Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase.

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N-acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C-acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.

Rapid Detection of Psychrotrophic Bacteria In Manufacturing Grade Raw Milks.

Methods to rapidly assess the bacteriological quality of raw milk were investigated. Whereas direct microscopic count, modified psychrotrophic plate count, and direct epifluorescent filter technique (DEFT) did not correlate well with initial psychrotrophic bacterial count of raw milk, improvements were obtained after preincubation of the milk samples. The best preincubation conditions were identified as 30°C for 6 h, 21°C for 10 h, 13°C for 15 h, 13°C for 20 h, or 7°C for 37 h. The "square root" equation was applied to the data, and a model was produced for predicting growth of the native microflora of raw milk. Using this equation, a DEFT count after preincubation of the milk at 21°C for 10 h could accurately predict the initial psychrotroph count and the count after storage of the milk at 6°C for 48 h.

Temperature-dependent malignant invasion in vitro by frog renal carcinoma-derived PNKT-4B cells.

The northern leopard frog, Rana pipiens, may be afflicted with a herpesvirus-transmitted renal carcinoma which has the interesting property that its metastatic behavior is temperature-related. PNKT-4B is a cell line derived from a pronephric carcinoma arising in a tadpole. We sought to ascertain if invasion of normal tissue by PNKT-4B cells in three-dimensional confrontation culture in vitro is similarly temperature-dependent. Normal fragments of tadpole and frog organs are invaded by PNKT-4B cells at 28 degrees C but not at 20 degrees C or 21 degrees C. PNKT-4B cells failed to invade tadpole tissues at 7 degrees C. A temperature critical for invasion was sought. Temperatures of 21 degrees C and cooler are invasion-restrictive and 23 degrees C and warmer are invasion-permissive under the conditions of this study. Identification of a critical permissive temperature allows for the characterization of biochemical events which may be activated at the same temperature. The biochemical changes, which are selectively activated and subsequently repressed as tumor cells are cycled through invasion-permissive and invasion-restrictive temperatures, become compelling candidates as reactions involved in, or causal for, malignant invasion.

Invasion in vitro by explants of Lucke renal carcinoma cocultured with normal tissue is temperature dependent.

Fragments of renal carcinoma of the northern leopard frog, Rana pipiens, were cocultured in vitro with small pieces of tadpole heart, frog heart and frog kidney with gyrotory shaking for up to 14 days at 21 degrees C and 28 degrees C. No invasion by renal carcinoma occurred in confrontation cultures at 21 degrees. However, the three normal tissues were invaded by renal carcinoma in confrontation cultures incubated at 28 degrees C. Invasion in vitro by histologically typical renal carcinoma is thus similar to temperature-dependent invasion by the renal carcinoma-derived cell line PNKT-4B and affords an opportunity for the identification of cell or biochemical events which may be activated at invasion-permissive temperature. Cell or biochemical events which are selectively activated and subsequently repressed as the renal tumor is incubated at invasion-permissive and invasion-restrictive temperatures become significant candidates as events involved in, or causal for, malignant invasion.