Disruption of blastomeric F-actin: a potential early biomarker of developmental toxicity in zebrafish

The expression of at least some biomarkers of toxicity is generally thought to precede the appearance of frank pathology. In the context of developmental toxicity, certain early indicators may be predictive of later drastic outcome. The search for predictive biomarkers of toxicity in the cells (blastomeres) of an early embryo can benefit from the fact that for normal development to proceed, the maintenance of blastomere cellular integrity during the process of transition from an embryo to a fully functional organism is paramount. Actin microfilaments are integral parts of blastomeres in the developing zebrafish embryo and contribute toward the proper progression of early development (cleavage and epiboly). In early embryos, the filamentous actin (F-actin) is present and helps to define the boundary of each blastomere as they remain adhered to each other. In our studies, we observed that when blastomeric F-actin is depolymerized by agents like gelsolin, the blastomeres lose cellular integrity, which results in abnormal larvae later in development. There are a variety of toxicants that depolymerize F-actin in early mammalian embryos, the later consequences of which are, at present, not known. We propose that very early zebrafish embryos (~5-h old) exposed to such toxicants will also respond in a like manner. In this review, we discuss the potential use of F-actin disruption as a predictive biomarker of developmental toxicity in zebrafish.     

Targeting Cdk5 Activity in Neuronal Degeneration and Regeneration

The major priming event in neurodegeneration is loss of neurons. Loss of neurons by apoptotic mechanisms is a theme for studies focused on determining therapeutic strategies. Neurons following an insult, activate a number of signal transduction pathways, of which, kinases are the leading members. Cyclin-dependent kinase 5 (Cdk5) is one of the kinases that have been linked to neurodegeneration. Cdk5 along with its principal activator p35 is involved in multiple cellular functions ranging from neuronal differentiation and migration to synaptic transmission. However, during neurotoxic stress, intracellular rise in Ca²⁺ activates calpain, which cleaves p35 to generate p25. The long half-life of Cdk5/p25 results in a hyperactive, aberrant Cdk5 that hyperphosphorylates Tau, neurofilament and other cytoskeletal proteins. These hyperphosphorylated cytoskeletal proteins set the groundwork to forming neurofibrillary tangles and aggregates of phosphorylated proteins, hallmarks of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease and Amyotropic Lateral Sclerosis. Attempts to selectively target Cdk5/p25 activity without affecting Cdk5/p35 have been largely unsuccessful. A polypeptide inhibitor, CIP (Cdk5 inhibitory peptide), developed in our laboratory, successfully inhibits Cdk5/p25 activity in vitro, in cultured primary neurons, and is currently undergoing validation tests in mouse models of neurodegeneration. Here, we discuss the therapeutic potential of CIP in regenerating neurons that are exposed to neurodegenerative stimuli.     

Analysis of chromatin of the brain of young and old rats by nick-translation

Chromatin of the brain of young (22-23 week) and old (118-119 week) rats has been analysed by nick-translation reaction following its digestion by DNaseI, EcoRI, MspI and HpaII. The incorporation of (3H)-dTMP in the old is only about 50 percent of that of the young. The difference in the incorporation following digestion of nuclei by MspI and HpaII that quantitate the degree of methylation of internal cytosines in the 5' CCGG 3' sequences, is nearly two-fold higher in the old. These data indicate that the chromatin undergoes increasing condensation as a function of age. One of the contributory factors may be increasing methylation of DNA. This may decrease the active fraction of chromatin.     

Conformational changes in the chromatin of the brain of developing rats and its modulation by zinc chloride

Conformational changes in the chromatin of the brain were studied during the development of the rat (3-, 14-and 30-day old) using microccoccal nuclease (MCN) and DNase I. The rate and extent of digestion of chromatin by MCN is not altered during development. However, pre-incubation of slices of the cerebral cortex with ZnCl₂ increases the initial rate of digestion by MCN by 2–3-fold, and also enhances the production of monomer DNA. The rate and extent of digestion of chromatin by DNase I is greater in an early stage of development. The initial rate of digestion by DNase I is stimulated by 3–4-fold after ZnCl₂ treatment. These data show that changes occur in the conformation of chromatin, particularly in the internucleosomal region of brain cells as they pass from dividing to the non-dividing state.     

Fundulus heteroclitus vitellogenin: The deduced primary structure of a piscine precursor to noncrystalline,liquid-phase yolk protein

We have cloned and sequenced a cDNA encoding a vitellogenin (Vtg) from the mummichog, Fundulus heteroclitus, an estuarine teleost. We constructed a liver cDNA library against RNA from estrogen-treated male mummichogs. Five overlapping cDNA clones totalling 5,197 by were isolated through a combination of degenerate oligonucleotide probing of the library and PCR. The cDNA sequence contains a 5,112 by open reading frame. The predicted primary structure of the deduced 1,704-amino-acid protein is 30–40% identical to other documented chordate Vtgs, establishing this Vtg as a member of the ancient Vtg gene family. Of the previously reported chordate Vtg sequences (Xenopus laevis, Gallus domesticus, Ichthyomyzon unicuspis, and Acipenser transmontanus), all four act as precursor proteins to a yolk which is eventually rendered insoluble under physiological conditions, either as crystalline platelets or as noncrystalline granules. The yolk of F. heteroclitus, on the other hand, remains in a soluble state throughout oocyte growth. The putative F. heteroclitus Vtg contains a polyserine region with a relative serine composition that is 10–20% higher than that observed for the other Vtgs. The trinucleotide repeats encoding the characteristic polyserine tracts of the phosvitin region follow a previously reported trend: TCX codons on the 5 end and AGY codons toward the 3 end. Whether the difference in Vtg primary structure between F. heteroclitus and that of other chordates is responsible for the differences in yolk structure remains to be elucidated. As the first complete teleost Vtg to be reported, these data will aid in designing nucleotide and immunological probes for detecting Vtg as a reproductive status indicator in F. heteroclitus and other piscine species.Abbreviations AGY AG(T or C) - TCX TC(AGC or T) - Lv lipovitellin - Pv phosvitin - Vtg vitellogenin Correspondence to: G.J. LaFleur, Jr.     

Induction and properties of pyruvate kinase of the cerebral hemisphere of rats of various ages

The induction of pyruvate kinase (ATP: pyruvate 2-O phosphotransferase, EC 2.7.1.40; PK) by estradiol or testosterone in the cerebral hemisphere of male and female rats of different ages was studied. There is a marked decrease in the activity of the enzyme of normal male and female rats with increasing age. Orchiectomy decreases its activity in young and old rats, but not in adult rats. Ovariectomy decreases its activity significantly in all the ages. Estradiol (50μg/100g body wt.) induces its activity in castrated male and female rats, but its effect is greater in female rats. A higher dose of estradiol (100μg/100g body wt.) has age- and sex-dependent induction. Testosterone (50 and 100μg/100g body wt.) has very little effect on its activity in castrated male and female rats of the three ages. The K_m values for PEP and ADP, and the K_t values for ATP and l -phenylalanine for the partially purified enzyme of the cerebral hemisphere of normal young and old rats are the same. Preincubation of the enzyme with l -alanine reverses the l -phenylalanine induced inhibition. The concentration of l -alanine required for this effect is the same for both ages. The concentration of Mg²⁺ required to reverse the inhibitory effect of ATP is the same for young and old rats. Estradiol and testosterone added directly to the incubation mixture do not have any effect on the enzyme activity. These data suggest that the nature of the enzyme molecule does not change with age, but that its induction properties change with age.