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When mouse thymocytes were incubated with staurosporine at low doses (1-100 nM), increased tyrosine phosphorylation of proteins with a molecular weight of 31-34 kD in the detergent-soluble fraction was observed, together with an increase in internucleosomal DNA fragmentation. Incubation with staurosporine for 6 h was enough to trigger and progress of apoptosis. Herbimycin A, a specific inhibitor of protein tyrosine kinases, and cycloheximide, an inhibitor of protein synthesis, reduced the amount of the phosphotyrosine proteins, this being followed by the inhibition of DNA fragmentation and cell death, suggesting a close relationship between protein tyrosine phosphorylation and the induction of apoptosis by staurosporine.

We studied the effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and cell death in CTLL-2 cells, whose growth is IL-2-dependent. The Flavonoids genistein, biochanin A, and apigenin inhibited topoisomerase II to the same extent as etoposide, a specific inhibitor of the enzyme. Methyl 2,5-dihydroxycinnamate (2,5-MeC) also inhibited topoisomerase II, but was less potent than genistein. Herbimycin A and staurosporine did not inhibit topoisomerase II. None of the inhibitors of protein tyrosine kinases examined inhibited topoisomerase I activity. All the inhibitors induced cell death with internucleosomal DNA fragmentation in the presence of IL-2. Genistein, biochanin A, and apigenin induced DNA fragmentation and cell death early in the incubation period and did not alter the profiles of phosphotyrosine proteins in either the lysate or pelleted fractions, indicating that the early cell death was induced by the inhibition of topoisomerase II activity rather than by the inhibition of protein tyrosine kinase activity. 2,5-MeC similarly induced early cell death and DNA fragmentation, but to a lesser extent than genistein presumably due to the inhibition of topoisomerase II activity. Herbimycin A induced a slow increase in DNA fragmentation and cell death, accompanied by a decrease in phosphotyrosine proteins in the pelleted fraction, suggesting that the inhibition of protein tyrosine phosphorylation, presumably of the nuclear proteins, is related to cell death and DNA fragmentation. Staurosporine-induced DNA fragmentation appeared to be due to mechanism(s) other than the inhibition of topoisomerases and protein tyrosine kinases, since it neither altered the profiles of phosphotyrosine proteins nor inhibited topoisomerase activity.

When mouse thymocytes were incubated with staurosporine at low doses (1-100 nM), apoptosis was induced dose- and time-dependently. Staurosporine-induced apoptosis was dependent on macromolecular synthesis, and it was also dependent on protein phosphorylation sensitive to 1-(5-isoquinolinesulfonyl-2-methylpiperazine dihydrochloride (H-7). Whereas, staurosporine at high doses (above 500 nM) did not induce significant DNA fragmentation, rather it inhibited the DNA fragmentation induced by 12-O-tetradecanoyl-13-acetate, A23187, and dibutyrylcyclic AMP, as H-7 did. K252a, a derivative of staurosporine, induced apoptosis, which was inhibited by H-7, even at high doses. These results indicate that staurosporine had a biphasic effect on thymocyte apoptosis, a stimulatory effect at low concentration, and an inhibitory effect at high concentration. K252a had only the former action.

Apoptosis is a controlled death process under regulation by multiple death and survival genes. In the immune systems, apoptosis has important roles in establishment of the mature immune system (embryological programmed cell death), in dynamic maintenance of functional cell number (physiological programmed cell death) and in elimination of unwanted cells (biochemical programmed cell death). The process of apoptosis is regulated by multiple genes and their products depending on different stimuli. The current knowledge on the molecular events of apoptotic cell death, particularly in thymocytes, is reviewed.

Apoptosis, a form of cell death defined by morphological and biochemical characteristics, is involved in many important physiological phenomena including embryogenesis, organ involution, maintenance of homeostasis and biodefense systems of the body. Impaired regulation of apoptosis may play a part in the etiology of malformation and disfunction of various organs causing autoimmune diseases, immunodeficiency diseases or neurodegenerative diseases. Apoptosis is also related to cancer. Elucidation of the mechanism of apoptosis may provide new insights into the pathophysiology of a variety of diseases, and its specific regulation may lead to exciting new therapies for these diseases.

Multiplication of Clostridium botulinum in honey-bees was examined to explain the heavy contamination of honey which may occur with this pathogen. When dead bees were inoculated with C. botulinum spores at levels of 10(2)-10(3) and incubated aerobically for 10 days, the organisms increased to 10(4)-10(5). When botulinum spores were inoculated together with Bacillus alvei, the growth of most strains was significantly enhanced (10(5)-10(7)). Similar results were obtained in bee pupae, but not in bee larvae. The heavy contamination of honey with botulinum spores that we have sometimes encountered may have been caused by contamination from dead bees in which C. botulinum had proliferated.

Internucleosomal DNA fragmentation and cell death were induced dose- and time-dependently by incubation of mouse thymocytes with bis(2,6-dioxopiperazine) derivatives, ICRF-154 and MST-16, inhibitors of topoisomerase II, which do not induce cleavable complex formation. The process was inhibited by actinomycin D and cycloheximide, indicating that the process was an active apoptotic process. Bis(2,6-dioxopiperazine) derivatives have been known to inhibit the etoposide-induced DNA cleavage, but ICRF-154 did not inhibit etoposide-induced apoptosis in thymocytes at 6 h incubation, suggesting that DNA cleavage is not essential for induction of apoptosis by topoisomerase II inhibitors. The alteration of DNA helicity induced by a subtle inhibition of topoisomerase II activity may have an important role in the induction of apoptosis in thymocytes, since topoisomerase II is a major component of the nuclear matrix that can regulate gene expression.

A gene encoding a phosphomannose isomerase from Streptococcus mutans GS-5 was identified immediately downstream from the fructokinase gene, scrK. Nucleotide sequence analysis of this region revealed an open reading frame (ORF) specifying a putative protein of 316 amino acids. The gene cloned in Escherichia coli expressed strong phosphomannose isomerase activity. The deduced amino acid sequence of the pmi gene has no significant similarity with any of the previously reported phosphomannose isomerase enzymes. Insertional inactivation of the upstream gene, scrK, in S. mutans also drastically reduced phosphomannose isomerase activity and the ability of the organism to utilize mannose as a sole carbon source. These results suggest that the S. mutans pmi gene constitutes an operon with the scrK gene.

Previous studies demonstrated that arabinosylcytosine (ara-C) induced internucleosomal DNA fragmentation and cell death in mouse thymocytes and that those were inhibited by 1-(5-iso-quinoline-sulfonyl)-2-methylpiperazine hydrochloride, an inhibitor of protein kinases. In the present study, we examined the relationship between the DNA fragmentation induced by ara-C and that by agents which activate intracellular signaling and induce apoptosis in mouse thymocytes. 12-O-tetradecanoyl 13-acetate, a phorbol ester capable of activating protein kinase C or A23187, a calcium ionophore, had no effect on ara-C induced DNA fragmentation. However, ara-C induced DNA fragmentation was synergistically enhanced by cAMP and cAMP receptor agonists. Ara-C inhibited the incorporation of choline into the acid soluble and lipid fractions, and cAMP enhanced this inhibition, suggesting that ara-C metabolites interfere with membrane phospholipid metabolism, partly evoking a certain cellular signaling for apoptosis and interacting with cAMP-evoked signaling.

Contrast and glare tests were performed on small intraocular lenses (IOLs) using a new eye model, which was based on and about the same size as the Gullstrand eye model. Lenses with a small optic diameter were inserted and retinal images were observed under the operating microscope. Contrast and glare disability tests were done using the MCT-8000 contrast tester and the Miller-Nadler glare tester. No effect on contrast sensitivity attributable to glare was observed, but under severe glare light conditions a faint reflection around the circumference of the optic was found. Questionnaires from patients implanted with small optic IOLs confirmed that this phenomenon was similar to their symptoms.

Incubation of mouse thymocytes with the protein tyrosine kinase inhibitors herbimycin A and methyl-2,5-dihydroxycinnamate induced a decreased and altered profile of nuclear phosphotyrosine proteins in parallel with an increase in internucleosomal DNA fragmentation and cell death dose-dependently. No change in the profile of cytoplasmic phosphotyrosine proteins was observed. DNA fragmentation was dependent on the synthesis of RNA and protein, suggesting that the inhibition of tyrosine phosphorylation of the nuclear proteins induces apoptosis. DNA fragmentation was enhanced by simultaneous incubation with phorbol esters capable of activating protein kinase C. Genistein, another inhibitor of protein tyrosine kinase, induced DNA fragmentation more rapidly than herbimycin A, but there was no predominant alteration of phosphotyrosine proteins in early incubation, suggesting that genistein may induce apoptosis by a mechanism other than direct inhibition of protein tyrosinekinase activity.

During T-lymphocyte differentiation in the thymus, the majority of thymocytes die by apoptosis in situ. This process is characterized by internucleosomal DNA fragmentation and is induced by a number of stimuli including glucocorticoids, calcium ionophore, cAMP and 12-o-tetradecanoylphorbol 13-acetate (TPA). In this study, the effect of cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma) on the programmed cell death of thymocytes was examined by measuring DNA fragmentation and LDH release. TNF-alpha and IFN-gamma had no effect on DNA fragmentation in control and TPA, or A23187-treated thymocytes. Both human and murine rTNF-alpha enhanced cAMP-induced programmed cell death dose-dependently, but IFN-gamma had no effect on the process. TNF-alpha did not stimulate cAMP accumulation in control or 2-chloroadenosine-treated thymocytes. TPA markedly stimulated cAMP-induced DNA fragmentation as a result of 6 h incubation, whereas TNF-alpha did not. Thus TNF-alpha did not appear to activate protein kinase C directly. The effect of TNF-alpha was observed in the cell preparations from which adherent cells had been removed, suggesting that cytokines secreted by adherent cells in response to TNF-alpha are not involved in the process. The enhancement of cAMP-induced DNA fragmentation was observed in CD4+CD(8+)-double positive cells, but not in CD4+CD(8-)-single positive cells. The results of the present study indicate that a physiological cytokine, TNF-alpha, may modulate programmed cell death in immature thymocytes in concert with cAMP.

The crystal structure of an oligo-1,6-glucosidase from Bacillus cereus ATCC7064 was determined by the X-ray diffraction method at 3.0 A resolution. The structure was solved by the multiple isomorphous replacement method and refined to a crystallographic R-factor of 0.208, using the molecular dynamics refinement program, X-PLOR. The electron density map revealed the folding of a polypeptide chain consisting of 558 amino acid residues. The molecule can be subdivided into three domains (N-terminal domain, subdomain, and C-terminal domain). The N-terminal domain has an (alpha/beta)8-barrel structure called the TIM-barrel structure. The C-terminal domain is characterized by a beta-barrel structure composed of eight antiparallel beta-strands, while the subdomain has a loop-rich structure with a small alpha-helix and a beta-sheet. The enzyme has a large cleft between the N-terminal domain and the subdomain. The cleft leads from the molecular surface to the molecular center. The bottom of the cleft is at the C-terminal end of the parallel beta-strands of the (alpha/beta)8-barrel. These structural features closely resemble those of alpha-amylases from Aspergillus oryzae and pig pancreas.

The effects of the inhibitors of topoisomerase I and II, camptothecin and etoposide, as well as novobiocin and adriamycin, on the DNA fragmentation and viability of mouse thymocytes in primary culture were examined. All inhibitors were shown to produce dose-dependent internucleosomal DNA cleavage by resolving isolated DNA by agarose-gel electrophoresis. The DNA fragmentation seemed to precede cell death, determined on the basis of LDH release, by a few hours. Etoposide-induced DNA fragmentation progressively increased after incubation and was enhanced by pretreatment with phorbol 12,13-dibutyrate, a phorbol ester capable of activating protein kinase C, whereas camptothecin-induced DNA fragmentation increased progressively after 12 h incubation and was unaffected by phorbol 12,13-dibutyrate-pretreatment. The process was also energy-dependent and required RNA and protein synthesis and protein phosphorylation, since it was inhibited by sodium azide, actinomycin D, cycloheximide and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine hydrochloride, a protein kinase inhibitor. DNA fragmentation was also inhibited by zinc ions, suggesting the involvement of a specific endonuclease in DNA cleavage. These phenomena are similar to those detected in thymocytes undergoing apoptosis following exposure to glucocorticoids (Cohen, J.J. and Duke, R.C. (1984) J. Immunol. 132, 38-42). Considering that topoisomerases function in cellular proliferation and differentiation by altering DNA topology, the results suggest that topoisomerases have important roles in T-lymphocyte ontogeny in the thymus and are in part involved in the elimination of autoreactive or harmful cells by an apoptotic process.

Incubation of mouse thymocytes with arabinosylcytosine or 5-azacytidine induced dose-dependent internucleosomal DNA cleavage followed by cell death. This process was RNA- and protein synthesis-dependent, since DNA fragmentation and cell death was inhibited by actinomycin D and cycloheximide. The results suggest that the cytidine analogs induce apoptosis, a programmed cell death, in thymocytes. The DNA cleavage induced by arabinosylcytosine and 5-azacytidine was inhibited by deoxycytidine and cytidine, respectively, suggesting that phosphorylation of these antimetabolites is required to induce DNA cleavage. DNA fragmentation was unaffected by the addition of aphidicolin or 3-aminobenzamide, indicating that DNA cleavage is not due to the inhibition of DNA synthesis or repair. Other antimetabolites, including methotrexate, fluoropyrimidines and thiopurines, failed to induce DNA fragmentation. Arabinosylguanine induced DNA fragmentation similar to that produced by the cytidine analogs, suggesting similarity to the selective sensitivity of T lymphocytes to deoxyguanosine toxicity. The precise mechanism by which DNA cleavage is induced remains unclear, but the present study shows that certain antimetabolites act on cells not only by inhibiting proliferation, but by inducing apoptosis with internucleosomal DNA fragmentation.

Incubation of mouse thymocytes with arabinosylcytosine or 5-azacytidine induced dose-dependent internucleosomal DNA cleavage followed by cell death. This process was RNA and protein synthesis-dependent, since DNA fragmentation and cell death was inhibited by actinomycin D and cycloheximide. The results suggest that the cytidine analogs induce apoptosis, a programmed cell death, in thymocytes. The DNA cleavage induced by arabinosylcytosine and 5-azacytidine was inhibited by deoxycytidine and cytidine, respectively, suggesting that phosphorylation of these antimetabolites is required to induce DNA cleavage. DNA fragmentation was unaffected by the addition of aphidicolin or 3-aminobenzamide, indicating that DNA cleavage is not due to the inhibition of DNA synthesis or repair. Other antimetabolites including methotrexate, fluoropyrimidines and thiopurines failed to induce DNA fragmentation. Arabinosylguanine induced DNA fragmentation similar to that produced by the cytidine analogs, suggesting similarity to the selective sensitivity of T lymphocytes to deoxyguanosine toxicity. The precise mechanism by which DNA cleavage is induced remains unclear, but the present study shows that certain antimetabolites act on cells not only by inhibiting proliferation, but by inducing apoptosis with internucleosomal DNA fragmentation.

In order to mutagenize Streptococcus mutans a marker rescue plasmid, pVA891, was employed. The plasmid was ligated with Sau3AI digested chromosomal DNA fragments from S. mutans GS-5IS3 and the resultant plasmids were amplified in Escherichia coli. These plasmids were then randomly integrated into the chromosome of strain GS-5IS3 following transformation. Lactose-negative transformants were isolated as white colonies on lactose-BTR-Xgal agar plates containing erythromycin. Six lactose-negative mutants representing three different chromosomal sites of integration were isolated from about eight thousand transformants. Mutant chromosomal DNA fragments flanking the plasmids were recovered by a marker-rescue method in E. coli and exhibited phospho-beta-galactosidase activity.

Isoproterenol and prostaglandin E2 increased cAMP levels in mouse thymocytes transiently, but failed to induce significant internucleosomal DNA fragmentation in thymocytes with a 24-hr incubation. However, these substances showed synergistic interaction with forskolin in the accumulation of cAMP and DNA fragmentation. The addition of 12-O-tetradecanoyl 13-acetate, an activator of protein kinase C, with isoproterenol or particularly with prostaglandin E2 enhanced DNA fragmentation. These results indicate that an increase in cAMP mediated by isoproterenol or prostaglandin receptor is involved in thymocyte apoptosis through internucleosomal DNA fragmentation in concert with a second signal, the activation of protein kinase C.

The scrA gene coding for sucrose EnzymeII of the phosphoenolpyruvate dependent phosphotransferase system previously isolated from Streptococcus mutans was fused in vitro to the promoterless lacZ' gene to monitor the expression of the scrA gene. The scrA::lacZ gene fusion was introduced back into S. mutans GS-5IS3 by two independent transformation procedures involving either linear or plasmid DNA to produce both scrA and scrA+ mutants. These mutants should prove useful for analyzing the regulation of sucrose transport in S. mutans.

12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the action of cAMP in DNA cleavage in thymocytes induced by a low concentration of adenosine receptor-site agonists such as adenosine, 2-chloroadenosine and forskolin. The enhancement of DNA cleavage by TPA was also observed in dibutyryl cAMP-treated thymocytes. On the other hand, TPA suppressed accumulation of cAMP by the adenosine receptor-site agonists. These results suggest that activation of protein kinase C inhibits cAMP production, but stimulates cAMP-triggered process to induce DNA cleavage and death of thymocytes.

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18-24 h resulted in 70%-80% fragmentation of DNA into 180-200-bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4+CD8+ cells and in a portion of CD4-CD8+ cells. To investigate whether DNA cleavage is also inducible via antigen-specific receptors, thymocytes were incubated in wells precoated with anti-CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti-CD3 antibody. In this anti-CD3-induced DNA degradation, CD4+CD8+ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti-CD3-induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti-CD3-coated wells revealed that CD4+CD8+ cells were preferentially decreased. Further experiments with purified CD4+CD8+ cells, however, could not define a clear-cut increase of DNA fragmentation when isolated CD4+CD8+ cells were stimulated with anti-CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon-gamma to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.

Two experiments were conducted to assess the usefulness of repeated questioning for the psychophysiological detection of deception. Twenty-four female subjects were instructed to conceal the critical item which they had previously selected, and their skin conductance response (SCR) was simultaneously recorded. In Experiment 1, the amplitudes of SCR to both critical and noncritical items tended to decrease. However, responses to the critical item was consistently larger than that to other noncritical ones during the total of nine presentations in this series where the critical item was pseudo-randomly inserted among the four non-critical items. In Experiment 2, the difference of SCR to the critical and noncritical items was enhanced compared with Experiment 1 at the first exposure in three successive presentations, though this difference was diminished at the following 2nd and 3rd presentations. These results suggest that the successive presentations of stimulus under the detection of deception would be effective to discriminate the responses to critical item from noncritical items, and they are discussed in terms of stimulus significance.

The cellular proteins of hamster tongue squamous cell carcinoma and epithelial dysplastic lesions induced by 9,10-dimethyl-1,2-benzanthracend (DMBA) were analyzed by two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis. Many of the polypeptides were common to these lesions. However, there were thirteen main polypeptides which differed between normal tongue mucosa and tongue carcinoma. In the carcinoma, eight polypeptides (5.2/53 K, 5.8/47 K, 6.9/46 K, 6.9/50 K, 7.1/55 K, 7.2/46 K, 7.3/75 K, 7.4/48 K, pI/MW) were decreased and three polypeptides (4.9/32 K, 6.3/50 K, 7.2/60 K) were increased in an amount. The remaining two polypeptides (4.8/37 K, 4.9/36 K) that were not normally present were appeared in carcinoma, but these two new polypeptides were not specific to carcinoma because they were also present in dysplastic lesions. There were marked differences in the polypeptide between various degree of dysplastic lesions and carcinoma on the one hand and normal mucosa on the others. These results suggests that two-dimensional electrophoresis may have usefulness in further research on transformations from normal mucosa to malignant cancer cells.

Xanthine oxidase, guanase, 5'-nucleotidase, and adenosine deaminase in human epidermis were demonstrated and accurately assayed with about 20 microng. of tissue by the new micro-assay methods which rely on the isolation of isotopically labeled end products from the substrates by electrophoresis on cellulose acetate membranes. These assay methods are rapid, reliable, and sensitive and are highly suitable for studies of small amount of human tissue. Theses methods for the separation of purine derivatives with cellulose acetate membrane will also permit the assays of purine nucleoside phosphorylase and nucleoside kinase.

Purine phosphoribosyltransferase activities in normal and experimental hyperkeratotic epidermis of guinea pig skin were demonstrated quantitatively by a new microassay method. The ratio of HGPRTase with hypoxanthine as a substrate to APRTase activity in normal and hyperkeratotic epidermis was found to be 0.94 and 0.60, respectively. The HGPRTase and APRTase activities expressed as micromoles per gram wet weight per min. were increased in experimental hyperkeratotic epidermis and it is suggested that the salvage pathway for purine nucleotide biosynthesis is activated in experimental hyperkeratotic epidermis. The pH optimum of these enzymes and their stability in the frozen state were also demonstrated.

In the hexokinase isoenzyme pattern on the cellulose acetate membrane electropherogram of the supernatant fraction of Ehrlich ascites tumor cells, a band was detected moving slower than of common Type I isoenzyme. This band was assumed to be due to the presence of a variant of the hexokinase isoenzymes in the cells, since such a band was not detected in the normal tissues, and a change in the intensity of the band in response to a sulfhydryl agent, etc., differed from that of the common Type I and II isoenzymes. The band was also found in AH-7974 and MH-134 ascites tumor cells, in spite of the difference in the species of the tumor-bearing animals and originating tissue.

CTP synthetase (UTP: glutamine ligase (ADP-forming), EC 6.3.4.2) was purified from Ehrlich ascites tumor cells to near homogeneity and found to be a dimer composed of two seemingly identical 66 kDa subunits. The formation of CTP was accompanied by the production of equivalent amounts of ADP from ATP and glutamate from glutamine. The reaction product, CTP, was a potent inhibitor generating sigmoidal kinetics as a function of UTP with an n value of 2.0. UTP and CTP pools in the ascites cells were elevated in an early period (12-16 h) following implantation into the intraperitoneal cavity of mice, whereas ATP, GTP and glutamine pools did not change. Kinetic data and analysis of the nucleotide pools in the cells growing in vivo suggested that the biosynthesis of CTP is regulated at the level of CTP synthetase by UTP and CTP.

An improved radioassay for cytidine 5-triphosphate synthetase is reported which employs thin-layer chromatographic methods and provides a number of advantages over previously available techniques. (i) The method resolves the nucleotides and the degradation products generated during the time course of the enzymatic reaction by ascending chromatography employing polyethyleneimine cellulose plastic-backed sheets. (ii) Determinations of CTP formed and all nucleotide pairs generated during kinetic analysis of CTP synthetase are greatly simplified, further facilitating the detection of extraneous enzymatic activities. (iii) The sensitivity of the assay is enhanced and as little as 50 pmol of product formed was readily detected in supernatant fluids. This was made possible, in part, by the addition of NaF and phosphoenolpyruvate which together maintain the nucleotide triphosphates in the reaction mixture. (iv) A large number of samples can be handled at one time with highly reproducible results. The synthesis of CTP from UTP by enzyme preparations from rat liver, hepatomas, and Salmonella typhimurium LT2 was quantitated with this method.

Xanthine oxidase and guanase activities in normal and psoriatic epidermis were demonstrated and accurately assayed by the new micro-assay methods which rely on the isolation of 14C-labelled end-products, xanthine and uric acid, from the substrates, hypoxanthine and guanine, by electrophoresis on cellulose acetate membrane using 0.1 M borate buffer, pH 9.0. The average specific activities of xanthine oxidase and guanase in normal epidermis were 10.2 and 55.0 pmol/min/mg protein, respectively, and in psoriatic epidermis, 52.1 and 980.6 pmol/min/mg protein, respectively. Increased activities of these enzymes in psoriatic epidermis suggested that urate formation might be enhanced in the psoriatic lesions resulting from an increased turn-over of nucleic acids.

A phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), induced the cleavage of nuclear DNA at linker regions in cultured mouse thymocytes. Similar DNA fragmentation was induced by 1-oleoyl-2-acetyl-glycerol, a synthetic diacylglycerol, but not by 4 alpha-phorbol-12,13 didecanoate. The DNA fragmentation was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine dihydrochloride, an inhibitor of protein kinase C, as well as actinomycin D and cycloheximide. It appears that TPA induces DNA cleavage through activation of protein kinase C and synthesis of yet unidentified protein(s). That the inhibition of DNA fragmentation was accompanied by a reduction in cell lysis suggests a causal relationship between DNA fragmentation and cell death.

The antiglutamine agent acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, inhibited the growth of hepatoma 3924A cells in culture. After 7 days of incubation with the drug, an LC50 of 1.4 microM was observed by determination of colony forming ability. A combination of cytidine (1 mM), deoxycytidine (10 microM) and guanosine (10 microM) completely protected the hepatoma cells against the cytotoxic action of acivicin, but each nucleoside by itself had no effect. Acivicin (0.1 mM) inhibited the incorporation of uridine and thymidine into macromolecules, but not that of leucine. Acivicin depressed the pools of CTP, GTP, dCTP, dGTP and dTTP to 46, 62, 40, 64 and 53%, respectively, but it increased UTP level to 152% of the values of untreated cancer cells. The activity of a highly purified CTP synthetase (EC 6.3.4.2) from rat liver and hepatoma 3924A was inhibited by acivicin. The inhibition was competitive with respect to L-glutamine, and the Ki values with liver and hepatoma enzymes, determined by Dixon and reciprocal plots, were 1.1 and 3.6 microM respectively. The hydroxy analog of acivicin was also a competitive inhibitor, but it was less effective than acivicin, with a Ki value of 1.8 mM for the hepatoma enzyme. Our observations on the impact of acivicin on the behavior of pools of ribonucleotides and deoxyribonucleotides and the competitive inhibition of purified CTP synthetase from hepatoma cells suggest that a major mechanism of action for this drug is the inhibition of CTP synthetase and GMP synthetase (EC 6.3.5.2).

The activities of the enzymes involved in purine nucleoside metabolism, adenosine deaminase (ADA), adenosine kinase (AK), purine nucleoside phosphorylase (PNP) and deoxycytidine kinase (deoxyCRK), were determined in mouse thymocytes at various stages of differentiation and maturation, and compared with those in other tissues. The thymocytes were characterized by high ADA and deoxyCRK activities with high ADA/AK and ADA/PNP ratios and low PNP/deoxyCRK ratio. In fetal thymocytes of 16 gestational days, ADA activity was lower, and PNP, AK and deoxyCRK activities were higher than those in the adult thymocytes. During differentiation of fetal thymocytes, ADA activity increased while PNP and AK activities decreased. DeoxyCRK activity decreased after birth. In spleen T lymphocytes, ADA and deoxyCRK activities were lower and PNP activity was about 2.5-fold higher than in the thymocytes. Thus the differentiation stages of T lymphocytes may be characterized by the absolute levels and the ratios of these enzymes.

CTP synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) was purified 200-fold from Ehrlich ascites tumor cells with about 50% purity as judged by polyacrylamide gel electrophoresis. The molecular weight estimated to be 118 000 by gel filtration. The optimal pH was 8.6. 2-Mercaptoethanol was required for optimal activity and stabilization of the enzyme. Magnesium was essential for the reaction and other divalent cations were ineffective. Ammonia could replace glutamine as the amino donor. When other substrates were at saturating concentrations, Michaelis-Menten kinetics were observed yielding Km values for UTP, ATP and glutamine of 0.18, 0.8 and 0.13 mM, respectively. With ATP at subsaturating concentration, the double-reciprocal plot for UTP saturation was sinusoidal and the Hill plot showed an n value of 1.3. The double-reciprocal plot for ATP saturation, when UTP was at subsaturating concentration, departed from Michaelis-Menten kinetics with an n value of 1.4. These data suggest the existence of cooperative interactions between enzyme and substrates at subsaturating concentrations of ATP or UTP. GTP was not essential, but it acted as an activator on the glutamine reaction; optimal activation was observed at 1 mM GTP. The affinity for glutamine was not affected by GTP.

An insight into the ordered pattern of enzymatic and biochemical imbalance of cancer cells was made possible, at least in part, by the molecular correlation concept, the concept of key enzymes, and the use of biological model systems. With these approaches the pattern of gene expression in neoplasia can be studied in terms of the activities of key enzymes and their linking with neoplastic transformation and progression. An ordered pattern of biochemical imbalance was recognized in carbohydrate, pentose phosphate, purine, pyrimidine, DNA, and polyamine metabolism and in other biochemical areas. Current work is directed to clarifying the enzymology and metabolic pattern of thymidine metabolism and CTP biosynthesis since these areas are of particular importance in selective chemotherapy and rescue. The activities of key enzymes in thymidine metabolism have been correlated with the growth rates in a spectrum of hepatomas. Increases in the activities of four key enzymes in CTP biosynthesis appear to be specific to neoplasia because no similar pattern was observed in the normal adult resting or regenerating liver or in the fetal and developing liver. The overall pattern discovered in transplantable rat hepatomas applies to other rodent tumors. It is of particular importance that the pattern of biochemical imbalance is also applicable to human hepatocellular carcinomas. With the recognition of the ordered pattern of reprogramming of gene expression in hepatomas, the path is open for the development of sensitive assays for biochemical diagnosis of liver tumors and for a rational design of selective chemotherapy and rescue.