Zymogram Technique Research Articles

HUMAN PERIPHERAL BLOOD RIBONUCLEASES REACTIVATION AFTER SORPTION ON NANOPLATELETS OF LAPONITE®

Summary. Aim: to investigate the possibility of enzymatic reactivation of RNase activity of peripheral blood cells of patients with colorectal cancer (CRC) after sorption on nanoplates Laponite® RD (Lap). Objects and methods: the study was performed with the cell suspension of peripheral blood of CRC patients. Samples of cell lysates were combined with a 1% suspension of Lap nanoplates. Then RNase was extracted with 0,25 N H2SO4 or 2% solution of sodium dodecyl sulfate (DDS). The zymogram technique was used to analyze RNase activity. Results: it was found that RNases bind with nanoplates Lap and form complexes with loss of enzymatic activity. It is known that RNase can be released from the complex by extraction with 0,25 N H2SO4 or 2% sodium DDS solution. RNase is able to restore its enzymatic activity when extraction from the complex with a 2% sodium DDS solution is used. But with the extraction of 0,25 N H2SO4, the enzymatic activity is irreversibly lost. Conclusion: RNase extracted from the nanoplates Lap can be active again as an enzyme that catalyzes the cleavage of RNA and hybrid RNA/DNA molecules, depending on the method of extraction.

Economical alternatives for the production of fungal β-1,3-glucanase using easily obtainable industrial substrates

The β-1,3-glucanases synthesized by filamentous fungi have wide applicability in the food, chemical, and pharmaceutical industries. However, its obtainment can be costly, especially due to substrates used to induce its synthesis. Therefore, the objective of this work was to produce β-1,3-glucanase by T. harzianum Rifai using free and immobilized cells in synthetic and plant sponges, using different inducing substrates that could provide better cost-effectiveness for the industrial production of the enzyme. The Petri dish zymogram technique proved to be efficient for screening substrates inducing β -1,3-glucanases against species of filamentous fungi. It was possible to perform the immobilization of T. harzianum in a synthetic sponge allowing the realization of repetitive batches for enzymatic production. All tested substrates resulted in the synthesis of β-1,3-glucanase, including succinoglycan, proposed innovatively in this study. Fungal biomass resulted in the best inducing substrate under conditions of free and immobilized cells, with a production of β-1,3-glucanases of 0.73 U and 0.80 U of β -1,3-glucanases. The substrates corn starch and cassava showed promise in the production of β-1,3-glucanase and maintained production until the fourth batch was evaluated, with values of 0.51 U and 0.46 U of β-1.3-glucanases, respectively. The results obtained in this study showed that the zymogram is a practical method for screening substrates induced by the fungus T. harzianum. Corn starch and cassava are accessible and low-cost sources for β-1,3-glucanase synthesis in repetitive batches, including the use of immobilized and free cells.

A Zymogram technique for preliminary screening and characterization of feruloyl esterases.

We report here, for the first time, a zymogram technique designed for rapid screening of feruloyl esterase using ethyl ferulate as enzyme substrate and casein precipitation as enzyme activity indicator.

Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents.

The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.

Partial characterization of digestive proteases in tropical gar Atractosteus tropicus juveniles

Tropical gar (Atractosteus tropicus) is an economically and socially important freshwater species from Southeastern Mexico, with a high aquaculture potential. With this in mind, the purpose of this study was to characterize the digestive proteases of tropical gar juveniles through biochemical and electrophoretic analyses. Twenty specimens with an average weight of 73.6±12.7g were used to obtain stomach and intestinal tissue from which multienzymatic extracts were prepared. The general activities of the acid and alkaline proteases were evaluated, as well as the specific activities of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A. The effect of the pH and temperature on the proteases was also analyzed, together with the composition of the multienzymatic extracts using protease inhibitors and electrophoretic tests. Results showed that A. tropicus have a functional stomach in which protein hydrolysis starts with pepsin and which contains endo- and exopeptidases (trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A) and proteases that are resistant to high temperatures (45 and 55°C for alkaline and acid proteases, respectively) and pH values. Using zymogram technique, we found two acid protease isoforms (0.35 and 0.71rf) and five alkaline protease isoforms (83.7, 43.7, 27.5, 24.0 and 19.4kDa), which decrease or disappear with the different inhibitors. Thus, this species is considered to be a carnivore capable of adapting to its environment by consuming different types of proteins from preys and also could adapt rapidly to consume a compound diet with different animal protein sources.

IDENTIFICATION AND PARTIAL CHARACTERIZATION OF CRUDE EXTRACELLULAR ENZYMES FROM BACTERIA ISOLATED FROM SHRIMP WASTE PROCESSING

Attention on chitin degrading enzymes has been growing since their ability to reduce the waste of shrimp/other crustaceans processing industries and converting them into value added products such as biologically active chitin and chitosan oligomer. Previous experiment found that KLU 11.16 isolate was one of the potential bacteria isolated from shrimp waste producing chitinolytic enzymes including chitosanases. A study on the identification of KLU 11.16 extracellularcrude enzyme was carried out by cultivating the bacteria on chitin medium. Due to the wide application of chitosanase, the characterization of the crude chitosanase was carried out after an identification of the enzymes secreted. Based on assessment using zymogram technique, this bacteria secreted a mixed extracellular chitinolytic enzyme and other hydrolytic enzyme. The crudechitinolytic enzyme degraded 85% deacetylated (DA) better than 100% DA chitosan, and slightly degraded glycol chitin, indicating that KLU 11.16 secreted chitosanases and chitinases enzyme. In addition to the chitinolytic enzyme, the bacteria also secreted protein and carbohydrate degrading enzymes when running at SDS-PAGE enriched with casein, soluble starch and CMC substrates.Crude chitosanases enzyme was performed well at pH 6 and temperature of 300C, and the activity can be increased by addition of 1 mM Fe 2+ in form of chloride salt. Addition of detergent, i.e1mM of Triton X-100 and SDS slightly decreased the activity. Future application of the crude chitosanase from KLU 11.16 was on producing chitosan derivative such as chitosan oligomer using substrateof 85% DA chitosan, which is more digestable by other enzymes secreted by KLU 11.16

Characterization of Chitosanase from Indralaya Swamp Bacteria, South Sumatera, Indonesia

The extracellular chitosanase-production capacity of 14 bacterial isolates taken from Indralaya Swamp South Sumatera, was evaluated in liquid cultures. The GB2 isolate was selected due to its high chitosanase activity. The optimum pH and temperature of chitosanase produced by GB2 were 8 and 30 oC, respectively. Ca2+ and Fe2+ increased GB2 chitosanase whereas Na+ and K+inhibited the enzyme.Study on the effect of metals ion indicated that chitosanase GB2 was metaloenzyme.Moleculer weights were determined by using SDSPAGE and zymogram technique. Moleculer weight of chitosanase isolate GB2 was 36-110 kD. Key Words: Chitosanase, Characterization, Bacteria, Indralaya swamp.

Influence of up-regulation of Notch ligand DLL4 on biological behaviors of human gastric cancer cells

To investigate the potential roles of Delta-like ligand 4 (DLL4) on the biological behavior of gastric cancer cells and its molecular mechanisms. A recombinant eukaryotic expression vector containing human DLL4 gene was constructed and transfected into the human gastric cancer cell line SGC7901. Clones with up-regulated DLL4 were selected and amplified. The effect of DLL4 up-regulation on gastric cancer cell growth was assessed using cell growth assay. The migration and invasion were assessed using a transwell migration assay and matrigel invasion assay. Matrix metalloproteinases were detected using the zymogram technique. Cells were implanted subcutaneously into male BALB/c nu/nu mice. Tumor volumes were then calculated and compared. DLL4 staining in the implanted tumor was performed using immunohistochemistry technique. Growth curves over a six-day time course showed significantly promoted cell proliferation of SGC7901 cells with up-regulated DLL4. DLL4 up-regulation in SGC7901 cells promoted the migration (205.4 ± 15.2 vs 22.3 ± 12.1, P < 0.05) and invasion (68.8 ± 5.3 vs 18.2 ± 6.0, P < 0.05) in vitro and tumorigenicity in vivo (2640.5 ± 923.6 mm(3) vs 1115.1 ± 223.8 mm(3), P < 0.05). Furthermore, significantly increased mRNA level and increased secretion of matrix metalloproteinase-2 (MMP-2) proenzyme were observed in SGC7901 cells with up-regulated DLL4. However, increased MMP-9 mRNA level but decreased extracellular MMP-9 proenzyme level was observed. Our observations indicated a mechanism by which activation of DLL4-mediated Notch signaling promotes the expression and secretion of MMP-2 proenzyme and influences the progress of gastric cancer.

Isolasi Bakteri dan Karakterisasi Protease dari Sumber Air Rawa Indralaya

The aim of this study was to bacteria isolation and characterization proteases from water swamp. The water swamp samples collected fromIndralaya for microbial isolation. Three isolates showed proteolytic index &gt;1.00. The optimum pH of extraceluller proteases from A6S3,A4S3 and A15S3 were 7.5; 8.0; 8.0, respectively. The optimum temperature of A6S3, A4S3 and A15S3 protease were 40; 50; 50 oC,respectively. Effect of metal ion (Fe, K, Mn, and Zn) shown Fe and K were inhibit protease A6S3, all metal ion were inhibit protease A4S3and K, Mn and Zn inhibit protease A15S3. Study on the effect of metals ion and spesific inhibitors indicated that all protease weremetaloprotease. Their moleculer weights were determined by using SDS-PAGE and zymogram technique for A6S3 isolate were 70, 88, 106and 121 kD respectively. Whereas for isolate A4S3 was 138 kD and isolate A15S3 was 131 kD.

Catalase and superoxide dismutase double staining zymogram technique for Deinococcus and Kocuria species exposed to multiple stresses

Superoxide dismutase (SOD) and catalase expression is associated with oxidative stress. Existing techniques for the individual staining of SOD and catalase have been described in the past. The objective of this study was to achieve a simple and rapid technique for the double staining of bacterial SOD and catalase on the same polyacrylamide gel. SOD detection was carried out using nitro-blue tetrazolium (NBT) dye reduction followed by ferricyanide precipitation for negative staining of the catalase enzyme on the same gel. The staining procedure resulted in pale blue SOD bands while catalase appeared as yellow bands against a greenish blue background on the same gel. This technique was used to detect changes in the polymorphic forms of these enzymes in Deinococcus radiodurans R1 and Kocuria sp. C2 subjected to stresses like UV and gamma radiation and desiccation.

Enzymatic Activity in Body and Fecal Extracts of the Storage Mite Chortoglyphus arcuatus

Background:Chortoglyphus arcuatus has been described in many countries. Many allergens are potent enzymes, which may promote a Th2 immune response. The aim of this study was to evaluate the enzymatic activity of body and fecal extracts of C. arcuatus. Material and Methods: Feces and bodies of full-grown C. arcuatus cultures were separated by sieving, extracted in PBS, dialyzed and lyophilized. The antigenic profile of both extracts was determined by SDS-PAGE. Immunoblot experiments were conducted using a pool of sera from allergic individuals residing in Galicia, a region of Spain, where this species is abundant. The enzymatic activity of the extracts was evaluated by the zymogram technique. Serine and cysteine protease activity was measured using in vitro methods. The API Zym® system was used to determine the enzymatic properties of the extracts. Results: The antigenic profile showed that the body extract contained more and better defined bands than the fecal extract. Allergens were detected in both extracts in a molecular weight range between 14 and 100 kDa. Gelatinolytic gels confirmed that fecal extracts contain more hydrolytic enzymatic activity than body extracts. Serine protease activity in fecal extracts was higher than in body extracts (5.98 vs. 2.701 IU of trypsin/mg of freeze-dried material). No cysteine protease activity was detected. Conclusion:C. arcuatus extracts contain several allergens and proteins with high enzymatic activity, especially in the feces. Some of these allergens may be enzymes. Fecal extracts have more enzymatic activity than body extracts.

Paenibacillus sp. DG-22에서의 β-xylosidase 생합성 조절

효소생산을 최적화하기 위해서 Paenibacillus sp. DG-22에서의 <TEX>${\beta}-xylosidase$</TEX> 생합성 조절을 연구하였다. Paenibacillus sp. DG-22의 <TEX>${\beta}-xylosidase$</TEX>는 배양액에 존재하는 탄소원에 의해 조절되는 것으로 관찰되었다. <TEX>${\beta}-Xylosidase$</TEX>의 합성은 xylan과 methyl <TEX>${\beta}-D-xylopyranoside$</TEX> (<TEX>${\beta}MeXyl$</TEX>)에 의해 유도되었으나 쉽게 대사되는 단당류에 의해서는 약간 억제되었다. <TEX>${\beta}MeXyl$</TEX>가 <TEX>${\beta}-xylosidase$</TEX>의 유도를 위한 최적의 기질임을 확인하였고 가장 효과적인 유도는 10 mg/ml의 농도에서 얻어졌다. <TEX>${\beta}-Xylosidase$</TEX>의 생산은 세포의 생장과 연관된 양상을 나타내었으며, 대수기 말에 최대양이 형성되었다. Glucose와 xylose가 존재하면 <TEX>${\beta}-xylosidase$</TEX>의 활성 수준이 감소하는 것으로 보아 이 효소의 생합성은 catabolite repression을 받는것으로 보인다. SDS-PAGE와 활성염색 기술을 이용하여 <TEX>${\beta}Mexyl$</TEX>가 이 효소의 생합성을 유도하며 약 80 kDa 크기의 하나의 <TEX>${\beta}-xylosidase$</TEX>가 존재함을 알 수 있었다. Regulation of <TEX>${\beta}-xylosidase$</TEX> synthesis in Paenibacillus sp. DC-22 was studied to optimize the enzyme production. <TEX>${\beta}-Xylosidase$</TEX> synthesis of the Paenibacillus sp. DG-22 was observed to be regulated by carbon sources present in culture media. The synthesis of <TEX>${\beta}-xylosidase$</TEX> was induced by xylan and methyl <TEX>${\beta}-D-xylopyranoside$</TEX> (<TEX>${\beta}MeXyl$</TEX>) but slightly repressed by readily metabolizable monosaccharides. <TEX>${\beta}MeXyl$</TEX> was found to be the best substrate for the induction of <TEX>${\beta}$</TEX>-xylosidase and the most effective induction was obtained at a concentration of 10 mg/ml. <TEX>${\beta}-Xylosidase$</TEX> production showed a cell growth associated profile with the maximum amount formed during the late exponential phase of growth. The presence of glucose and xylose decreased the level of <TEX>${\beta}-xylosidase$</TEX> activity indicating that its production was subjected to a form of carbon catabolite repression. SDS-PAGE and zymogram techniques demonstrated the induction by <TEX>${\beta}MeXyl$</TEX> and revealed the presence of one <TEX>${\beta}-xylosidase$</TEX> of approximately 80 kDa.

A phosphate‐binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.

Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M r polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.

Antibody zymography: a novel adaptation of zymography to determine the protease-neutralising potential of specific antibodies and snake antivenoms

A common problem in the development of antibody-based therapeutics is the selection, usually from a large population, of specific antibodies with the desired function. One of our research objectives is to identify antibodies capable of neutralising the most important haemorrhagic and haemostasis-disruptive proteases from viper venom. Here, we describe a modification of conventional gelatin-zymography that permits the identification of antibodies capable of neutralising gelatinolytic proteases. We demonstrate that the gelatinolytic activity of viper venom proteases is neutralised by addition of viper antivenom to the matrix of conventional gelatin-zymograms. Venom protein gelatinolytic activity was unaffected by inclusion of antibody from control, non-immunised animals or immunoglobulin-depleted serum. The application of this antibody zymogram technique for future research on snake venoms is evaluated in the context of identified limitations.

Production of beta-xylanase and beta-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis.

The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed for beta-xylanase activity between 5% and 15% NaCl, while beta-xylosidase activity was highest at 5% NaCl. Almost half of the maximum activities remained at 27%-30% NaCl for both enzyme activities. When dialyzed culture supernatant and culture broth were employed for determination of beta-xylanase and beta-xylosidase stabilities, approximately 55% and 83% of the initial beta-xylanase and beta-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl. The enzymes were also shown to be slightly thermophilic; beta-xylanase activity exhibiting two optima at 55 degrees and 70 degrees C, while beta-xylosidase activity was optimal at 65 degrees C. SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants. To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon.

Combination of zymography and immunodetection to analyze proteins in complex culture supernatants.

The physiological function of an allergen might be an important factor for the allergenicity. The major grass pollen allergen Phl p 1 shows sequence similarities to the consensus sequences of cysteine proteases. However, up to now, the proteolytic activity of Phl p 1 is controversial. The culture supernatant of Phl p 1-transfected clones from Pichia pastoris showed a proteolytic activity but this might be due to Phl p 1 or irrelevant yeast contaminants. To solve this question, we made use of the zymogram technique and improved it. Substrate as well as substrate concentration was changed from 1% casein to 0.25% skimmed milk powder. For staining, we used a colloidal Coomassie stain (RotiBlue) with a higher sensitivity and better practicability than the conventional Coomassie staining. The proteins in the zymogels and in the SDS-PAGE gels showed similar electrophoretic mobility. Furthermore, the zymogels could be blotted and immunostained. Thus, the molecular mass of the proteolytic bands could be determined and directly compared with immunoblotting results. To clearly assign the protease, we separated the culture supernatant of the Phl p 1-transfected P. pastoris clone by affinity chromatography with monoclonal antibody. Our studies demonstrate that the proteolytic activity did not belong to the recombinant allergen but to the yeast proteins. The enzyme was classified by zymogram inhibition tests as a strong serine protease.

Transformants of Metarhizium anisopliae sf. anisopliae overexpressing chitinase from Metarhizium anisopliae sf. acridum show early induction of native chitinase but are not altered in pathogenicity to Manduca sexta.

Extracellular chitinase activity has been implicated in the pathogenesis of several fungal infections. Following induction with chitin, the insect pathogens Metarhizium anisopliae sf. acridum ARSEF strain 324 and Metarhizium anisopliae sf. anisopliae ARSEF strain 2575 secrete 44-kDa basic and acidic isoforms of endochitinase, respectively. The gene from strain 324 (Chit1) was cloned and inserted into the genome of strain 2575 under the control of Aspergillus regulatory elements such that transgenic 2575 (2575-Chit(+)) expressed CHIT1 in a noninducing medium (i.e., not containing chitin). Isoelectric focusing followed by a zymogram technique revealed that neither wild-type 2575 nor 2575-Chit(+) produced significant amounts of the native 2575 acidic chitinase in a noninducing medium. However, in a chitin-containing medium, 2575-Chit(+) produced the native chitinase earlier than strain 2575, soon after secretion of CHIT1. We hypothesize that this is due to the production of soluble inducers following chitin hydrolysis by CHIT1 and that M. anisopliae uses enzymes expressed at low levels to sense the nature of the polymeric nutrient present in the immediate environment. However, the chitinase overproducers did not show altered virulence to caterpillars (Manduca sexta) compared to the wild-type fungus, suggesting that wild-type levels of chitinase are not limiting for cuticle penetration.

Multiple endoxylanases ofButyrivibrio sp.

Butyrivibrio sp. Mz 5 with a high xylanolytic activity was isolated. Four major xylanases were detected in the cell-associated fraction using the zymogram technique. The xylanolytic activity was inducible with the oat spelts xylan; two endoxylanases (51 and 145 kDa) were formed constitutively. The bulk of the xylanolytic activity was cell-bound and growth-phase dependent; the maximum activity in the cell-associated fraction was achieved after 16 h of incubation. The highest xylanolytic activity was determined in a medium with 0.5% oat spelts xylan. Under optimum conditions (the highest xylanolytic activity produced), the two cell-bound xylanases (51 and 58 kDa) were isolated by anion exchange chromatography on CIM DEAE 8 tubes attached to a MPLC system, and gel filtration.

Different sublines of Jurkat cells respond with varying susceptibility of internucleosomal DNA degradation to different mediators of apoptosis.

The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.