A role for calcium-calmodulin-dependent protein kinase II in the consolidation of visual object recognition memory.

The aim was to investigate the role of calcium-calmodulin-dependent protein kinase (CAMK)II in object recognition memory. The performance of rats in a preferential object recognition test was examined after local infusion of the CAMKII inhibitors KN-62 or autocamtide-2-related inhibitory peptide (AIP) into the perirhinal cortex. KN-62 or AIP infused after acquisition impaired memory tested at 24 h, indicating an involvement of CAMKII in the consolidation of recognition memory. Memory was impaired when KN-62 was infused at 20 min after acquisition or when AIP was infused at 20, 40, 60 or 100 min after acquisition. The time-course of CAMKII activation in rats was further examined by immunohistochemical staining for phospho-CAMKII(Thre286)alpha at 10, 40, 70 and 100 min following the viewing of novel and familiar images. At 70 min, processing novel images resulted in more phospho-CAMKII(Thre286)alpha-stained neurons in the perirhinal cortex than did the processing of familiar images, consistent with the viewing of novel images increasing the activity of CAMKII at this time. This difference was eliminated by prior infusion of AIP. These findings establish that CAMKII is active within the perirhinal region between approximately 20 and 100 min following learning and then returns to baseline. Thus, increased CAMKII activity is essential for the consolidation of long-term object recognition memory but continuation of that increased activity throughout the 24 h memory delay is not necessary for maintenance of the memory.

How negotiators get to yes: predicting the constellation of strategies used across cultures to negotiate conflict.

Individualism, hierarchy, polychronicity, and explicit-contracting values explain why managers from Germany, Japan, and the United States use a different mix of strategies to negotiate workplace conflict. Hypotheses extend prior research in showing that conflict behavior is multiply determined and that each culture uses a variety of interests, regulations, and power-based conflict management strategies. Results of actual (rather than survey-based) conflict resolution behavior suggest several fruitful avenues for future research, including examining the inferred meaning of negotiation arguments, analyzing interaction effects of cultural value dimensions, studying the effectiveness of different strategies across cultures, and examining whether strategic adjustments are made during intercultural conflict management.

Meningococcal pathogenesis: at the boundary between the pre- and post-genomic eras.

Meningococcal disease remains an important public health burden worldwide and, indeed, cause of death, particularly in poorer countries. The rapidly progressive nature of infections means that antibiotic therapy often comes too late. Vaccines are of limited efficacy in infants, one of the most vulnerable age groups, and do not exist for bacteria of serogroup B. Hence, much remains to be achieved in terms of vaccine design and the understanding of the pathogenesis of meningococcal disease. The causative bacterium, Neisseria meningitidis, is usually a commensal of the nasopharynx. Factors that lead to the invasion of the bloodstream, often followed by the crossing of the blood-brain barrier and meningitis, may be partly host- and partly bacterium-dependent, but are ill-understood. It is hoped that, taken together with the fundamental knowledge gained from biochemical and genetic studies, the huge amount of new information made available with the recent publication of the genome sequences will help to unlock more of the secrets of the lifestyle and pathogenic potential of this still poorly understood pathogen.

Stereological analysis of regional brain volumes and neuron numbers in rats displaying a spontaneous hydrocephalic condition.

Stereological methods were employed to investigate a novel spontaneously occurring brain mutation in an inbred colony of Wistar rats. These mutants displayed changes (enlarged cerebral ventricles and malformed hippocampi) similar to those seen in H-Tx hydrocephalic rats. Mutant and control rats were studied at three postnatal ages: 4, 7, and 13 weeks. Brain weight in the mutant animals was significantly (P < 0.05) increased when compared to age-matched controls. Using systematic random sampling and the Cavalieri principle we estimated the volumes of various brain compartments, including the cerebral ventricles, forebrain, and cerebral cortex. We found that ventricular volume (P < 0.001) and forebrain volume (P < 0.05) were significantly increased in mutant rats when compared to control rats. Total numbers of nucleoli, estimated using the physical fractionator, were obtained for neurons in the cerebral cortex and granule cells in the dentate gyrus. Numbers were not altered significantly in mutant rats. Nor were mean soma volumes as estimated from total volumes and numbers. The changes in brain and ventricle volumes provide quantitative evidence that these animals display a hydrocephalic condition. This condition appears not to compromise cell number or mean soma size in the brain regions examined.

Invasion of the Central Nervous System by Neisseria meningitidis.

Neisseria meningitidis is an extracellular pathogen responsible for septicemia and meningitis. The occurrence of meningitis requires that bacteria cross the blood-brain barrier (BBB) and induce an inflammatory response within the sub arachnoid space. The mechanisms that lead to the development of cerebrospinal fluid (CSF) pleocytosis once bacteria have reached the CSF have been studied using several animal models. These mechanisms are similar among extracellular pathogens responsible for meningitis (i.e., Haemophilus influenzae type b, Streptococcuspneumoniae, and N. meningitidis). The in situ production of cytokines is the primary event leading to transmigration of leucocytes through the BBB (1-4).

Scanning electron microscopic changes in the morphology of rabbit pulmonary tissue biopsied following ischemia and reperfusion: a window of opportunity?

Reperfusion is known to cause tissue damage in ischemic pulmonary tissue. We investigated the time frame of this occurrence by examining electron microscopic changes in lung tissue. Isolated, perfused, and ventilated rabbit lungs (and heart) were placed en bloc in a 37 degrees C chamber and perfused through the pulmonary artery at 15 mm Hg pressure with oxygenated Krebs-Henseleit buffer, pH 7.4, 70 ml min(-1), for 20 min and the pulmonary pump and ventilator were stopped. The resultant ischemic state was maintained for 2 h, and reperfusion resumed with the same buffer. The lungs of four groups of rabbits (n = 5 per group) were each subjected to 30 min, 1, 2, and 4 h of reperfusion respectively. Upon completion, lungs were biopsied for scanning electron microscopy. Ischemic damage including the loss of lung architecture, and edema were seen. Reperfusion restored some of the tissue anatomy and the return to normalcy increased up to 1 h of reperfusion after which the damage increased with time. Results suggest that damage due to ischemia alone may be reversible. Initial recovery is due to the re-establishment of circulation. However, with time, the damage seen may be due to free radicals and with 4 h of reperfusion, cell death may have occurred.

Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae.

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.

Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains.

Specific virulence factors associated with the pathogenesis of Escherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E. coli strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The sfa and ibe10 genes were located in regions 2 and 6, respectively. A group of genes (cnf1, hra, hly, and prs) known to be present in a pathogenicity island of the uropathogenic strain E. coli J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitis-associated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E. coli to invade the meninges of neonates.

Identification of regions of the chromosome of Neisseria meningitidis and Neisseria gonorrhoeae which are specific to the pathogenic Neisseria species.

Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as Neisseria lactamica. Their common properties may be determined by chromosomal regions found only in the pathogenic Neisseria species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109-11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in N. lactamica. Sequences present in N. lactamica were physically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish these pathogens from the commensal N. lactamica. The sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by N. meningitidis and N. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.

Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: two closely related bacteria expressing two different pathogenicities.

We have investigated genetic differences between the closely related pathogenic Neisseria species, Neisseria meningitidis and Neisseria gonorrhoeae, as a novel approach to the elucidation of the genetic basis for their different pathogenicities. N. meningitidis is a major cause of cerebrospinal meningitis, whereas N. gonorrhoeae is the agent of gonorrhoea. The technique of representational difference analysis was adapted to the search for genes present in the meningococcus but absent from the gonococcus. The libraries achieved are comprehensive and specific in that they contain sequences corresponding to the presently identified meningococcus-specific genes (capsule, frp, rotamase, and opc) but lack genes more or less homologous between the two species, e.g., ppk and pilC1. Of 35 randomly chosen clones specific to N. meningitidis, DNA sequence analysis has confirmed that the large majority have no homology with published neisserial sequences. Mapping of the cloned DNA fragments onto the chromosome of N. meningitidis strain Z2491 has revealed a nonrandom distribution of meningococcus-specific sequences. Most of the genetic differences between the meningococcus and gonococcus appear to be clustered in three distinct regions, one of which (region 1) contains the capsule-related genes. Region 3 was found only in strains of serogroup A, whereas region 2 is present in a variety of meningococci belonging to different serogroups. At a time when bacterial genomes are being sequenced, we believe that this technique is a powerful tool for a rapid and directed analysis of the genetic basis of inter- or intraspecific phenotypic variations.

Measurement of oxygen free radicals in a rabbit shock lung model. Effect of superoxide dismutase and retinol.

OBJECTIVES: To develop an isolated rabbit lung model in which oxygen free radical activity could be measured and to examine the effects on the model of oxygen free radical scavengers. DESIGN: Prospective, randomized study. SETTING: A clinical and basic research facility attached to a teaching hospital. PARTICIPANTS: Twenty-five New Zealand white rabbits weighing 3.5 to 4.5 kg. INTERVENTIONS: The mechanism of lung injury by oleic acid or by phorbol myristate acetate (20 ng/mL) plus polymorphonuclear neutrophils (PMA-PMN) in an ex vivo rabbit shock lung model may be the production of oxygen free radicals. Using a standard heart-lung preparation from these rabbits, baseline mean pulmonary artery pressure was maintained at 15 mm Hg and the mean airway pressure at 10 mm Hg. Experimental perfusates were infused over 30 minutes, followed by Krebs-Henseleit solution, pH 7.4. Dimethyl pyrroline oxide trapped oxygen free radicals, levels of which were measured by electron paramagnetic resonance spectroscopy. Lung injury was assessed by light and scanning electron microscopy and by lung weight. RESULTS: A 5-fold increase in pulmonary artery pressure (P < .001) and a nearly 3-fold increase in mean airway pressure (P < .001) were observed in both the oleic acid and PMA-PMN models. Superoxide dismutase (20,000 U/kg), but not retinol palmitate (2000 U), prevented lung injury, the increases in pulmonary artery pressure and mean airway pressure, and the increase in oxygen free radicals in the PMA-PMN model. There were no increases in oxygen free radicals in the control, oleic acid, or PMA-PMN/superoxide dismutase groups (n = 5 in each group). Maximum mean +/- SD increases in oxygen free radicals were 112 +/- 22 nmol/L in the PMA-PMN group (P < .003, n = 5) and 108 +/- nmol/L in the PMA-PMN/retinol group (P < .003, n = 5). CONCLUSIONS: The mechanism of lung injury in the PMA-PMN model is an increase in oxygen free radicals, because superoxide dismutase prevents both the rise in oxygen free radicals and lung injury. Administration of retinol does not prevent lung injury. Oleic acid produces injury not by an increase in oxygen free radicals but rather by another, unknown mechanism.

Variation in the latencies and amplitudes of N400 and NA as a function of semantic priming.

The purpose of this study was to determine whether the latencies of two event-related potential (ERP) components, the NA and N400, were sensitive to semantic priming. Subjects performed a semantic judgment task, which was designed in such a way that the N400 could be examined without overlap from the P3. Priming effects on the latencies of both NA and N400 were most apparent at frontocentral sites. The amplitude of NA was not significantly affected by priming. The amplitude of N400 was smaller for primed than for unprimed words, but the effect was significant only at centroposterior sites. Current source density (CSD) analyses performed on the ERP data suggested the activation of multiple generators in the N400 time region. The ERP and CSD data were consistent with the existence of two types of N400, a frontal N400 that varies in latency as a function of semantic priming, and a posterior N400 that varies in amplitude.

Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants.

The pathogenic Neisseria species accumulate polyphosphate to levels between 10 and 20% of their total phosphate content. However, the significance of this compound for the growth and pathogenicity of these species is not understood. A previous report (C.R. Tinsley, B.N. Manjula, and E.C. Gotschlich, Infect. Immun. 61:3703-3710, 1993) describes the purification of polyphosphate kinase, the enzyme responsible for synthesis of polyphosphate, from Neisseria meningitidis BNCV. By use of probes based on the amino acid sequence of the purified enzyme, the structural gene ppk has been cloned and sequenced. The coding sequence is 2,055 bp long and codes for a protein of 77.2 kDa. The open reading frame of the cloned gene was interrupted by the insertion of a kanamycin resistance cassette, and ppk mutants were obtained in both Neisseria gonorrhoeae and N. meningitidis by transformation with the recombinant plasmid. Amounts of polyphosphate in the ppk mutants were reduced to between 2 and 10% of wild-type levels. The mutants grew less vigorously than wild-type organisms in vitro and showed a striking increase in sensitivity to killing by human serum.

Novel lipoprotein expressed by Neisseria meningitidis but not by Neisseria gonorrhoeae.

The ppk gene, which codes for the enzyme polyphosphate kinase in Neisseria meningitidis strain BNCV, is preceded by an open reading frame coding for a protein with a predicted size of 19.2 kDa with a typical lipoprotein signal sequence of 21 amino acids. The protein has significant homology to the N-terminal portion of an outer membrane protein from Haemophilus somnus (J. Won and R. W. Griffith, Infect. Immun. 61:2813-2821, 1993). Sequencing of the same open reading frame from meningococcus strain M1080 predicted an almost identical protein. Antisera were raised against the lipoprotein, expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The antisera reacted with meningococcal membrane fractions on a Western blot (immunoblot) but did not elicit complement-dependent bactericidal activity. Restriction enzyme digestion demonstrated conservation of this portion of the meningococcal and gonococcal chromosomes. However, antisera raised to the recombinant protein showed that the protein was absent from all strains of gonococcus tested. The sequences of the gene from several strains of Neisseria gonorrhoeae and N. meningitidis were compared and found to be almost identical, except that the coding sequences from all of the gonococcal strains were terminated prematurely as a result of a frameshift mutation. The significance of the remarkable conservation of these gonococcal genes is discussed.

Purification and characterization of polyphosphate kinase from Neisseria meningitidis.

The important human pathogens Neisseria meningitidis and Neisseria gonorrhoeae accumulate phosphate in the form of polyphosphate (A. Noegel and E. C. Gotschlich, J. Exp. Med. 157:2049-2060, 1983), and the localization of more than half of this long-chain polymer on the exterior of the cells suggests a function as a protective, capsule-like coating. To enable further genetic investigation of the role of polyphosphate in Neisseria spp., the enzyme polyphosphate kinase (PPK), which catalyzes the synthesis of polyphosphate from ATP, was purified from N. meningitidis BNCV. The activity is dependent on Mg2+ and phosphate or polyphosphate and is inhibited by ADP. The Km for ATP is 1.5 mM, and the turnover number is 47 phosphate residues per polypeptide per s. Analysis of PPK labelled with [gamma-32P]ATP indicates that the enzyme is phosphorylated during the reaction, probably at an arginine residue. N-terminal and two internal amino acid sequences were derived from the purified protein and will allow the design of synthetic oligonucleotides for cloning and genetic manipulation of the ppk gene.

Antibodies recognizing a variety of different structural motifs on meningococcal Lip antigen fail to demonstrate bactericidal activity.

The neisserial Lip antigen is a conserved antigen associated with the pathogenic Neisseria species, and is composed of multiple repeats of a consensus pentapeptide. A series of monoclonal antibodies reacting with meningococcal Lip antigen were subjected to epitope mapping, using solid-phase synthetic peptides based on the consensus repeat sequence. The antibodies were found to recognize different continuous epitopes based on the consensus sequence. One monoclonal antibody was utilized in affinity chromatography to obtain purified Lip antigen and the antigen was used for immunization of mice. The resulting antisera did not recognize Lip antigen on Western blots but reacted specifically with Lip antigen in immune precipitation experiments, indicating that the predominant polyclonal immune response was directed against conformational epitopes. Despite the diversity of both continuous and conformational epitopes recognized by the antibodies produced, none of the antibodies demonstrated the ability to promote complement-mediated bactericidal activity. Thus despite its initial apparent promise as a potential vaccine candidate the case for the inclusion of Lip antigen in vaccine formulation cannot be supported at present.

Blocking of bactericidal killing of Neisseria meningitidis by antibodies directed against class 4 outer membrane protein.

The class 4 protein of Neisseria meningitidis is a highly conserved outer membrane protein, closely related to the protein PIII of Neisseria gonorrhoea. Monoclonal antibodies SM50 and SM54 raised against PIII also react with class 4 protein but do not promote complement mediated bactericidal killing of meningococci. In addition, mAb SM50 inhibits the anti-meningococcal bactericidal activity both of normal human sera and of mAb SM300, directed against a protective epitope on the class 1 outer membrane protein. The ability of class 4 protein to induce such 'blocking' antibodies suggests that its presence in experimental vaccines, based on meningococcal outer membranes, may be antagonistic to the development of effective bactericidal immunity.

Vaccination against gonorrhoea: the potential protective effect of immunization with a synthetic peptide containing a conserved epitope of gonococcal outer membrane protein IB.

Monoclonal antibody SM24 recognizes a protective, highly conserved but non-immunogenic epitope on outer membrane protein PIB of Neisseria gonorrhoeae. A series of overlapping synthetic peptides, spanning the deduced amino acid sequence of PIB from strain R10, have been synthesized on solid phase supports. Monoclonal antibody SM24 reacted with two adjacent decapeptides corresponding to residues 191-200 and 196-205, containing the common sequence TYSIP. Following localization of the epitope recognized, a peptide was synthesized corresponding to residues 193-204. The peptide was coupled to a carrier protein (KLH) and both the free peptide and peptide-KLH conjugate were used for immunization of rabbits. The resulting antisera reacted with the immunizing peptide, with denatured PIB on Western blots and, in addition, with native PIB in outer membranes of both the homologous and a heterologous strain. In the presence of human complement the sera were bactericidal for both the homologous and the heterologous strain. Thus synthetic peptides may be used to induce a protective polyclonal immune response against epitopes on gonococcal PI which are normally only weakly or non-immunogenic.

Proton-linked L-fucose transport in Escherichia coli.

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.