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Female rats were fed semi-purified diets containing 10% safflower oil or 10% soybean oil for six weeks prior to mating and through-out pregnancy and lactation. The progeny were weaned to the diet of the dam. Physical, neuromotor and reflex development was monitored in the progeny prior to weaning and learning ability of the mature progeny was assessed in a simple Y-maze test. Brain lipid analyses were conducted in the progeny at birth, 21 and 210 days of age. Inclusion of soybean oil in the diet resulted in higher levels of 22:6omega3 and lower levels of 22:5omega6 in the brain ethanolamine glycerophosphatides. The nature of the dietary fat exerted no effect on the physical development, onset of reflexologic responses or onset of neuromotor co-ordination in the pups. The soybean oil-fed animals spent more time in certain neuromotor activities possibly associated with explorative drive than did their safflower oil-fed counterparts. The performance of the mature soybean oil-fed progeny in the discrimination-learning test was superior to that of progeny fed safflower oil. The association of superior learning capacity with dietary soybean oil-induced incorporation of omega3 fatty acids into the brain glycerophosphatides is offered as support for an essential role for dietary linolenic acid for the young rat.

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The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu 340 is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4 region of the PI-9 RCL is important for interaction with granzyme B, and that the P4 residue ( ). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-GluPro-Asp) fused to the PI-9 P1-P4 sequence was efficiently cleaved by granzyme B (k cat /K m 7.5 ؋ 10 5 s ؊1 M ؊1 ) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k cat /K m 6.7 ؋ 10 4 s ؊1 M ؊1 ) and confirms that P residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu 344 forms a salt bridge with Lys 27 of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4 Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4 substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.