EFFECT OF SOME IMMUNOMODULATORY DRUGS ON EMBRYONIC DEVELOPMENT OF DANIO RERIO FISH.

At the current stage of healthcare development, the inclusion of immunomodulators in the complex pharmacotherapy of various immunoinflammatory and viral diseases is widely discussed, but due to the lack of sufficient research and a broad evidence base, not all drugs with similar properties are used in medicine. According to the information obtained from the instructions for the use of immunomodulators, it was obtained that the main contraindications to their use include the prescription of children, pregnant women, and women during breastfeeding. In this study, we evaluated the effects of immunomodulatory drugs: aminodihydrophthalazindione sodium and meglumine acridonacetate, on the early developmental stages of Danio rerio (Zebrafish) embryos.

Describing motivation for health and treatment decisions and communication choices of calf-care workers on western United States dairies.

On large dairy farms, animal health assessments and treatments are made by farm employees. Little is known about how employees make decisions about illness detection or treatment, information critical to improving antimicrobial stewardship. The objectives of this study were to describe calf-care employee motivations for decisions associated with preweaned calf health and treatments, describe on-farm worker communication networks, and determine information sources used by these employees to support their decisions. Personal interviews were conducted with 103 calf-care employees on 28 farms in the western United States. The interview consisted of 10 motivation source type (MST) questions and questions about training, communication and educational opportunities. A latent class analysis created a summary for MST and resulted in 4 classes. Forty-three percent of calf-care employees fell into a class where responses were a combination of internal and intrinsic (personal beliefs or values and task fulfillment, respectively) and 23% were a combination of internal and goal internal (aligned with organizational goals). This latter class aligned health decisions with internal motivation and treatment decisions with goal internal. A network analysis summarized dominant communication relationships and established that feeders and treaters perceived more communication with supervisors than was reciprocated by supervisors, and that there was less communication between workers and management for tasks relative to daily work. Employee training was primarily done by herdsman, calf manager, or coworkers, and information for skill improvement and problem solving was sought from these individuals. Although veterinarians were not often involved in employee training, when they were involved, employees were likely to use them as an information source for skill improvement and problem solving. Few participants had ever used social media, but almost all had a device that could access the internet; more than 60% indicated interest in a social media platform for work-related information. Work motivation for many calf caretakers appeared to be sourced from personal beliefs, values, and job fulfillment, particularly when deciding to treat a sick calf. Investigation and incorporation of beliefs and values in training programs could help with alignment of protocols with actual treatment and further efforts to implement judicious use of antimicrobials.

Calf care personnel on dairy farms and their educational opportunities.

As dairy herd sizes become larger and the organization of the business more complex, targeting communication and education to enhance animal care becomes more difficult. The purpose of this study was to describe selected demographics of calf care employees on large (>500 animals) and small (<501 animals) dairy farms that raise their own calves. Two to 8 individuals per farm involved with calf care, including owners, veterinarians, and calf managers, feeders, and treaters, were interviewed in either English or Spanish. Interviews were conducted in person on 53 dairy farms located in Arizona, Idaho, New York, Oregon, and Washington State. The number of preweaned calves on the farm ranged from 9 to 1,500 (median = 93). A total of 224 individuals were interviewed across 8 job titles. As farm size increased, personnel structure became more complex. Farms with >100 preweaned calves were 15 times more likely to have a calf manager title compared with farms with ≤100 preweaned calves. Eight farms designated the same person as calf manager, treater, and feeder, all with ≤100 preweaned calves. Thirty-two (60%) of the farms had at least 1 full-time calf feeder. Almost 30% of owners and over 40% of veterinarians interviewed were over 50 yr of age, whereas over 40% of the calf managers, feeders, and treaters were under 30 yr of age. Seventy-three percent of feeders and 72% of treaters spoke Spanish at home. For languages in which interviewees were comfortable speaking, more than 30% of owners and 33% of veterinarians were comfortable communicating in Spanish. For calf care employees, 60% of calf managers, 42% of feeders, and 38% of treaters were bilingual (English and Spanish), but most (72%) preferred to be interviewed in Spanish. The level of education varied by job title for those interviewed, but most of the calf care team had high school or less education. However, some diversity was observed in educational background within job title with almost 38% of the calf managers having at least some college education. The majority of feeders (88%) and treaters (83%) reported being trained by another employee and 66 and 58%, respectively, had not received any continuing education in the previous year. With the amount of diversity seen on these farms, understanding employees' educational backgrounds, language, and generational differences may be valuable when developing training for new procedures for animal health or other aspects of animal care.

Heart transplantation in the United States, 1999-2008.

This article features 1999-2008 trends in heart transplantation, as seen in data from the Organ Procurement and Transplantation Network (OPTN) and the Scientific Registry of Transplant Recipients (SRTR). Despite a 32% decline in actively listed candidates over the decade, there was a 20% increase from 2007 to 2008. There continues to be an increase in listed candidates diagnosed with congenital heart disease or retransplantation. The proportion of patients listed as Status 1A and 1B continues to increase, with a decrease in Status 2 listings. Waiting list mortality decreased from 2000 through 2007, but increased 18% from 2007 to 2008; despite the increase in waiting list death rates in 2008, waiting list mortality for Status 1A and Status 1B continues to decrease. Recipient numbers have varied by 10% over the past decade, with an increased proportion of transplants performed in infants and patients above 65 years of age. Despite the increase in Status 1A and Status 1B recipients at transplant, posttransplant survival has continued to improve. With the rise in infant candidates for transplantation and their high waiting list mortality, better means of supporting infants in need of transplant and allocation of organs to infant candidates is clearly needed.

Comparison of transcranial color Doppler imaging (TCDI) and transcranial Doppler (TCD) in children with sickle-cell anemia.

BACKGROUND: Transcranial Doppler (TCD) has been demonstrated to identify those at highest risk of stroke among children with sickle-cell disease. Based on a randomized clinical trial [Stroke Prevention in Sickle-Cell Anemia Trial (STOP)], which ended in 1997, the National Heart Lung and Blood Division of NIH has recommended TCD screening and chronic blood transfusion based on Nicolet TC 2000 dedicated Doppler (TCD). Studies performed using TCD imaging modalities need to be correlated to that used in the clinical trial to provide information for treatment decisions when screening with TCDI. OBJECTIVE: To correlate transcranial arterial time-averaged mean velocities obtained from an Acuson Transcranial Doppler Imaging to those obtained using the TCD as the gold standard for treatment decisions based on STOP. MATERIALS AND METHODS: A total of 29 children with sickle-cell disease, age 3-16 years, were studied at one of two scanning sessions using both techniques and a scanning protocol based on that used in STOP performed and read independently. The average difference in the measured velocities for each arterial segment was tested to determine difference from zero. Differences were compared before and after modifications to the TCDI technique were made to mimic the STOP protocol more closely. RESULTS: TCDI velocities were generally lower than TCD velocities for the same segment, but the difference was reduced (from 15 % to 10% for the middle cerebral artery) by modifications to the TCDI protocol. CONCLUSIONS: Measurements using the Acuson system are modestly lower than those obtained with dedicated Doppler using the Nicolet TCD.

Intracranial resistive index (RI) values in normal term infants during the first day of life.

PURPOSE: The purpose of this study was to determine normal resistive index (RI) values for term neonates during the first day of life as part of an ongoing prospective study of RI values in term infants with perinatal asphyxia. MATERIALS AND METHODS: Forty normal term neonates underwent cranial sonography and Doppler during the first 24 h after birth. Transfontanelle Doppler was performed of the internal carotid, anterior cerebral, and middle cerebral arteries bilaterally. In addition, transtemporal Doppler was performed of the middle cerebral arteries bilaterally. Mean and median RI values were calculated in all vessels interrogated. The transfontanelle and transtemporal middle cerebral artery measurements were compared using paired t-tests. RESULTS: The overall mean RI of all interrogated vessels was 0.726 with a standard deviation of 0.057. The mean RI value in the middle cerebral arteries was not significantly different with the two different measurement techniques. CONCLUSION: Normal intracranial RI values for a term infant in the first day of life were calculated for comparison with RI values in term infants with perinatal asphyxia.

Dual-energy x-ray absorptiometry in osteopetrosis.

We have used dual-energy x-ray absorptiometry (DXA) in evaluation and follow-up of a patient with osteopetrosis, before and after cord blood transplantation. Other methods of follow-up in such cases have been described, but the use of DXA has not previously been reported. We have shown that DXA offers a safe means of assessing disease progression, the timing of treatment, and response after therapy for osteopetrosis.

Bcl-2-mediated resistance to apoptosis is associated with glutathione-induced inhibition of AP24 activation of nuclear DNA fragmentation.

Studies on the mechanism of apoptosis in this laboratory support a model in which signal transduction involving caspase 3 leads to activation of a serine protease called Mr 24,000 apoptotic protease (AP24), which then induces internucleosomal DNA fragmentation in the nucleus. This study examined the effect of Bcl-2 overexpression on activation of AP24 and the induction of DNA fragmentation by AP24 in isolated nuclei. It was demonstrated that overexpression of Bcl-2 in either HL-60 or PW leukemia cell lines suppressed activation of AP24 induced by either tumor necrosis factor or UV light and protected cells from apoptosis. Furthermore, nuclei isolated from Bcl-2-overexpressing cells were relatively resistant to internucleosomal DNA fragmentation induced by AP24 isolated from apoptotic cells. Bcl-2-overexpressing cells that were nutritionally depleted of glutathione (GSH) became sensitive to tumor necrosis factor- or UV light-induced activation of AP24 and underwent apoptotic cell death. Moreover, nuclei isolated from Bcl-2-overexpressing cells that were depleted of GSH became sensitive to AP24-induced DNA fragmentation. The addition of exogenous GSH blocked the proteolytic activity of AP24, as well as its ability to induce DNA fragmentation in normal isolated nuclei. These results indicate that Bcl-2 can attenuate at least two events in the AP24 apoptotic pathway: activation of AP24 and induction of DNA fragmentation by activated AP24. Furthermore, agents that deplete intracellular levels of GSH may have therapeutic use in the sensitization of Bcl-2-overexpressing cancer cells to apoptotic cell death.

Use of power Doppler in pediatric neurosonography: a pictorial essay.

In pediatric neurosonography, conventional color Doppler imaging has been the primary adjunct to routine gray-scale imaging. Power Doppler sonography is a relatively recent development that does not have the limitations of conventional color Doppler ultrasound. The power Doppler technique measures the energy of moving red blood cells instead of the velocity and direction of flow. Advantages of this technique include increased sensitivity for identifying flow in slow-flow states, more complete evaluation of a vessel, and more accurate evaluation of the course of the vessel. Power Doppler sonography is helpful in evaluation of the neonatal brain in a variety of clinical situations: identifying the exact locations of extraaxial fluid collections, differentiating intraventricular clot from normal choroid plexus, detecting intraventricular hemorrhage, and demonstrating asymmetries in cerebral perfusion. However, in certain difficult cases, use of both conventional color Doppler sonography and power Doppler sonography produces increased diagnostic accuracy because these techniques furnish complementary information.

Chemotherapeutic drug activation of the AP24 protease in apoptosis: requirement for caspase 3-like-proteases.

AP24 is a serine protease that is activated during TNF or UV light-induced apoptosis and stimulates DNA fragmentation in isolated nuclei. The present study determined whether apoptosis induced by chemotherapeutic drugs resulted in activation of AP24 and examined the possible relationship to caspase activity. We showed that an inhibitor of AP24, DK120, could block DNA fragmentation induced in three leukemia cell lines (U937, HL-60, and CEM) by various DNA-damaging drugs including etoposide, camptothecin, chlorambucil, and the CC1065-related drug, YW201. Etoposide-induced activation of intracellular DEVD-pNa cleaving activity and apoptosis was suppressed by low micromolar concentrations of cell-permeable inhibitors of caspase-3. Furthermore, these inhibitors also suppressed activation of AP24. In contrast, DK120 did not prevent etoposide activation of DEVD-pNa cleaving activity, nor did it prevent cleavage of poly(ADP-ribose) polymerase. AP24 isolated from apoptotic cells following treatment with etoposide activated DNA fragmentation in isolated normal nuclei and was inhibited by DK120, but not by caspase inhibitors. This evidence shows that activation of caspase 3-like proteases generates signals that contribute to the activation of AP24 which may then induce nuclear DNA fragmentation in chemotherapeutic drug-induced apoptosis.

Transcranial Doppler, MRA, and MRI as a screening examination for cerebrovascular disease in patients with sickle cell anemia: an 8-year study.

OBJECTIVE: The authors previously reported five transcranial Doppler ultrasonography (TCD) findings as significant in detecting clinical cerebrovascular disease in a 4-year study in patients with sickle cell disease. This is a follow-up to evaluate the validity of the original findings over another 4-year period during which the study population doubled. A clinical follow-up of the original asymptomatic sickle cell patients with positive TCD, MRA, and MRI was also made. MATERIALS AND METHODS: Over an 8-year period TCD, MRI, and MRA were prospectively performed in 90 sickle cell patients who were clinically asymptomatic for stroke and in 27 sickle cell patients with clinical stroke. RESULTS: Of the 4 out of original 46 control patients in 1992 who had positive MRA and TCD, 3 have subsequently had clinical stroke. None of the 9 original patients with positive TCD and positive MRI but negative MRA have developed stroke. All five original TCD indicators of disease were still significant (P < 0.05) for detecting clinical disease: maximum velocity in ophthalmic artery (OA) > 35 cm/s, mean velocity in middle cerebral artery (MCA) > 170 cm/s, resistive index (RI) in OA < 50, velocity in OA greater than in MCA, and velocity in posterior cerebral (PCA), vertebral, or basilar arteries greater than in MCA. An RI of < 60 in the DA was also now found to be significant [corrected]. Four additional factors were also significant: turbulence, PCA or ACA without MCA, RI < 30, and maximum velocity in MCA > 200 cm/s. CONCLUSION: Positive MRA with a positive TCD in an asymptomatic patient in long-term follow-up suggests a trend for developing clinical stroke. A 4- to 8-year follow-up of nine patients with positive TCD, positive MRI, but not positive MRA did not show development of clinical stroke. Nine Doppler findings are significant in screening for clinically symptomatic vascular disease in sickle cell patients. It is recommended that children with sickle cell disease be screened for cerebrovascular disease with TCD. If one or two indicators of abnormality are present, MRA is recommended. If the MRA is positive, the patient may be considered for transfusion therapy or other treatment for prevention of stroke.

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity.

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.

Solid-phase synthesis of hydroxyethylamine angiotensin analogues.

Three hydroxyethylamine analogues of angiotensins II, III, and IV were prepared by solid-phase methods. The resin-bound peptide was alkylated with the iodomethylketone derivative of the N-terminal amino acid, followed by reduction to the alcohol using sodium borohydride. The iodomethylketones can be made in good yields from commercially available N-protected amino acids. The compounds were evaluated for their ability to displace labeled angiotensins from bovine adrenal membranes, and their metabolic stability tested in kidney homogenates and aminopeptidase M preparations. The hydroxyethylamine amide bond replacement reduced the affinity of the analogues; however, they were substantially more stable to enzymatic degradation.

Isotope or mass encoding of combinatorial libraries.

BACKGROUND: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds. RESULTS: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution. CONCLUSIONS: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice.

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.

Lipid peroxidation and formation of 8-hydroxydeoxyguanosine from acute doses of halogenated acetic acids.

Chlorinated, brominated, and mixed bromochloro acetates are major by-products of water disinfection by chlorine or ozone. The chlorinated acetates, trichloroacetate (TCA) and dichioroacetate (DCA), are carcinogenic in rodents. Brominated analogs of TCA and DCA have received little study. TCA and DCA induce lipid peroxidation in the livers of rodents when administered acutely. Oxidative stress can also result in oxidative damage to DNA, most commonly measured as increases in 8-hydroxydeoxyguanosine (8-OHdG) adducts. In this study, the ability of acute doses of TCA, DCA, dibromoacetate (DBA), bromodichloroacetate (BDCA), and bromochloroacetate (BCA) to induce lipid peroxidation and 8-OHdG formation was examined. Male B6C3F1 mice developed significant increases in 8-OHdG/dG ratios in nuclear DNA isolated from livers when treated with haloacetates. The extent of 8-OHdG formation appeared to be related to the ability to induce thiobarbituric acid-reactive substances (TBARS). The order of potency was DBA = BCA > BDCA > DCA > TCA. The induction of 8-OHdG was found to be generally more sensitive to treatment with haloacetates than the TBARS response. Significantly elevated levels of 8-OHdG were observed at doses of DBA, BCA, and BDCA as low as 30 mg/kg. We suggest that formation of 8-OHdG by brominated haloacetates may contribute to their toxicological effects.

Biochemical pathways of apoptosis: nicotinamide adenine dinucleotide-deficient cells are resistant to tumor necrosis factor or ultraviolet light activation of the 24-kD apoptotic protease and DNA fragmentation.

The function of nicotinamide adenine dinucleotide (NAD) and adenosine diphosphate (ADP) ribosylation reactions in the mechanism of apoptotic cell death is controversial, although one theory postulates an essential role for NAD depletion by poly-ADP-ribose polymerase. The present study examined the role of intracellular NAD in tumor necrosis factor (TNF) and ultraviolet (UV) light-induced activation of the 24-kD apoptotic protease (AP24) leading to internucleosomal DNA fragmentation and death. Our results demonstrate that nutritional depletion of NAD to undetectable levels in two leukemia lines (U937 and HL-60) renders them completely resistant to apoptosis. This was attributed to a block in the activation of AP24 and subsequent DNA cleavage. Normal cells show an elevation of ADP-ribosyl transferase (ADPRT) in both the cytosol and nucleus after exposure to TNF, but before DNA fragmentation. ADPRT activity as well as cell death was suppressed by an inhibitor specific for mono-ADPRT. Nuclei from NAD-depleted cells were still sensitive to DNA fragmentation induced by exogenous AP24, indicating a selective function for NAD upstream of AP24 activation in the apoptotic pathway. We confirmed a requirement for intracellular NAD, activation of ADPRT, and subsequent NAD depletion during apoptosis in KG1a, YAC-1, and BW1547 leukemia cell lines. However, this mechanism is not universal, since BJAB and Jurkat leukemia cells underwent apoptosis normally, even in the absence of detectable intracellular NAD. We conclude that TNF or UV light-induced apoptotic cell death is not due to NAD depletion in some leukemia cell lines. Rather, NAD-dependent reactions which may involve mono-ADPRT, function in signal transduction leading to activation of AP24, with subsequent DNA fragmentation and cell death.

Peptide inhibitors of src SH3-SH2-phosphoprotein interactions.

Activated pp60c-src has been implicated in a number of human malignancies including colon carcinoma and breast adenocarcinoma. Association of the src SH2 domain with tyrosine-phosphorylated proteins plays a role in src-mediated signal transduction. Inhibitors of src SH2 domain-phosphoprotein interactions are, thus, of great interest in defining the role(s) of src in signal transduction pathways. To facilitate such studies, an enzyme-linked immunosorbent assay (ELISA) was developed to detect inhibitors of src SH2-phosphoprotein interactions. This assay measures inhibition of binding of a fusion construct (glutathione S-transferase src SH3-SH2) with autophosphorylated epidermal growth factor receptor tyrosine kinase domain. Activities of phosphopeptide segments derived from potential src SH2 cognate phosphoprotein partners were determined, with the focal adhesion kinase-derived segment VSETDDY*AEIIDE yielding the highest inhibitory activity. Structure activity studies starting from acetyl (Ac)-Y*EEIE have identified Ac-Y*Y*Y*IE as the most active compound screened in the ELISA. This compound is at least 20-fold more active than the parent peptide Ac-Y*EEIE. A high resolution (2 A) crystal structure of human src SH2 complexed with Ac-Y*EEIE was obtained and provided a useful framework for understanding the structure-activity relationships. Additionally, Ac-Y*EEIE was able to block interactions between src and its cellular phosphoprotein partners in vanadate-treated cell lysates from MDA-MB-468 breast carcinoma cells. However, it is unable to abrogate proliferation of MDA-MB-468 cells in culture, presumably because of poor cell penetration and/or lability of the phosphate group on tyrosine.

Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation.

We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1-tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala-borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24-kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis.