Rat cathepsin H-catalyzed transacylation: comparisons of the mechanism and the specificity with papain-superfamily proteases.

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

Induction of differentiation of HL-60 cells by protein kinase C inhibitor, K252a

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH)2D3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH)2D3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.     

Genetic polymorphisms of blood proteins in the troops of Japanese macaques,Macaca fuscata: IV. Erythrocyte esterase polymorphism inMacaca fuscata

Genetic variation at the locus controlling A₁ band of erythrocyte esterase was found in the Japanese macaque,Macaca fuscata. Existence of four alleles,Es-A ₁ ¹ ,Es-A ₁ ² ,Es-A ₁ ³ , andEs-A ₁ ⁴ , controlling the mobility of the band and codominance relation between them were postulated. A majority of the troops examined were monomorphic inEs-A ₁ 1-1 phenotype, and the variant phenotypes were observed to occur only in Yugawara-Ihama, Arashiyama, and Koshima areas.     

Genetic polymorphisms of blood proteins in the troops of Japanese macaques,Macaca fuscata: III. Erythrocyte carbonic anhydrase polymorphism inMacaca fuscata

In order to clarify the genetic relationships between troops of Japanese macaques, the authors have been looking for the genetic variation of blood proteins by electrophoretical technique. In this work the genetic variants of erythrocyte carbonic anhydrase isozymes of 1273 blood samples from 34 troops of this species were examined. The genetic polymorphisms were observed in several troops. The variable troops of CA I were Fukushima, Takasakiyama, Kohchi, Shimane, Shodoshima I, K, and T, Kashima, Kawara, Takasakiyama A, B, and C, and Tomogashima. The variant allele found in these troops is onlyd ₂ allele, which was probably identical with that reported byTashian et al. (1971). The frequencies ofd ₂ allele in the Shodoshima and Kashima troops were very high, and this phenomenon was interpreted as being a result of the founder effect and the random fluctuation of gene frequencies in these troops.     

Carbohydrate composition of the isolated cell walls of dermatophytes

In an attempt to evaluate taxonomic character of sugar composition of dermatophytes, the purified cell walls from 13 species are analyzed on neutral sugar composition by gas liquid chromatography. The results were principally compatible with those obtained by conventional morphological examination. Neutral sugar components of dermatophytes cell walls were mannose and glucose in the ratio of 1∶2.7 for Epidermophyton and 1∶1.4 for Microsporum. There were two types in Trichophyton, in which the ratios of mannose to glucose were 1∶1.6 and 1∶3.8. The cases of Trichophyton ferrugineum and Trichophyton mentagrophytes were exceptional. The ratio of the former was 1∶1.4, which implied the relation to Microsporum group, and the ratio of the latter was 1∶2.3, which was supposed to be the intermediate of two types of Trichophyton group. Albino type cell wall of Epidermophyton floccosum was more rich in glucose than pigmented type one.     

Molecular cloning and expression analysis of novel wheat cysteine protease

A cDNA clone encoding a novel papain-like cysteine protease was isolated from wheat germ (Triticum aestivum). This cDNA encoded a 371-residue protein, designated WCP2, composed of signal peptide followed by a propeptide and a mature protease containing active site residues that are highly conserved among the papain family. The mature WCP2 protein (26 kDa) was detected in the quiescent embryo and its level of expression in the germinating embryo was greatly increased.