A 1-step silver-staining technique, requiring only 2 min to perform, is described for the differential staining of nucleolus organizer regions. A protective colloidal developer is used to control the reduction of the silver.

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The Fenholloway River near Perry, Florida, receives effluent from a paper mill and contains populations of masculinized female eastern mosquitofish, Gambusia holbrooki. A previous study identified the androgen precursor androstenedione at a low concentration (0.14 nM) in water samples from the river. The present study makes use of a toxicity identification and evaluation approach that includes solid phase extraction and high pressure liquid chromatography purification, androgen receptor transcription assays, and liquid chromatography mass spectroscopy to identify and characterize steroids in the Fenholloway River sediment. Androstenedione (2.4 nM) and progesterone (155 nM) were identified in the river sediment at concentrations greater than in the river water column (0.14 nM androstenedione, and 6.5 nM progesterone). Spring Creek, a comparison stream that does not receive mill effluent, contained low levels of progesterone (0.3 nM) but no androstenedione in the sediment. The data are consistent with the hypothesis that pine pulp-derived phytosteroids in the paper mill effluent accumulate in river sediment where they are converted by microbes into progesterone and this into androstenedione and other bioactive steroids. Equally important is that normal streams with much less organic matter still contain progesterone, but at dramatically lower levels. The presence of androgens and androgen precursors in the river water and sediment likely contributes to the masculinized phenotype of the female Gambusia holbrooki in the Fenholloway River.

Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription. The silver preferentially stained the known locations of 18S + 28S ribosomal cistrons. Cytochemical tests revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA. As rRNA transcription proceeds, the quantity of silver stainable proteins progressively increases. The silver procedure can be used to visualize gene activity at the rDNA sites with conventional light microscopy.

Effluent from a paper mill discharging into the Fenholloway River, Taylor County, Florida, USA, contains chemicals that masculinize females of the resident population of eastern mosquitofish (Gambusia holbrooki), as evidenced in females by elongated anal fins, which is normally a male-specific trait. To identify androgenic components in the effluent, water collected from the Fenholloway River and a control tributary was fractionated using solid-phase extraction and reverse-phase high-performance-liquid chromatography. Two Fenholloway River fractions induced androgen receptor-dependent transcriptional activity in transient transfection cell culture assays. Of these, androstenedione was confirmed by liquid chromatography-mass spectrometry with multiple reaction monitoring.

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