The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).

SummaryThe role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca 2 +-activated potassium channels (Pc,c,) and voltage-gated potassium channels (PK,v) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (Ib2 and interferon 3') are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,C~ and Pg.v. we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for Pg, v. Theses studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca2+-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

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Ninety-six 7-month-old Hereford heifers (two replicates of 48, one year apart) were individually fed for 2 years a basal ration containing .14% phosphorus on an "as fed" basis, which approxiamted 66% NRC recommendations. Forty-eight of the animals were limited to this low phosphorus (P-) diet. The other 48 (P+) received sufficient monosodium phosphate, top dressed on the basal ration, to elevate phosphorus intake to .36% on an "as fed" basis (174% NRC recommendations). The average daily weight gain for all groups was .45 kg. Feed efficiency was similar for the two groups. There was no evidence of lack of appetite or depraved appetite in either group; nor was there a difference in age of puberty. The P-cattle had a 96% pregnancy rate with 91% live calves compared to 100% and 93%, respectively, for the P+ cattle. These differences were not significant. After 9 months on trial (approximately 16 months of age) no difference in rib bone morphology was discernable from microradiographs. Blood and serum levels of phosphorus temporarily increased in the P+ cattle, but at 16 months of age were comparable to the less fluctuating values in the P-cattle. The phosphorus levels for bone and muscle were similar each year and for each treatment. Urine and fecal phosphorus excretion was significantly higher in the P+ than in the P-cattle.

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Accurate quantitative determination of oxalate in chemical analyses of edible plants is important because oxalate is a poisonous compound. The traditional KMn04-oxalate reduction-oxidation (Dye) method of analyzing for plant oxalate content presents two problems: (a) Plant pectins are not entirely separated in the determination of watersoluble oxalate and interfere in the oxidation of oxalate; and (b) a brownish-black precipitate containing ferrous iron is precipitated at the same pH as calcium oxalate and the precipitate interferes in determination of both water- and acid-soluble oxalate. Our modification of the Dye method consists of (a) using double-distilled water as the diluting medium, (6) increasing the acidity of the water-soluble oxalate medium after plant digestion to obtain complete removal of pectins, and (c) eliminating the ferrous iron-containing precipitate by filtration. Oxalate in the ferrous iron-containing precipitate is determined as calcium and magnesium salts by atomic absorption spectrophotometry; the value thus obtained is added to the oxalate value from the oxidation- reduction method. An indirect analytic method for oxalate, in which all of the oxalate was precipitated as the calcium salt and then measured by atomic absorption spectrophotometry, proved to be inaccurate

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