Architectural organisation of neuronal activity-associated magnetic fields: a hypothesis for memory

Despite intensive investigation into the mechanisms underlying the memory process, the physical bases for this superior cognitive function remain elusive. Neuronal activity-associated magnetic fields (NAAMFs) hypothesis of memory suggests that items of information are stored as three-dimensional bundles of magnetic fields associated to the complex but extremely organised cerebral cortex. The present paper proposes a plausible architectural organisation of neuronal activity-associated magnetic fields that may explain how information could be stored in the human cerebral cortex. Magnetic fields generated as consequence of neuronal minicolumns activation could modify the basal "electromagnetic status" of the closest astrocytes allowing codification and storage of information.     

Wilson disease: novel mutations in the ATP7B gene and clinical correlation in Brazilian patients

Wilson disease (WD) is a rare inherited autosomal recessive disorder caused by a defect in a metal transporting P-type ATPase, resulting in copper overload in various tissues and cells. The aim was to assess both the phenotype in Brazilian WD patients and the corresponding ATP7B genotype. Sixty subjects belonging to 46 pedigrees diagnosed as WD were included in this study. Direct sequencing of all 21 exons within ATP7B and their flanking introns was performed. Demographic, clinical, laboratory and histopathological data at the time of diagnosis were obtained. We identified twenty-five mutations, twelve of them reported for the first time. The c.3402delC mutation had the highest allelic frequency (30.8%), followed by the c.2123T>C (p.L708P) (16.7%). Exons 8 and 15 were the site of 62.5% of the mutations. The common European mutation c.3207C>A (p.H1069Q) was not present at all. Phenotype varied greatly among individuals with the same ATP7B genotype. Our data confirm the heterogeneity of ATP7B genotype in Brazilian WD patients. The mutational spectrum is compatible with the Brazilian history of Mediterranean immigration; however, new mutations, and different frequencies and phenotype associated with the previously known mutations characterize this population. Exons 8 and 15 should be preferentially screened in WD cases from Brazil. Phenotype variation among subjects with the same ATP7B genotype suggests that modifying factors play an additional role in the pathogenesis of WD.     

Synaptonemal complex analysis in spermatocytes and oocytes of rainbow trout,Oncorhynchus mykiss (Pisces,Salmonidae): the process of autosome and sex chromosome synapsis

The surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of rainbow trout in order to visualize the process of autosome and sex chromosome synapsis in this species. The structure of lateral elements (LEs) of the SC and the chromosome synapsis process at the stages of leptotene, zygotene and pachytene are described. Comparative analysis of SCs of spermatocytes and oocytes showed a difference in the synaptic process, i.e. in spermatocytes all LEs were synapsed before the appearance of centromeric regions in the biarmed elements, while in the oocytes some fully synapsed LEs, including the centromeric region of the biarmed elements, were found together with fully or partially unsynapsed LEs. In males the sex chromosome synapsis starts only after all autosomes have synapsed. Irregular synapses involving three or four LEs were found in 3.4% of the cells analyzed in mid or late zygotene. Multivalents were found in males and females. Some aspects of initial meiotic development and their implications in rainbow trout cytogenetics, genetics and evolution are discussed.     

Indirect enzyme‐linked Immunosorbent assay for detection of cow's milk in goat's milk

An indirect enzyme‐linked immunosorbent assay (ELISA) has been developed for the specific detection of cow's milk (1–25%) in goat's milk. The test uses polyclonal antibodies raised in rabbits against bovine whey proteins (BWP). The anti‐BWP antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized BWP. The anti‐BWP antibodies were biotinylated and rendered cow's milk specific by mixing them with lyophilized ovine and caprine whey proteins. Streptavidin‐peroxidase was used to detect the biotinylated anti‐BWP antibodies bound to bovine milk proteins immobilized on 96‐well plates. The colour developed by the subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of goat's milk containing variable amounts of cow's milk.     

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Micronuclei induced by alachlor, mitomycin-C and vinblastine in human lymphocytes: presence of centromeres and kinetochores and influence of staining technique

Antikinetochore antibodies and fluorescence in situ hybridizationwith an alphoid centromeric probe were applied to the cytokinesis-blockmicronucleus (MN) assay to study the suitability of these methodologiesto detect clastogenic/aneugenic activity in isolated human lymphocytes.The chemicals selected for this study were the herbicide alachlor,the clastogen mitomycin-C (MMC), and the aneugen vinblastinesulphate (VBL). Futhermore, MN frequencies obtained from slidesstained with May–Grünwald–Giemsa (MGG) andwith the DNA fluorochrome 4', 6'diamidino-2-phenylindole (DAPI)were compared to check if the DNA-specific DAPI facilitateda more accurate recording of MN than the unspecific MGG. Theresults showed that the detection of kinetochores (KC) or centromeres(CM) within MN are equally reliable and sensitive techniquesto study the mode of action of clastogenic and aneugenic agents.The comparison of CM and KC detection in control cultures suggestedthat up to 17% of spontaneous chromosomecontaining MN may bedue to KC disruption, whereas the majority are caused by dysfunctionin other components of the mitotic apparatus. Alachlor (7.5–20µg/ml) and MMC (0.6 µM) acted as pure clastogenswithout aneugenic activity, inducing exclusively KC- and CM-negativeMN. VBL produced primarily KC- and CM-positive MN, in accordancewith its known mechanism of action. A comparison between CMand KC data in the VBL treatment suggested that some 7% of KC-containingMN may not be detected by the probe. The frequencies of MN weregenerally higher in slides stained with DAPI than in those stainedwith MGG, especially in controls and clastogen-treated cultures.This finding probably reflects an underestimation with MGG ofsmall, light MN indistinguishable from the cytoplasmic background. ⁴To whom correspondence should be addressed