-unit T-maze to investigate whether phenserine (PHEN), a long-acting acetylcholinesterase inhibitor, could overcome a learning deficit in rats induced by the NMDA receptor antagonist, 3-(+/-) 2-carboxypiperzin-4-yl) propyl phosphonic acid (CPP). Prior to drug treatment, 3-month-old male Fischer-344 rats were trained to criterion (13 of 15 shock avoidances) in a straight runway. Twenty-four hours later, rats were given i.p. injections of saline (SAL), CPP (9 mg/kg) + SAL or CPP + PHEN (0.25, 0.5 or 0.75 mg/kg) and received 15 massed training trials in a 14-unit T-maze. CPP significantly increased the number of errors made in the maze relative to controls, and phenserine significantly reduced the number of errors made relative to rats receiving CPP only, with the lowest dose being the most effective. These results provide further support of phenserine's potent, cognitive-enhancing properties, and suggest that combined modulation of glutamatergic and acetylcholinergic systems may be of potential benefit in developing new pharmacotherapies for Alzheimer's disease and age-related cognitive decline.

Learning in a 14-unit T-maze is impaired in rats following systemic treatment with N omega-nitro-L-arginine.

We examined whether inhibition of nitric oxide synthase (NO synthase) impairs learning in male Fischer-344 rats (9 mo) in a shock-motivated 14-unit T-maze. Rats were pretrained in one-way active avoidance of foot shock to a criterion of 13/15 avoidances in a straight runway. The next day, rats received intraperitoneal (i.p.) injections of 0.9% NaCl as controls or Nomega-nitro-L-arginine (N-Arg: 3.0. 4.5, or 6.0 mg/kg) to inhibit NO synthase 30 min before maze training. During 15 trials, rats were required to negotiate each of 5 segments within 10 s to avoid footshock. Performance variables included errors (deviations from the correct pathway), runtime from start to goal, shock frequency and duration. N-Arg treatment impaired performance on all variables in a dose-dependent manner. Specifically, only the 6 mg/kg N-Arg dose significantly increased errors compared to controls over the last 10 trials but not the first 5 trials. Controls and rats treated with 3 or 4.5 mg/kg N-Arg were retested in the maze 7-10 days following training, with half receiving N-Arg (6 mg/kg i.p.) 30 min in advance. In this retention test, maze performance was not significantly affected; thus, these results indicated that NO synthase inhibition primarily impaired acquisition without impacting upon noncognitive aspects of performance. This conclusion was further reinforced by the demonstration that 6 mg/kg N-Arg did not significantly affect sensorimotor performance in a rotarod task. When rats were treated with sodium nitroprusside, an NO donor, at 1 min, but not 30 min, prior to training, the N-Arg induced impairment (6 or 8 mg/kg i.p.) in maze learning was significantly attenuated.

Impaired learning in rats in a 14-unit T-maze by 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, is attenuated by the nitric oxide donor, molsidomine.

In previous experiments, it was demonstrated that systemic or central administration of the nitric oxide synthase (NO synthase) inhibitor, NG-nitro-L-arginine (N-Arg), produced dose-dependent learning impairments in rats in a 14-unit T-maze; and that sodium nitroprusside, a NO donor, could attenuate the impairment. Since N-Arg is not specific for neuronal NO synthase and produces hypertension, it is possible that effects on the cardiovasculature may have contributed to the impaired maze performance. In the present experiment, we have investigated the maze performance of 3-4 months old male Fischer-344 rats following treatment with 7-nitroindazole, a NO synthase inhibitor that is selective for neuronal NO synthase and does not produce hypertension. In addition, we examined the effects of the NO donor, molsidomine, which is much longer acting than sodium nitroprusside. Rats were pretrained to avoid footshock in a straight runway and received training in a 14-unit T-maze 24 h later. In an initial dose-response study, rats received intraperitoneal (i.p.) injections of either 7-nitroindazole (25, 50, or 65 mg/kg) or peanut oil 30 min prior to maze training. 7-nitroindazole produced significant, dose-dependent maze acquisition deficits, with 65 mg/kg producing the greatest learning impairment. This dose of 7-nitroindazole had no significant effect on systolic blood pressure. Following the dose-response study, rats were given i.p. injections of either 7-nitroindazole (70 mg/kg) plus saline, 7-nitroindazole (70 mg/kg) plus the NO donor, molsidomine (2 or 4 mg/kg), or peanut oil plus saline as controls. Both doses of molsidomine significantly attenuated the learning deficit induced by 7-nitroindazole relative to controls. These findings represent the first evidence that impaired learning produced by inhibition of neuronal NO synthase can be overcome by systemic administration of a NO donor.

Intracerebroventricular injection of N omega-nitro-L-arginine in rats impairs learning in a 14-unit T-maze.

We investigated whether intracerebroventricular (i.c.v.) infusion of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine (N-Arg), impairs learning in male Sprague-Dawley rats (2-3 months old) in a 14-unit T-maze. Rats were pretrained in one-way active avoidance to a criterion of 13/15 avoidances of foot shock in a straight runway. The next day, rats received i.c.v. injections of either artificial cerebrospinal fluid (aCSF) as controls or N-Arg (12 microg or 15 microg) 30 min before training in the 14-unit T-maze. The learning contingency was to negotiate each of 5 segments within 10 s to avoid footshock during 15 trials. Performance variables included errors (deviations from the correct pathway), runtime from start to goal, and shock frequency and duration. Compared to controls, the number of errors over the last 10 trials was higher in rats receiving 15 microg N-Arg and over the last 5 trials for those given 12 microg. Runtime, shock frequency and duration were increased in both N-Arg groups. The N-Arg-induced (15 microg i.c.v.) impairment could be attenuated when the nitric oxide donor, sodium nitroprusside (1 mg/kg), was administered intraperitoneally 1 min prior to maze learning. In a retention test, rats were treated with either aCSF or 15 microg N-Arg i.c.v. 30 min before being retested in the maze 7-10 d following acquisition training. Under these conditions, maze performance was not significantly affected. These results confirmed previous findings that inhibition of nitric oxide synthase impairs acquisition but not retention. Moreover, the N-Arg-induced learning impairment does not appear to be related to noncognitive aspects of performance.

Combined stimulation of the glycine and polyamine sites of the NMDA receptor attenuates NMDA blockade-induced learning deficits of rats in a 14-unit T-maze.

The present study examined the effects of multi-site activation of the glycine and polyamine sites of the NMDA receptor on memory formation in rats learning a 14-unit T-maze task. The competitive NMDA receptor antagonist, (+/-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP, 9 mg/kg), was used to impair learning. The objectives were two-fold: (1) to investigate the effects of independent stimulation of the strychnine-insensitive glycine site or the polyamine site; (2) to investigate the effects of simultaneous activation of these two sites. Male, Fischer-344 rats were pretrained to a criterion of 13 out of 15 shock avoidances in a straight runway, and 24 h later were trained in a 14-unit T-maze that also required shock avoidance. Prior to maze training, rats received intraperitoneal (i.p.) injections of saline, saline plus CPP, CPP plus the glycine agonist, D-cycloserine (DCS, 30 or 40 mg/kg), CPP plus the polyamine agonist, spermine (SPM, 2.5 or 5 mg/kg), or CPP plus a combination of DCS (7.5 mg/kg) and SPM (0.625 mg/kg). Individual administration of either DCS or SPM attenuated the CPP-induced maze learning impairment in a dose-dependent manner. However, the combined treatment with both DCS and SPM completely reversed the learning deficit at doses five-fold less than either drug given alone. These findings provide additional evidence that the glycine and polyamine modulatory sites of the NMDA receptor are involved in memory formation. Furthermore, the potent synergistic effect resulting from combined activation of the glycine and polyamine sites would suggest a stronger interaction between these two sites than previously considered, and might provide new therapeutic approaches for enhancing glutamatergic function.

Age-associated memory impairment. Assessing the role of nitric oxide.

Several neurotransmitter systems have been investigated to assess hypothesized mechanisms underlying the decline in recent memory abilities in normal aging and in Alzheimer's disease. Examining the performance of F344 rats in a 14-unit T-maze (Stone maze), we have focused on the muscarinic cholinergic (mACh) and the N-methyl-D-aspartate (NMDA) glutamate (Glu) systems and their interactions. Maze learning is impaired by antagonists to mACh or NMDA receptors. We have also shown that stimulation of mACh receptors can overcome a maze learning deficit induced by NMDA blockade, and stimulation of the NMDA receptor can overcome a similar blockade of mACh receptors. No consistent evidence in rats has been produced from our laboratory to reveal significant age-related declines in mACh or NMDA receptor binding in the hippocampus (HC), a brain region that is greatly involved in processing of recent memory. Thus, we have directed attention to the possibility of a common signal transduction pathway, the nitric oxide (NO) system. Activated by calcium influx through the NMDA receptor, NO is hypothesized to be a retrograde messenger that enhances presynaptic Glu release. Maze learning can be impaired by inhibiting the synthetic enzyme for NO, nitric oxide synthase (NOS), or enhanced by stimulating NO release. However, we have found no age-related loss of NOS-containing HC neurons or fibers in rats. Additionally, other laboratories have reported no evidence of an age-related loss of HC NOS activity. In a microdialysis study we have found preliminary evidence of reduced NO production following NMDA stimulation. We are currently working to identify the parameters of this phenomenon as well as testing various strategies for safely stimulating the NO system to improve memory function in aged rats.

Laparoscopic cholecystectomy can disseminate in situ carcinoma of the gallbladder.

BACKGROUND: Early case reports suggest more frequent and rapid recurrences of carcinoma of the gallbladder after laparoscopic cholecystectomy (LC) than after open cholecystectomy. This cancer has a poor prognosis and occurs in 1 percent of patients who undergo cholecystectomies. STUDY DESIGN: A recent community hospital series of gallbladder carcinoma (GBC) was reviewed and the total reported experience of GBC after LC was compiled. Diagnostic findings were compared for patients with GBC and a consecutive series of 24 patients who had LC for benign disease. RESULTS: Nine patients with GBC were found among 928 patients who had undergone cholecystectomy (0.97 percent incidence). Compared to patients without GBC, patients with carcinoma were older, had thicker gallbladder walls, and had more abnormalities detected intraoperatively (all p < or = 0.05). Recurrence of GBC occurred more rapidly after LC, and in diffuse peritoneal and port sites when compared with recurrence patterns after open cholecystectomy. CONCLUSIONS: In patients with GBC, LC may be sufficient when the disease is confined to the gallbladder mucosa and the gallbladder is excised intact without bile spillage. However, patients whose gallbladders are torn during dissection or patients who have invasive tumors should undergo laparotomy and local reexcision. In situ GBC can be implanted if the organ is torn during dissection. When gallbladders with suspicious wall thickening or adhesions are noted at LC, especially in older patients, the procedure should be converted to open cholecystectomy.

Double dissociation of passive avoidance and milk maze performance deficits with discrete lesions of substantia innominata or globus pallidus of rats.

In three experiments, small bilateral lesions of the substantia innominata (SI), globus pallidus (GP) and central nucleus of the amygdala (ACe) produced deficits in passive avoidance of drinking (dPA) or escape performance in a milk maze (MM). Severe deficits in dPA were produced by electrolytic lesions in lateral SI or rostral ACe, and by electrolytic or ibotenic acid lesions in the heart of the SI. Such lesions produced no effects on MM performance. Lesions of the rostral SI produced no, or mild, deficits in dPA and MM performance. However, lesions of the rostral GP produced an extreme deficit in MM performance but not dPA. The milder MM deficits produced by rSI lesions appeared to reflect a spatial navigation deficit, while the more severe impairment produced by rGP lesions appeared to represent a broader disruption of instrumental behavior. SI lesions also produced a temporary cessation of drinking and a chronic decrease in body weight, both of which were associated with impaired oromotor function. Eating and drinking deficits were less severe when lesions were more lateral or rostral in SI, and absent with lesions in rostral GP or amygdala. The most important finding, however, was a double dissociation of MM performance deficits following rostral GP lesions versus passive avoidance deficits produced by SI lesions.

Bilateral destruction of the ventrolateral orbital cortex produces allocentric but not egocentric spatial deficits in rats.

Previous studies have implicated the ventrolateral orbital cortex (VLO) in spatial attention and orientation. Unilateral destruction of the VLO has been found to produce severe multimodal neglect to unilateral stimulation which is qualitatively quite similar to that found following unilateral destruction of either the medial agranular or posterior parietal cortices. A series of anatomical studies have shown that the VLO is reciprocally interconnected with both the medial agranular cortex and the posterior parietal cortex, which are involved in egocentric and allocentric spatial processing respectively. However, the role of the VLO in either egocentric or allocentric spatial processing has never been directly examined. The present study directly examined the role of the VLO in spatial learning by examining the effects of bilateral VLO destruction on performance in both egocentric (adjacent-arm maze task) and allocentric (cheeseboard task) spatial tasks. Subjects in either the cheese board task or the adjacent arm maze were given presurgical maze training and then were assigned to one of three surgical groups: a bilateral VLO group, a lesion control group which received bilateral destruction of the laterally adjacent lateral orbital cortex which has a quite different pattern of connectivity than the VLO, or a sham operated control group. The results indicated that the VLO operates were significantly impaired in the cheeseboard task (allocentric task) relative to controls, but displayed no deficits in the adjacent-arm maze (egocentric task), a pattern of results similar to those found for the posterior parietal cortex. The results of the present study strongly support the contention that the VLO is a component of the cortical circuitry for spatial processing in rodents.

An ethylene-responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases.

Carnation flower petal senescence is associated with the expression of specific senescence-related mRNAs, several of which were previously cloned. The cDNA clone pSR8 represents a transcript which accumulates specifically in senescing flower petals in response to ethylene. Here we report the structural characterization of this cDNA. A second cDNA clone was isolated based on shared sequence homology with pSR8. This clone, pSR8.4, exhibited an overlapping restriction endonuclease map with pSR8 and contained an additional 300 nucleotides. Primer extension analysis revealed the combined cDNAs to be near full-length and the transcript to accumulate in senescing petals. Analysis of the nucleotide sequence of SR8 cDNAs revealed an open reading frame of 220 amino acids sufficient to encode a 25 kDa polypeptide. Comparison of the deduced polypeptide sequence of pSR8 with other peptide sequences revealed significant similarity with glutathione s-transferases from a variety of organisms. The predicted polypeptide sequence shared 44%, 53% and 52% homology with GSTs from maize, Drosophila and man, respectively. We discuss our results in relation to the biochemistry of flower petal senescence and the possible role of glutathione s-transferase in this developmental process.

Body weight, total body water and hematocrit in diarrheic piglets.

Piglets aged 12 to 72 h in which diarrhea had been induced by enteric Escherichia coli infection or sucrose gavage were studied with respect to body weight, total body water concentration (determined by tritiated-water dilution) and hematocrit. Sucrose-induced diarrhea reduced body weight by 13 to 17%, and E. coli diarrhea, by 8 to 9%. Neither age nor diarrheal treatment affected total body water concentration, although diarrheic piglets tended to have higher hematocrit values at all ages. There was a significant daily cycle in the piglets' hematocrit values, so hematocrit might be a less valid reflector of neonates' whole body hydration status than of adults'. It was concluded that diarrheic neonatal piglets lose body water and dry matter in a ratio similar to that of normal body water and dry matter concentrations, thus their bodies have normal total body water concentrations and normal average specific heat values.

Cold resistance and environmental temperature preference in diarrheic piglets.

Piglets aged 12 to 72 h in which diarrhea had been induced by enteric Escherichia coli infection or sucrose gavage were studied with respect to cold resistance and thermal-circulation index in a 90-min test in a 6 C environment (Exp. 1) and free-choice environmental-temperature preference during a 60-min test in a 24 to 44 C thermocline (Exp. 2). In Exp. 1, diarrhea lowered the piglet's ability to maintain body temperature during the cold test. Also, diarrheic piglets tended to have lower thermal circulation index values at the end of the cold test, indicative of a greater vasoconstrictive response to the cold environment. In Exp. 2, mean preferred environmental temperatures were 35.7, 34.9 and 34.5 C, respectively, for piglets in sham-control, E. coli-infected and sucrose-gavaged groups. For reason(s) still unknown, diarrheic piglets did not choose to locate themselves in a warmer niche than did normal piglets; in fact, they did the opposite. Results of the two experiments indicate that diarrheic neonatal piglets need even more attention and care in terms of the thermal environment than do healthy ones.