Increased posthatching mortality and loss of sexually dimorphic gene expression in alligators (Alligator mississippiensis) from a contaminated environment

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.     

Unexpected ceiling of genetic differentiation in the control region of the mitochondrial DNA between different subspecies of the ayu Plecoglossus altivelis

Sequence analyses of the non-coding, control region (CR) and coding region of the ND4-tRNA(Ser) genes in the mitochondrial DNA (mtDNA) were conducted for populations of the ayu Plecoglossus altivelis altivelis and the Ryukyu-ayu P. a. ryukyuensis. The level of genetic differentiation between the two subspecies evaluated from the CR data was substantially low, when comparing with that estimated from ND4-tRNA(Ser) gene region data, as well as those from nuclear genome data sets. By contrast, the differentiation between subspecies in the ND4-tRNA(Ser) gene region was substantial, being consistent with the results from the previous nuclear genome analyses. Results of UPGMA and minimum spanning network analyses also implied the unexpected ceiling of genetic differentiation in the CR. These results suggest that the CR does not reflect accurately the level of overall genetic differentiation between the populations of the ayu, but other coding regions of the mtDNA do reflect it so that the mtDNA on the whole may function as a rich source of useful markers for genetic assessment of populations of this species.     

An improved enrichment broth for isolation of Escherichia coli O157, with specific reference to starved cells,from radish sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

The role of circulating ghrelin in growth hormone (GH) secretion in freely moving male rats

To examine the physiological significance of plasma ghrelin in generating pulsatile growth hormone (GH) secretion in rats, plasma GH and ghrelin levels were determined in freely moving male rats. Plasma GH was pulsatilely secreted as reported previously. Plasma ghrelin levels were measured by both N-RIA recognizing the active form of ghrelin and C-RIA determining total amount of ghrelin. Mean +/- SE plasma ghrelin levels determined by N-RIA and C-RIA were 21.6 +/- 8.5 and 315.5 +/- 67.5 pM, respectively, during peak periods when plasma GH levels were greater than 100 ng / ml. During trough periods when plasma GH levels were less than 10 ng / ml, they were 16.5 +/- 4.5 and 342.1 +/- 29.8 pM, respectively. There were no significant differences in plasma ghrelin levels between two periods. Next, effect of a GH secretagogue antagonist, [D-Lys-3]-GHRP-6, on plasma GH profiles was examined. There were no significant differences in both peak GH levels and area under the curves of GH (AUCs) between [D-Lys-3]-GHRP-6-treated and control rats. These findings suggest circulating ghrelin in peripheral blood does not play a role in generating pulsatile GH secretion in freely moving male rats.     

T-588 Protects Motor Neuron Death Against Glutamate-Induced Neurotoxicity

To examine the possible neuroprotective effect of T-588 against glutamate-induced neurotoxicity, we analyzed the pharmacological utility of T-588 in a postnatal organotypic culture model of motor neuron degeneration. Treatment with 10^–5 M of glutamate resulted a motor neuron loss and decreased activity of choline acetyltransferase (ChAT). Cotreatment of 10^–5 M of glutamate and T-588 revealed a protective effect against motor neuron death and decreased ChAT activity. We concluded that T-588 may play important roles in the survival and maintenance of spinal motor neurons in its neuroprotection against glutamate-induced neurotoxicity. Our data may provide a rationale for designing a therapeutic strategy for protection against pathologically induced motor neuron damage or cell death such as amyotrophic lateral sclerosis and motor neuropathy.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.     

Imidazole-induced cell death,associated with intracellular acidification,caspase-3 activation,DFF-45 cleavage,but not oligonucleosomal DNA fragmentation

Intracellular acidification is known to be involved in the initiation phase of apoptosis. However, the necessity of intracellular acidic conditions in the execution phase of apoptosis remains unknown. In this study, we found that in HL-60 cells imidazole induces cell death, associated with intracellular acidification, caspase-3 activation and DFF-45 cleavage, but not oligonucleosomal DNA fragmentation. A caspase inhibitor prevented cell death but not intracellular acidification. When pHi was neutralized by changing from imidazole-containing medium to fresh medium, oligonucleosomal DNA fragmentation and increased caspase-3 activity was observed in the imidazole-treated HL-60 cells. Furthermore, the DNA fragmentation induced by intracellular neutralization was inhibited by caspase inhibitor treatment. These results indicate that imidazole induces caspase-dependent cell death, and suggest that maintaining pHi in the neutral range is essential for the induction of oligonucleosomal DNA fragmentation in the execution phase of apoptosis.