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Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.

Alzheimer's disease (AD) is associated with a progressive loss of locus ceruleus neurons. These noradrenergic neurons receive a major afferent projection from epinephrine neurons in epinephrine cell groups in the brainstem. The epinephrine neurons have a specific enzymatic marker, phenylethanolamine N-methyltransferase (PNMT), which allows them to be identified chemically and immunohistochemically. We have previously reported a decrease in PNMT in brains of patients with AD. We now report that the decrease in PNMT activity in projections to the locus ceruleus is not due to the loss of epinephrine neurons, although up to 33% of these neurons are atrophic. The decrease in presynaptic PNMT does, however, correlate with the loss of postsynaptic locus ceruleus neurons in brains from AD patients. The percentage of degenerating neurons in the epinephrine nuclei also correlates significantly with the amount of loss of locus ceruleus neurons in AD. In addition, there is a 55% decrease in mitogen activity, a nonspecific measure of growth or maintenance factors, in dialysed locus ceruleus extracts from the AD patients compared to those from control subjects. The mitogen activity in the locus ceruleus was significantly correlated with PNMT activity and with the density of locus ceruleus neurons in all cases examined. These findings provide evidence for the hypothesis that retrograde degeneration is a mechanism of neuronal degeneration in AD and suggest that trophic factors may play a role in this process.

Bovine brain-derived growth factor (BDGF), whose biochemical properties resemble those of endothelial cell growth factor (ECGF) and brain-derived acidic fibroblast growth factor (acidic FGF), is able to promote colony formation of normal rat kidney fibroblasts (NRK cells) in soft agar. As in the case of transforming growth factor beta (TGF beta), EGF potentiates the anchorage-independent growth promoting activity of BDGF. In the presence of EGF (5 ng/ml), the optimal concentration of BDGF for stimulation of anchorage-independent of NRK cells is approximately 0.5 ng/ml. At higher concentrations, BDGF becomes inhibitory. The anchorage-independent cell growth promoting activity of BDGF differs from that of TGF beta in acid and reducing agent stability.

This paper reports a precise method that is shorter than previously reported methods for the quantitative determination of N1-methylnicotinamide (MNA) in urine. The method employs a single column chromatographic isolation step, followed by high-pressure liquid chromatographic (HPLC) analysis. Potential interfering substances present in urine are removed during the column chromatography step. The combined MNA fractions eluted from this column were collected and concentrated for quantitative assay of MNA by HPLC. HPLC analysis was effected in less than 15 min using a strong cation- exchange column eluted with 0.25 M ammonium dihydrogen phosphate (pH 4.3). Linearity of MNA detection by HPLC at 254 nm extended below 20 ng, with an average recovery of 101% for 150, 250 and 500 microgram MNA added to 5 ml or urine.

Nineteen of 20 healthy Oriental adults living in the United States developed abdominal cramps and diarrhea after ingesting an amount of lactose equivalent to that in one quart of milk; 14 reported similar symptoms after one or two glasses of milk; all had consumed milk as infants without having such symptoms. Two of 20 Caucasians tested were intolerant to milk and lactose. Many Orientals therefore may have a genetically determined lactase deficiency that may lead to intolerance to milk. Since lactase deficiency is also common among Negroes, the bulk of the world's adult population is probably intolerant to milk.