To test the reliability of children's reporting as compared with that of their mothers, a highly structured psychiatric diagnostic interview was used with 307 subjects, ages 6 through 16. Another interviewer gave each mother a similar interview about the child. Responses of each mother-child pair to 168 questions were compared using the kappa statistic. Highest agreement was found on questions concerning symptoms that are concrete, observable, severe, and unambiguous. Mothers tended to report significantly more behavioral symptoms, and children more subjective symptoms. Reasons for low kappas and asymmetrical reporting of symptoms are discussed.

We used a 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mouse to study postnatal subventricular zone (SVZ) progenitor fate, with a focus on the olfactory bulb (OB). The postnatal OB of the CNP-EGFP mouse contained EGFP+ interneurons and oligodendrocytes. In the anterior SVZ, the majority of EGFP+ progenitors were NG2+. These NG2+/EGFP+ progenitors expressed the OB interneuron marker Er81, the neuroblast markers doublecortin (DC) and Distalless-related homeobox (DLX), or the oligodendrocyte progenitor marker Nkx2.2. In the rostral migratory stream (RMS), EGFP+ cells displayed a migrating phenotype. A fraction of these cells were either NG2-/Er81+/DC+/DLX+ or NG2+/Nkx2.2+. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) injection into the lateral ventricle (LV) of early postnatal mice demonstrated that NG2+/EGFP+ progenitors migrate from the SVZ through the RMS into the OB. Moreover, fluorescence-activated cell-sorting-purified NG2+/CNP-EGFP+ or NG2+/beta-actin-enhanced yellow fluorescent protein-positive (EYFP+) progenitors transplanted into the early postnatal LV displayed extensive rostral and caudal migration. EYFP+ or EGFP+ graft-derived cells within the RMS were DLX+/Er81+ or Nkx2.2+, migrated to the OB, and differentiated to interneurons and oligodendrocytes. In the subcortical white matter (SCWM), grafted cells differentiated to either oligodendrocytes or astrocytes. Transplantation of NG2+/EYFP+ progenitors selectively purified from the SVZ showed that these cells were migratory and generated glia and neurons in the OB, hippocampus, and striatum. In contrast, cortical, OB, or cerebellar NG2+ cells had a very limited migratory potential and gave rise to glia in the SCWM and striatum. Our findings indicate region-specific differences between NG2+ progenitor cells and show that NG2+ cells can migrate throughout the RMS and contribute to both gliogenesis and neurogenesis in the postnatal OB.

BACKGROUND

An understanding of the relation of commensal microbiota to health is essential in preventing disease. Here we studied the oral microbial composition of children (N = 74, aged 3 - 18 years) in natural transition from their deciduous to a permanent dentition and related the microbial profiles to their oral health status. The microbial composition of saliva was assessed by barcoded pyrosequencing of the V5-V6 hypervariable regions of the 16 S rRNA, as well as by using phylogenetic microarrays.

RESULTS

Pyrosequencing reads (126174 reads, 1045 unique sequences) represented 8 phyla and 113 higher taxa in saliva samples. Four phyla--Firmicutes, Bacteriodetes, Proteobacteria and Actinobacteria--predominated in all groups. The deciduous dentition harboured a higher proportion of Proteobacteria (Gammaproteobacteria, Moraxellaceae) than Bacteroidetes, while in all other groups Bacteroidetes were at least as abundant as Proteobacteria. Bacteroidetes (mainly genus Prevotella), Veillonellaceae family, Spirochaetes and candidate division TM7 increased with increasing age, reflecting maturation of the microbiome driven by biological changes with age. Microarray analysis enabled further analysis of the individual salivary microbiota. Of 350 microarray probes, 156 gave a positive signal with, on average, 77 (range 48-93) probes per individual sample. A caries-free oral status significantly associated with the higher signal of the probes targeting Porphyromonas catoniae and Neisseria flavescens.

CONCLUSIONS

The potential role of P. catoniae and N. flavescens as oral health markers should be assessed in large-scale clinical studies. The combination of both open-ended and targeted molecular approaches provides us with information that will increase our understanding of the interplay between the human host and its microbiome.

Copper oxide (CuO) nanoparticles were characterised and investigated with respect to potential antimicrobial applications. It was found that nanoscaled CuO, generated by thermal plasma technology, contains traces of pure Cu and Cu2O nanoparticles. Transmission electron microscopy (TEM) demonstrated particle sizes in the range 20-95 nm. TEM energy dispersive spectroscopy gave the ratio of copper to oxygen elements as 54.18% to 45.26%. The mean surface area was determined as 15.69 m(2)/g by Brunau-Emmet-Teller (BET) analysis. CuO nanoparticles in suspension showed activity against a range of bacterial pathogens, including meticillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, with minimum bactericidal concentrations (MBCs) ranging from 100 microg/mL to 5000 microg/mL. The ability of CuO nanoparticles to reduce bacterial populations to zero was enhanced in the presence of sub-MBC concentrations of silver nanoparticles. Studies of CuO nanoparticles incorporated into polymers suggest release of ions may be required for optimum killing.

Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.

BACKGROUND

Mesial temporal lobe epilepsy (mTLE) with hippocampus sclerosis (HS) is an important cause for focal epilepsy. In this study, we explored the integrity of connecting networks using diffusion tensor imaging (DTI) and two whole-brain voxel-based methods: statistical parametric mapping (SPM) and tract-based spatial statistics (TBSS).

METHODS

Thirty-three consecutive patients with mTLE and HS undergoing presurgical evaluation were scanned at 3 T, a DTI data set was acquired and parametric maps of fractional anisotropy (FA) and mean diffusivity (MD) were calculated. Twenty-one patients had left hippocampal sclerosis (LHS) and 12 patients had right HS (RHS). These groups were compared to 37 normal control subjects using both SPM5 and TBSS.

RESULTS

The ipsilateral temporal lobe showed widespread FA reduction in both groups. The limbic system was clearly abnormal in the LHS group, also involving the arcuate fasciculus. In RHS, changes were more restricted but also showed involvement of the contralateral temporal and inferior frontal lobe. Increased MD was found in the ipsilateral hippocampus by SPM that was only marginally detected by TBSS. In white matter regions, however, TBSS was more sensitive to changes than SPM.

CONCLUSIONS

DTI detects extensive changes in mTLE with HS. The affected networks were principally in the ipsilateral temporal lobe and the limbic system but also the arcuate fasciculus. SPM and TBSS gave complementary information with higher sensitivity to FA changes using TBSS.

1. Many lines of evidence suggest that signals relayed by the magnocellular and parvocellular subdivisions of the primate lateral geniculate nucleus (LGN) maintain their segregation in cortical processing. We have examined two response properties of units in the striate cortex of macaque monkeys, latency and transience, with the goal of assessing whether they might be used to infer specific geniculate contributions. Recordings were made from 298 isolated units and 1,129 multiunit sites in the striate cortex in four monkeys. Excitotoxin lesions that selectively affected one or the other LGN subdivision were made in three animals to demonstrate directly the magnocellular and parvocellular contributions. An additional 435 single units and 551 multiunit sites were recorded after the ablations. 2. Most units in striate cortex had visual response latencies in the range of 30-50 ms under the stimulus conditions used. The earliest neuronal responses in striate cortex differed appreciably between individuals. The shortest latency recorded in the four animals ranged from 20 to 31 ms. Comparable values were obtained from both single unit and multiunit sites. After lesions were made in the magnocellular subdivision of the LGN in two animals, the shortest response latencies were 7 and 10 ms later than before the ablations. A larger lesion in the parvocellular subdivision of another animal produced no such shift. Thus it appears that the first 7-10 ms of cortical activation can be attributed to activation relayed by the magnocellular layers of the LGN. 3. The units with the shortest latencies were all found in layers 4C or 6 and their responses were among the most transient in striate cortex. Furthermore, their responses all showed a pronounced periodicity at a frequency of 50-100 Hz. This periodicity was stimulus locked, and the responses of all short-latency units oscillated in phase. 4. An index of response transience was computed for the units recorded in striate cortex. The distribution of this index was unimodal and gave no suggestion of distinct contributions from the geniculate subdivisions. Magnocellular and the parvocellular lesions affected the overall transience of responses in striate cortex. The changes, however, were very small; extremely transient responses and extremely sustained responses survived both types of lesions. 5. A characteristic profile was observed in the response latencies in superficial layers. Latencies appeared to increase monotonically from layer 4 toward the surface of cortex, with the most superficial neurons not becoming active until 15 ms after responses were observed in layer 4C.(ABSTRACT TRUNCATED AT 400 WORDS)

Keratinocytes electroporated with human papillomavirus (HPV) DNA (HPV-6, 11, 16 and 18) exhibited an increased cellular proliferation which was quantitated as microcolony and macrocolony formation. However, only macrocolonies induced by HPV-16 or HPV-18 DNA (the two viral types most commonly found in human cervical carcinomas) gave rise to proliferating, poorly-stratified colonies when grown in the presence of serum and calcium. Hydrocortisone increased the frequency of these differentiation-resistant colonies, and studies showed that they were immortalized, contained one copy of viral DNA per cell, expressed three discrete species of viral RNA and synthesized the viral E7 protein. HPV-induced cellular proliferation and altered differentiation are therefore separable events and may represent the activity of different viral genes.

The integrin alpha4beta7 binds to MAdCAM-1 and contributes to homing of lymphocytes to gut and other mucosal tissues. In humans, the alpha4beta7(hi) subset of circulating memory cells appears to have been primed in mucosal tissues. The factors that determine whether alpha4beta7(lo) naive cells become alpha4beta(hi) or alpha4beta7(-) cells upon differentiation are poorly understood but could include an influence of the activating antigen-presenting cell. To address this point, the induction of alpha4beta7 following activation of mouse cells with the APC-dependent stimulus soluble anti-CD3 has been examined. Almost all mouse T cells freshly isolated from mesenteric lymph nodes (MLN) and peripheral (PLN; axillary, brachial and inguinal) lymph nodes stained only weakly for alpha4beta7 but a subpopulation became alpha4beta7(hi) upon activation with anti-CD3 in a cell cycle- and accessory cell-dependent manner. A small proportion (approximately 1.5 %) of the starting cells gave rise to alpha4beta7(hi) cells after culture. A higher proportion of alpha4beta7(hi) cells were generated in MLN than PLN cultures. Peyer's patch cultures gave intermediate values. In crossover experiments, MLN dendritic cells (DC) induced higher proportions and numbers of alpha4beta7(hi) cells than PLN DC irrespective of the source of T cells. Therefore, in addition to their other immunoregulatory roles, DC have the potential to shape immune responses by influencing the homing of the lymphocytes they activate.

BACKGROUND

Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies.

METHODS

Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8-493 World Health Organization (WHO) units/ml and IA-2A 2-235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using (35)S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays.

RESULTS

The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001).

CONCLUSIONS

Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements.

Two mitochondrial genotypes are shown to exist within one Holstein cow maternal lineage. They were detected by the appearance of an extra Hae III recognition site in one genotype. The nucleotide sequence of this region has been determined and the genotypes are distinguished by an adenine/guanine base transition which creates the new Hae III site. This point mutation occurs within an open reading frame at the third position of a glycine codon and therefore does not alter the amino acid sequence. The present pattern of genotypes within the lineage demands that multiple shifts between genotypes must have occurred within the past 20 years with the most rapid shift taking place in no more than 4 years and indicates that mitochondrial DNA polymorphism can occur between maternally related mammals. The process that gave rise to different genotypes in one lineage is clearly of fundamental importance in understanding intraspecific mitochondrial polymorphism and evolution in mammals. Several potential mechanisms for rapid mitochondrial DNA variation are discussed in light of these results.

Mortality reports on asbestos exposed cohorts which gave information on exposure levels from which (as a minimum) a cohort average cumulative exposure could be estimated were reviewed. At exposure levels seen in occupational cohorts it is concluded that the exposure specific risk of mesothelioma from the three principal commercial asbestos types is broadly in the ratio 1:100:500 for chrysotile, amosite and crocidolite respectively. For lung cancer the conclusions are less clear cut. Cohorts exposed only to crocidolite or amosite record similar exposure specific risk levels (around 5% excess lung cancer per f/ml.yr); but chrysotile exposed cohorts show a less consistent picture, with a clear discrepancy between the mortality experience of a cohort of xhrysotile textile workers in Carolina and the Quebec miners cohort. Taking account of the excess risk recorded by cohorts with mixed fibre exposures (generally<1%), the Carolina experience looks uptypically high. It is suggested that a best estimate lung cancer risk for chrysotile alone would be 0.1%, with a highest reasonable estimate of 0.5%. The risk differential between chrysotile and the two amphibole fibres for lunc cancer is thus between 1:10 and 1:50. Examination of the inter-study dose response relationship for the amphibole fibres suggests a non-linear relationship for all three cancer endpoints (pleural and peritoneal mesotheliomas, and lung cancer). The peritoneal mesothelioma risk is proportional to the square of cumulative exposure, lung cancer risk lies between a linear and square relationship and pleural mesothelioma seems to rise less than linearly with cumulative dose. Although these non-linear relationships provide a best fit ot the data, statistical and other uncertainties mean that a linear relationship remains arguable for pleural and lung tumours (but not or peritoneal tumours). Based on these considerations, and a discussion fo the associated uncertainties, a series of quantified risk summary statements for different elvels of cumulative exposure are presented.

Flavonoids, a group of naturally occurring antioxidants and iron chelators, might be used as cardioprotective agents in doxorubicin-induced cardiotoxicity, which is believed to be caused by the formation of oxygen free radicals. To investigate the underlying molecular mechanism, we tested a large group of flavonoids from all major structural subclasses on their ability to inhibit doxorubicin (enzymatically)-induced and Fe2+/ascorbate (nonenzymatically)-induced microsomal lipid peroxidation (LPO) and to chelate Fe2+. In addition, we measured half peak oxidation potentials (Ep/2). LPO inhibition data gave a good qualitative correlation with the oxidation potentials. Most flavonoids tested chelated Fe2+, but there were large differences in the chelating capacity. For good scavenging activity, a catechol moiety on ring B is required. The 3-OH moiety can function as a chelation site and can also be oxidized. The 3-OH group in combination with a C2 C3 double bond, increases the scavenging activity. Fe2+ chelation only plays a role in the LPO inhibition by less active scavengers. Chelation can then raise the activity to the level of the most active scavengers, possibly by site-specific scavenging. It can be concluded that Ep/2 values and iron chelating activity can almost completely describe the LPO inhibiting behaviour of the flavonoids.

Epidemiological, clinical, and experimental data indicate that the risk of developing breast cancer is strongly dependent on the ovary and on endocrine conditions modulated by ovarian function, such as early menarche, late menopause, and parity. Women who gave birth to a child when they were younger than 24 years of age exhibit a decrease in their lifetime risk of developing breast cancer, and additional pregnancies increase the protection. The breast tissue of normally cycling women contains three identifiable types of lobules, the undifferentiated Lobules type 1 (Lob 1) and the more developed Lobules type 2 and Lobules type 3. The breast attains its maximum development during pregnancy and lactation (Lobules type 4). After menopause the breast regresses in both nulliparous and parous women containing only Lob 1. Despite the similarity in the lobular composition of the breast at menopause, the fact that nulliparous women are at higher risk of developing breast cancer than parous women indicates that Lob 1 in these two groups of women might be biologically different, or might exhibit different susceptibility to carcinogenesis. Based on these observations it was postulated that Lob 1 found in the breast of nulliparous women and of parous women with breast cancer never went through the process of differentiation, retaining a high concentration of epithelial cells that are targets for carcinogens and are therefore susceptible to undergo neoplastic transformation. These epithelial cells are called Stem cells 1, whereas Lob 1 structures found in the breast of early parous postmenopausal women free of mammary pathology, on the contrary, are composed of an epithelial cell population that is refractory to transformation, called Stem cells 2. It was further postulated that the degree of differentiation acquired through early pregnancy has changed the 'genomic signature' that differentiates Lob 1 of the early parous women from that of the nulliparous women by shifting the Stem cells 1 to Stem cells 2 that are refractory to carcinogenesis, making this the postulated mechanism of protection conferred by early full-term pregnancy. The identification of a putative breast stem cell (Stem cells 1) has, in the past decade, reached a significant impulse, and several markers also reported for other tissues have been found in the mammary epithelial cells of both rodents and humans. Although further work needs to be carried out in order to better understand the role of the Stem cells 2 and their interaction with the genes that confer them a specific signature, collectively the data presently available provide evidence that pregnancy, through the process of cell differentiation, shifts Stem cells 1 to Stem cells 2 - cells that exhibit a specific genomic signature that could be responsible for the refractoriness of the mammary gland to carcinogenesis.

BACKGROUND

Two recent studies with white males have shown that genotypes associated with high levels of monamine oxidase A (MAOA) protect against the impact of childhood maltreatment and adversity on the development of antisocial behavior and conduct disorder.

METHODS

Participants in a prospective cohort design study involving court substantiated cases of child abuse and neglect and a matched comparison group were followed up into adulthood and interviewed (N = 802). Eighty-two percent consented to provide blood and 631 gave permission for DNA extraction and analyses. A composite index of violent and antisocial behavior (VASB) was created based on arrest, self-report, and diagnostic information.

RESULTS

No main effect was found for the relationship between MAOA genotype and VASB. Genotypes associated with high levels of MAOA activity buffered abused and neglected whites from increased risk of becoming violent and/or antisocial in later life. This protective effect was not found for non-white abused and neglected individuals.

CONCLUSIONS

Possible explanations for this differential effect for whites and non-whites include differences in contextual factors (e.g., environmental stressors) and a question of the suitability of using the MAOA promoter VNTR polymorphism as a proxy for MAOA levels in non-white populations.

Monoclonal antibodies specific for gH of herpes simplex virus were shown previously to neutralize viral infectivity. Results presented here demonstrate that these antibodies (at least three of them) block viral penetration without inhibiting adsorption of virus to cells. Penetration of herpes simplex virus is by fusion of the virion envelope with the plasma membrane of a susceptible cell. Electron microscopy of thin sections of cells exposed to virus revealed that neutralized virus bound to the cell surface but did not fuse with the plasma membrane. Quantitation of virus adsorption by measuring the binding of purified radiolabeled virus to cells revealed that the anti-gH antibodies had little or no effect on adsorption. Monitoring cell and viral protein synthesis after exposure of cells to infectious and neutralized virus gave results consistent with the electron microscopic finding that the anti-gH antibodies blocked viral penetration. On the basis of the results presented here and other information published elsewhere, it is suggested that gH is one of three glycoproteins essential for penetration of herpes simplex virus into cells.

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO(3))(2) resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb(2+) and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P(i) on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P(i) is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.

OBJECTIVE

The neutral results of the SAINT II trial have again highlighted difficulties translating neuroprotective efficacy from bench to bedside. Animal studies are susceptible to study quality biases, which may lead to overstatement of efficacy. We report the impact of study quality on published estimates of the efficacy of NXY-059 in animal models of stroke.

METHODS

We conducted a systematic review and stratified meta-analysis of published studies describing the efficacy of NXY-059 in experimental focal cerebral ischemia.

RESULTS

Overall, NXY-059 improved infarct volume by 43.3% (95% CI, 34.7 to 52.8). Only 2 of 9 publications reported randomization, concealment of treatment allocation, and blinded outcome assessment. Studies not reporting these quality items gave substantially higher estimates of efficacy than did higher-quality studies.

CONCLUSIONS

The reported efficacy of NXY-059 in animal models of stroke is confounded by low study quality. The failure of SAINT II highlights the need for substantial improvements in the design, conduct, and reporting of animal studies; journals can play an important role in this by adopting standards for animal studies similar to those agreed over 10 years ago for clinical trials.

1. Potassium conductances were studied in large layer V neurons using an in vitro slice preparation of cat sensorimotor cortex. The kinetics and pharmacological sensitivity of K+ currents were studied directly using single microelectrode voltage clamp and indirectly by evoking single or multiple spikes and recording the spike repolarization and subsequent afterhyperpolarizations (AHPs). 2. A fast-decaying afterhyperpolarization (fAHP) and a subsequent medium-duration afterhyperpolarization (mAHP) followed a single spike. The amplitude and duration of the mAHP increased when multiple spikes were evoked at a fast rate (e.g., 100 Hz), and a slower afterhyperpolarization (sAHP) appeared only after sustained repetitive firing. 3. All AHPs were reduced by membrane potential hyperpolarization and raised extracellular K+ concentration, suggesting they were caused by an increased K+ conductance. Only the mAHP and sAHP reversed at the estimated value of potassium equilibrium potential (-100 mV), whereas the mean reversal potential of the fAHP was nearly identical to the mean value of resting potential (-71 mV). 4. Mechanisms underlying spike repolarization, the fAHP, and the mAHP were investigated. Two rapidly activating outward currents, a fast-inactivating current and a slowly inactivating delayed rectifier, were detected by voltage clamp. Both currents were reduced rapidly by tetraethylammonium (TEA). The fast transient current was reduced slowly after divalent cations were substituted for Ca2+ (through a mechanism unrelated to blockade of Ca2+ channels), whereas the delayed rectifier was unaffected. 5. Spike duration was increased and the fAHP was abolished only by blocking agents that reduced the fast outward currents. Effects of extracellular and intracellular TEA were similar. Effects of TEA and Ca2+-free perfusate were additive and resembled the effects of intracellular Cs+. The addition of apamin, d-tubocurare, or Cd2+ was ineffective. We conclude that the two fast outward currents reflect pharmacologically and kinetically separate K+ conductances that are primarily responsible for spike repolarization and the fAHP. 6. Voltage-clamp studies revealed two additional outward currents, which were persistent and Ca2+-mediated. Each current activated and deactivated slowly, but the kinetics of one component were approximately 10 times slower than the other. The decay of these currents gave rise to AHPs resembling the mAHP and the early sAHP. 7. Neither the mAHP nor the sAHP was reduced by TEA. The mAHP was reduced when divalent cations were substituted for Ca2+ or when Cd2+, apamin, or d-tubocurare were added.(ABSTRACT TRUNCATED AT 400 WORDS)

Native chemical ligation of unprotected peptide segments involves reaction between a peptide-alpha-thioester and a cysteine-peptide, to yield a product with a native amide bond at the ligation site. Peptide-alpha-thioalkyl esters are commonly used because of their ease of preparation. These thioalkyl esters are rather unreactive so the ligation reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts used to date have been either a mixture of thiophenol/benzyl mercaptan, or the alkanethiol MESNA. Despite the use of these thiol catalysts, ligation reactions typically take 24-48 h. To gain insight into the mechanism of native chemical ligaton and in order to find a better catalyst, we investigated the use of a number of thiol compounds. Substituted thiophenols with pK(a)>> 6 were found to best combine the ability to exchange rapidly and completely with thioalkyl esters, and to then act as effective leaving groups in reaction of the peptide-thioester with the thiol side chain of a cysteine-peptide. A highly effective and practical catalyst was (4-carboxylmethyl)thiophenol ('MPAA'), a nonmalodorous, water-soluble thiol. Use of MPAA gave an order of magnitude faster reaction in model studies of native chemical ligation and in the synthesis of a small protein, turkey ovomucoid third domain (OMTKY3). MPAA should find broad use in native chemical ligation and in the total synthesis of proteins.

Only small numbers of cells from solid tumours are needed for haematogenous metastasis. Detection is difficult because existing techniques are not sensitive enough. We have used reverse transcriptase to make complementary DNA from peripheral blood messenger RNA, and the polymerase chain reaction (PCR) to amplify cDNA specific for a gene actively transcribed only in the tumour tissue type. We prepared cDNA from peripheral blood of seven patients with malignant melanoma, four patients with other metastatic cancers, and four healthy subjects, as well as from several melanoma-derived cell lines. PCR was used to amplify the gene for tyrosinase, a tissue-specific gene in melanocytes. Since normal melanocytes are not thought to circulate in peripheral blood, detection of tyrosinase transcription in peripheral blood should indicate the presence of circulating cancer cells. The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood. Blood samples from four of the seven patients with malignant melanoma gave positive results, whereas all eight control subjects gave negative results. This method does not depend on the characterisation of cancer-specific genetic abnormalities and can be applied to any cancer for which tissue-specific genes can be identified, including epithelial cancers. It could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment.

BACKGROUND

We studied the relations between physical activity and changes in physical activity, all-cause mortality, and incidence of major coronary-heart-disease events in older men.

METHODS

In 1978-80 (Q1), 7735 men aged 40-59 were selected from general practices in 24 British towns, and enrolled in a prospective study of cardiovascular disease, which included physical activity data. In 1992 (Q92), 12-14 years later, 5934 of the men (91% of available survivors, mean age 63 years) gave further information on physical activity and were then followed up for a further 4 years. The main endpoints were all-cause mortality during 4 years of follow-up from Q92, and major fatal and non-fatal coronary-heart-disease events during 3 years of follow-up from Q92.

RESULTS

Among 4311 men with no history of coronary heart disease, stroke, or "other heart trouble" by Q92 and who did not report "poor health", there were 219 deaths. In the inactive/occasionally active, light, moderate, and moderately vigorous/vigorous activity groups there were 101 (18.5/1000 person-years) 48 (11.4), 23 (7.3), and 47 (9.1) deaths, respectively (adjusted risk ratios 1.00, 0.61 [95% CI 0.48-0.86], 0.50 [0.31-0.79], 0.65 [0.45-0.94]). Men who were sedentary at Q1 and who began at least light activity by Q92 had significantly lower all-cause mortality than those who remained sedentary, even after adjustment for potential confounders (risk ratio=0.55 [0.36-0.84]). Physical activity improved both cardiovascular mortality (0.66 [0.35-1.23]) and non-cardiovascular mortality (0.48 [0.27-0.85]). The relation between physical activity at Q92, changes in physical activity, and mortality were similar for men with pre-existing cardiovascular disease.

CONCLUSIONS

Maintaining or taking up light or moderate physical activity reduces mortality and heart attacks in older men with and without diagnosed cardiovascular disease. Our results support public-health recommendations for older sedentary people to increase physical activity, and for active middle-aged people to continue their activity into old age.

An in vivo screen was performed in search of chemicals capable of enhancing neuron formation in the hippocampus of adult mice. Eight of 1000 small molecules tested enhanced neuron formation in the subgranular zone of the dentate gyrus. Among these was an aminopropyl carbazole, designated P7C3, endowed with favorable pharmacological properties. In vivo studies gave evidence that P7C3 exerts its proneurogenic activity by protecting newborn neurons from apoptosis. Mice missing the gene encoding neuronal PAS domain protein 3 (NPAS3) are devoid of hippocampal neurogenesis and display malformation and electrophysiological dysfunction of the dentate gyrus. Prolonged administration of P7C3 to npas3(-/-) mice corrected these deficits by normalizing levels of apoptosis of newborn hippocampal neurons. Prolonged administration of P7C3 to aged rats also enhanced neurogenesis in the dentate gyrus, impeded neuron death, and preserved cognitive capacity as a function of terminal aging. PAPERCLIP:

The use of immuno-PET, the combination of PET with monoclonal antibodies (mAbs), is an attractive option to improve tumor detection and mAb quantification. The long-lived positron emitter (89)Zr has ideal physical characteristics for immuno-PET, such as a half-life of 3.27 d, which is compatible with the time needed for intact mAbs to achieve optimal tumor-to-nontumor ratios. Thus far, a major limitation in the use of (89)Zr has been the lack of suitable methods for its stable coupling to mAbs. In this article, practical protocols for reproducible isolation of highly pure (89)Zr and the production of optimal-quality mAb-(89)Zr conjugates are provided.

METHODS

(89)Zr was produced by a (p,n) reaction on natural yttrium ((89)Y), isolated with a hydroxamate column, and used for labeling of premodified mAbs. mAbs were premodified with a novel bifunctional derivative of the chelate desferrioxamine B (Df) via a new linker chemistry. To this end, Df was initially succinylated (N-sucDf), temporarily filled with Fe(III), esterified by use of tetrafluorophenol, and then directly coupled to mAbs. Chimeric mAb (cmAb) U36, directed against head and neck cancer, was used for in vitro and in vivo evaluation. The in vitro stability of cmAb U36-N-sucDf-(89)Zr was assessed in human serum, and its in vivo behavior was evaluated by biodistribution and PET imaging studies in tumor-bearing nude mice. A cmAb U36-Df-(89)Zr conjugate containing a previously described succinimide ring-thioether unit in the linker was used as a reference.

RESULTS

(89)Zr was produced in large batches (6.5-13.5 GBq) and isolated with improved radionuclidic purity (>99.99%) and high yield (>94%). The Df-premodified mAbs gave (89)Zr-labeling efficiencies of 80% within 30 min, resulting in conjugates with preserved integrity and immunoreactivity. With respect to stability, the novel cmAb U36-N-sucDf-(89)Zr conjugate appeared to be superior to the reference conjugate. In vivo, the novel conjugate demonstrated selective tumor targeting, and on PET images obtained at 24, 48, and 72 h after injection of this conjugate, small tumors in the range of 19-154 mg were readily visualized.

CONCLUSIONS

Methods were developed for improved purification of the long-lived positron emitter (89)Zr. Moreover, a novel bifunctional Df chelate was synthesized for the reproducible coupling of (89)Zr to mAbs. The suitability of such conjugates to detect millimeter-sized tumors in xenograft-bearing nude mice was demonstrated.