Neurotoxic effect of <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> by neurite inhibition on the neuroblastoma cell line

In the present study, antiproliferative, apoptotic and especially neurotoxic effects of Caulerpa racemosa var. cylindracea dry and wet extracts on mouse neuroblastoma cell line, NA2B were investigated by neurotoxicity screening test (NST). C. racemosa var. cylindracea wet and dry extracts were obtained by methanol (MT) extraction. The effect of the extracts on viability and proliferation was measured by MTT. NA2B cells were induced to differentiate using 1 μM dcAMP and the amount of inhibition of growing neurites in different dilutions (50, 35, 25, 15, 10 and 5 μl/ml) by extracts was measured. The number of apoptotic cells was computed by TUNEL method using cells in culture. It was found that majority of the cells died with dry extract above the level of 15 μl/ml due to the MT effect. Below this level, on the other hand, presence of cell death and antiproliferative effect was noted due to the toxic effects of C. racemosa var. cylindracea which was independent of MT. In all doses of wet extracts, similar but less prominent dose-dependent effects were observed. Below the level of 15 μl/ml, mild toxic effect presented itself with neurite inhibition. In addition to the toxic, apoptotic and antiproliferative effects of C. racemosa var. cylindracea, its neurotoxic effects possessing property at low concentrations which manifesting itself by neurite inhibition was also showed. This species offers a potential for developing new drugs due to its antiproliferative, toxic and apoptotic effects. Nevertheless, its neurotoxic effect is a factor to be considered as multifunctional agents especially in neuronal metabolism.     

Initial maternal meiotic I error leading to the formation of a maternal i(2q) and a paternal i(2p) in a healthy male

We report on the investigation of the parental origin and mode of formation of the two isochromosomes, i(2p) and i(2q), detected in a healthy adult male. Conventional cytogenetic analysis revealed the proband's lack of structurally normal chromosomes 2, these being replaced by an i(2p) and an i(2q). Investigation of the parental origin of the isochromosomes revealed a paternal origin of the i(2p) chromosome and a maternal origin of the i(2q) chromosome. Thus, the formation of both isochromosomes, or at least of the paternal i(2p), appears to have occurred postzygotically. Interestingly, whilst a paternal isodisomy was observed for the entire 2p, maternal heterodisomy was detected for two segments of 2q, separated by a segment showing isodisomy. The results are indicative of an initial error (non-disjunction or i(2q) formation) concerning the maternal chromosomes 2 during meiosis I, which likely favored the subsequent mitotic recombination event resulting in the presence of two isochromosomes. To the best of our knowledge this is the first case of an initial meiotic error, followed by postzygotic trisomy rescue through the formation of isochromosomes, resulting in a normal phenotype. A prenatal detection, by cytogenetic and molecular analysis, of such chromosome abnormality would have led to the incorrect conclusion of a most likely poor prognosis for the fetus.     

The immune system as a chronotoxicity target of the anticancer mTOR inhibitor everolimus

The circadian timing system controls many biological functions in mammals including xenobiotic metabolism, detoxification, cell proliferation, apoptosis and immune functions. Everolimus is a mammalian target of rapamycin inhibitor, whose immunosuppressant properties are both desired in transplant patients and unwanted in cancer patients, where it is indicated for its antiproliferative efficacy. Here we sought whether everolimus circadian timing would predictably modify its immunosuppressive effects so as to optimize this drug through timing. C57BL/6J mice were synchronized with light-dark 12h:12h, with L onset at Zeitgeber Time (ZT) 0. Everolimus was administered orally to male (5 mg/kg/day) and female mice (15 mg/kg/day) at ZT1, during early rest span or at ZT13, during early activity span for 4 weeks. Body weight loss, as well as hematological, immunological and biochemical toxicities, were determined. Spleen and thymus were examined histologically. Everolimus toxicity was less severe following dosing at ZT13, as compared to ZT1, as shown with least body weight inhibition in both genders; least reductions in thymus weight both in males (p < 0.01) and females (p < 0.001), least reduction in female spleen weight (p < 0.05), and less severe thymic medullar atrophy both in males (p < 0.001) and females (p < 0.001). The mean circulating counts in total leukocytes, total lymphocytes, T-helper and B lymphocytes displayed minor and non-significant changes following dosing at ZT13, while they were decreased by 56.9% (p < 0.01), 45.5% (p < 0.01), 43.1% (p < 0.05) and 48.7% (p < 0.01) after everolimus at ZT1, respectively, in only male mice. Chronotherapy of everolimus is an effective way to increase the general tolerability and decrease toxicity on the immune system.     

Conserved sequence motifs, alignment, and secondary structure for the third domain of animal 12S rRNA

Secondary structure models are an important step for aligning sequences, understanding probabilities of nucleotide substitutions, and evaluating the reliability of phylogenetic reconstructions. A set of conserved sequence motifs is derived from comparative sequence analysis of 184 invertebrate and vertebrate taxa (including many taxa from the same genera, families, and orders) with reference to a secondary structure model for domain III of animal mitochondrial small subunit (12S) ribosomal RNA. A template is presented to assist with secondary structure drawing. Our model is similar to previous models but is more specific to mitochondrial DNA, fitting both invertebrate and vertebrate groups, including taxa with markedly different nucleotide compositions. The second half of the domain III sequence can be difficult to align precisely, even when secondary structure information is considered. This is especially true for comparisons of anciently diverged taxa, but well-conserved motifs assist in determining biologically meaningful alignments. Patterns of conservation and variability in both paired and unpaired regions make differential phylogenetic weighting in terms of "stems" and "loops" unsatisfactory. We emphasize looking carefully at the sequence data before and during analyses, and advocate the use of conserved motifs and other secondary structure information for assessing sequencing fidelity.      

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.