RESUMEN
To investigate whether human immunodeficiency virus (HIV)-1 and HIV-1 antigens modulate surface and cytoplasmic CD8 or CD3, as well as CD4, we used cell permeabilization reagents, surface/cytoplasmic fluorescent staining, multiparameter flow cytometric techniques and an in vitro culture system in which relatively few lymphocytes are actively infected with HIV. Human peripheral blood lymphocytes were: not stimulated, not stimulated but HIV-inoculated, phytohaemagglutinin (PHA)-stimulated, PHA/HIV-inoculated (PHA/HIV), or placed into media with soluble gp120, Rev or Nef. HIV inoculation and Nef had striking modulatory effects on CD8. The cytoplasmic CD8 median fluorescent intensity (MFI) of positive lymphocytes was lower for cells in unstimulated/HIV-infected cultures than unstimulated cultures (44 versus 62% of ex vivo value, P = 0.032) and lower for cells in PHA/HIV cultures than in PHA cultures (56 versus 100% of ex vivo, P = 0.041). The surface CD8 MFI values for Nef were significantly lower than the ex vivo value (75% of ex vivo, P = 0.006). At days 2-7 of culture, Rev was associated with slight reductions in surface CD4 MFI (58% of ex vivo versus 78% of ex vivo for unstimulated cultures, P = 0.047) and greater effects on cytoplasmic CD3 MFI (131 versus 179% of ex vivo for unstimulated cultures, P = 0.035), and surface CD8 MFI (70% of ex vivo, P = 0.006 versus ex vivo value). The globality of Rev's effects suggests these are related to a shared processing pathway, i.e. not due to direct interaction with CD3, CD4 and CD8; the effects of HIV inoculation and Nef on CD8 expression appear to be more CD8 specific. Because CD8 is essential for cytotoxic T-cell function, its down-modulation could inhibit this activity, including anti-HIV cytotoxicity. Given the critical roles of CD3 and CD8 in T-lymphocyte signal transduction and antigen responsiveness, the effects of HIV, Rev and Nef on these molecules have clinically significant implications concerning the pathogenesis and treatment of HIV.
Asunto(s)
Complejo CD3/metabolismo , Antígenos CD8/metabolismo , Antígenos VIH/administración & dosificación , VIH-1/inmunología , Linfocitos/inmunología , Brefeldino A/farmacología , Antígenos CD4/metabolismo , Productos del Gen nef/administración & dosificación , Productos del Gen rev/administración & dosificación , Proteína gp120 de Envoltorio del VIH/administración & dosificación , Infecciones por VIH/inmunología , Humanos , Técnicas In Vitro , Activación de Linfocitos , Linfocitos/efectos de los fármacos , Fitohemaglutininas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
It is not clear whether CD3 contacts CD4 or CD8 directly, nor have the regulation and interregulation of expression of these three receptor molecules been determined. We explored these issues by first stimulating human peripheral blood lymphocytes in vitro with three well-characterized T-cell receptor-directed mitogens (phytohemagglutinin [PHA], concanavalin A [ConA], and anti-CD3 monoclonal antibody [alphaCD3]) and then using multiparameter flow cytometric techniques to investigate modulation of surface (sur) and cytoplasmic (c) CD3, CD4, and CD8. Cultures with alphaCD3 had a rapid, large, and persistent decline in surCD3; the cCD3 median fluorescent intensity (MFI) declined gradually, over the entire culture period. With alphaCD3, surCD4 MFI and cCD4 MFI declined by days 4 to 8 (31% of ex vivo value, p < 0.001 and 47%, p = 0.033), as did surCD8 MFI (58%, p = 0.010). PHA was associated with an increase in surCD8%, surCD8 MFI, and cCD8% at days 4 to 8 (178% of ex vivo, p = 0.003; 168%, p = 0.025; and 331%, p = 0.001). For PHA at days 4 to 8, cCD8 MFI was highly variable but always higher than in unstimulated cultures (5 of 5 experiments). With ConA, at 3 to 5 hours ex vivo, there was a decrease in surCD3 MFI relative to ex vivo (64%), surCD4% (83%), cCD4% (87%), surCD4 MFI (50%) and cCD4 MFI (48%), surCD8% (85%) and an increase in cCD8% (260%). As with PHA, at days 4 to 8, surCD8% was high relative to ex vivo (169%). Thus, we found that alphaCD3 had delayed effects on CD4 and CD8; PHA had delayed effects on CD8 only; and ConA had very rapid effects on CD3, CD4, and CD8, as well as a delayed effect on surface CD8. These effects involve both surface and cytoplasmic antigen expression and are more consistent with degradation or retention, rather than with shedding or increased production. They may reflect direct interactions between CD4 or CD8 and CD3 and/or interregulation of CD3 expression with expression of these coreceptor molecules.
Asunto(s)
Complejo CD3/biosíntesis , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Brefeldino A/farmacología , Concanavalina A/farmacología , Humanos , Mitógenos/farmacología , Fitohemaglutininas/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Determining the effects of HIV infection on the expression of cell surface molecules has been limited by an inability to differentiate between productively infected cells and those without productive infection. We inoculated human peripheral blood mononuclear cells from healthy, human immunodeficiency virus type 1 (HIV) antibody-negative donors with HIV; noninoculated cells were also examined. Using multiparameter flow cytometry, we differentiated cells actively producing HIV cytoplasmic p24 antigen during acute, in vitro HIV infection from those not producing detectable cytoplasmic p24. For both resting and PHA-stimulated cells inoculated with HIV (R/H and P/H), a higher proportion of p24+ cells expressed CD25, compared with p24-cells (p = 0.031 and p = 0.008, respectively), consistent with either increased viral replication in stimulated cells or increased stimulation secondary to productive HIV infection. Findings were similar for the expression of CD38, HLADR, and CD28. A striking proportion of p24+ cells expressed CD80 or CD86, antigens not usually expressed by CD3+ lymphocytes. The increased expression appeared to be independent of stimulation status in that it occurred in both the R/H and P/H treatment groups but not in resting or PHA-stimulated uninfected cells. CD28 expression was generally comparable between CD3+ cells that did and did not express CD80 or CD86. Multiparameter flow cytometry, in association with improved techniques for cell permeabilization and cytoplasmic fluorescent staining, should prove useful in examining the effects of productive HIV infection on surface and cytoplasmic cellular molecules. Using this approach, we found an association between productive infection and increased expression of CD80 and CD86. This association has implications for HIV disease pathogenesis and, potentially, HIV therapy.
Asunto(s)
Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Complejo CD3/metabolismo , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/metabolismo , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Glicoproteínas de Membrana/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígeno B7-1/inmunología , Antígeno B7-2 , Biomarcadores , Citometría de Flujo , Anticuerpos Anti-VIH/sangre , Seronegatividad para VIH , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Glicoproteínas de Membrana/inmunología , Receptores de Interleucina-2/metabolismoRESUMEN
We investigated the evidence for an infectious etiology of Kawasaki disease (KD), an acute vasculitis of unknown etiology, by assessing the effects of KD on the T cell antigen receptor variable beta region families (V beta). Using 3-color flow cytometry, we studied KD patients pre- and post-intravenous gamma globulin (IVIG) therapy and at > 40 days post therapy, additionally comparing them to matched pediatric control patients (PCC) and their own healthy parents (one parent/KD child). Of all the V beta families examined, only V beta 2 exhibited statistically significant differences, between the pre- and post-IVIG samples and preIVIG and parent samples. No associations were found between V beta 2 findings and T cell memory, activation, or adhesion markers. For 2 KD patients, 4 parents, and 1 PCC participant, > 15% of resting CD8+ lymphocytes and > 15% of blastic CD8+ lymphocytes expressed a single V beta family, which varied by individual, without similar expansions in the CD4+ cell populations. One of the participants with this abnormality was the only one with significant cardiac abnormalities. For all participants with the V beta abnormality, other T-cell abnormalities were extensive and involved both CD4+ and CD8+ cells. We suggest that V beta 2 changes do occur in KD, as previously reported. However, these may not be involved in disease pathogenesis. Other V beta changes also occur. Those occurring in parents may reflect asymptomatic reinfection with an infectious agent causing KD. Further, some KD patients may have restricted cytotoxic T-cell responses to that as yet unidentified agent; this restricted response may be associated with more severe cardiac involvement.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Síndrome Mucocutáneo Linfonodular/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Enfermedad Aguda , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Lactante , Selectina L/inmunología , Masculino , Síndrome Mucocutáneo Linfonodular/terapia , Estadísticas no Paramétricas , gammaglobulinas/administración & dosificaciónRESUMEN
Kawasaki disease (KD) is an acute vasculitis of unknown etiology, occurring in young children and treated with intravenous gamma globulin (IVIG) to prevent significant cardiac morbidity and mortality. We studied KD patients pre- and post-IVIG therapy and at >40 days posttherapy, additionally comparing them with matched pediatric control patients and parents. Using three-color flow cytometry, we examined immune changes in KD, especially previously unassessed markers of T-lymphocyte activation, memory, and adhesion. The percentage of cells positive for CD19, CD25, CD38, and CD71 was significantly lower during convalescence compared with pre-IVIG (medians: CD19, 18% vs 26%, P = 0.0004; CD25, 6% vs 9% for CD3(+) cells, P = 0.0074; CD38, 78% vs 89% for CD8(+) cells, P = 0.0015; CD71, 1% vs 6% for CD4(+) cells, P = 0.0024). The proportion of CD3(+) cells increased (medians: CD3, 66% vs 45%, P < 0.0001). Values for all parameters varied greatly pre- and post-IVIG, but not in a consistent direction. The sole patient with cardiac abnormalities had the greatest pre-/post-IVIG variability. These changes support the involvement of T-lymphocytes in the acute KD vasculitic process. They also suggest that T-lymphocytes involved in endothelial damage during acute KD may be subsequently removed or eliminated from the peripheral blood.
Asunto(s)
Síndrome Mucocutáneo Linfonodular/inmunología , Linfocitos T/inmunología , Antígenos CD/análisis , Antígenos CD19/análisis , Antígenos de Diferenciación de Linfocitos B/análisis , Biomarcadores/sangre , Crisis Blástica/inmunología , Crisis Blástica/patología , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/inmunología , Niño , Preescolar , Femenino , Humanos , Inmunoglobulinas Intravenosas/farmacología , Lactante , Integrina beta1/análisis , Masculino , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Interleucina-2/análisis , Receptores de Transferrina , Linfocitos T/química , Factores de TiempoRESUMEN
The authors investigated whether the human immunodeficiency virus (HIV) has restrictive effects on the variable region of the beta chain (V beta) of the T-cell antigen receptor (TCR), by in vitro cultivation of non-HIV-infected peripheral blood lymphocytes with one of six HIV antigens or heat-inactivated whole virus (HIV-HI). Resting and blastic CD4+ and CD8- cells were assessed with 3-colour cytofluorometry and monoclonal antibodies to various V beta families/subfamilies. The V beta families affected include V beta's 13.1/.3, 8, and 21 with gp 120; V beta 21 with gp 160 and RT; V beta 8 with p25; V beta's 8 and 21 with Rev; and V beta's 3 and 21 with HIV-2 Vpx. V beta family-specific effects with HIV-HI did not differ significantly from those found with IL-2 stimulation. Findings differed between CD4+ and CD8+ cells. For CD4+ lymphocytes, significant V beta-specific decreases were found, not the expansions found with superantigens or mitogens. CD8+ lymphocytes showed slight but significant expansions. The effects on V beta's 8, 13, and 21 are consistent with previous studies of HIV-infected persons. However, it is difficult to accept that antigens encoded by different HIV genetic regions cause proportionate diminutions of similar V beta families. The authors suggest that these effects may be secondary to changes in cytokine profiles rather than direct interactions with TCR V beta's.
Asunto(s)
Antígenos VIH/genética , Antígenos VIH/inmunología , Familia de Multigenes/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Homólogo de la Proteína Chromobox 5 , Enterotoxinas/inmunología , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/inmunología , Humanos , Lactante , Recién Nacido , Activación de Linfocitos/inmunología , Reacción en Cadena de la Polimerasa , Staphylococcus aureus/inmunología , Superantígenos/inmunologíaRESUMEN
Phytohaemagglutinin (PHA), Concanavalin A (Con A), interleukin-2 (IL-2), and monoclonal antibodies to CD3 (CD3MoAbs) are used for the assessment of the T-cell receptor (TCR) BV gene family expression in autoimmune disorders and multiple sclerosis, and to produce clones for assessment of cytokine profiles in progressive human immunodeficiency virus infection. The authors examined the effects of these stimulants on the TCR V beta repertoire of resting and blastic CD4+ and CD8+ normal human peripheral blood lymphocytes, using three-colour cytofluorometry and a panel of anti-TCR V beta monoclonal antibodies. IL-2 was associated with an increased percentage of blastic CD4+ cells expressing V beta 5.1 (from median of 3.7% to 8.0%, P = 0.0002) and blastic CD8+ cells expressing V beta 5.3 (1.0 to 1.5%, P = 0.0039). CD3MoAb caused a slight increase in V beta 6.7 + blastic CD4+ cells (4.5 to 6.9%, P = 0.0078). PHA did not alter the V beta repertoire of blastic cells. Con A caused skewing in CD8+ blastic cells, toward expression of V beta 5.2/5.3 (3.1 to 8.1%) and V beta 5.3 (0.8 to 4.8%) (P = 0.0020). Thus, IL-2 stimulation causes slight alterations in the V beta repertoire that should be taken into account in certain research settings. Con A produced skewing in CD8+ blastic cells suggesting that, in the presence of CD8, either Con A binds selectively to certain V beta or the three-dimensional complex created by Con A's binding to other T-cell surface molecules induces preferential V beta 5 stimulation.
Asunto(s)
Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Donantes de Sangre , Complejo CD3/fisiología , Enterotoxinas/inmunología , Humanos , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Mitógenos/farmacología , Transducción de Señal , Superantígenos/inmunologíaRESUMEN
NADPH-dependent superoxide generation was activated by anionic amphiphiles plus GTP gamma S in a cell-free system consisting of plasma membranes plus recombinant p47-phox, p67-phox, and the small GTP-binding protein Rac1. Rac1 was expressed in Escherichia coli both as the native form and as a mutant form (Rac1(C189S)) lacking the prenylation site. When preloaded with GTP gamma S, both Rac proteins supported activity to a level comparable to that seen using cytosol. A peptide corresponding to the carboxyl-terminal region of Rac1 was used to investigate oxidase assembly and activation. Rac1(178-188), but not several control peptides, inhibited activity. The peptide inhibited competitively (Ki = 15 microM) with respect to Rac1(C189S), while inhibition was noncompetitive or mixed with respect to p47-phox and p67-phox. This indicated specific inhibition of the interaction of the Rac protein with its target, possibly cytochrome b558. The peptide was effective only when added prior to activation with arachidonic acid, suggesting that it affects assembly rather than activity. Consistent with this possibility, the peptide prevented translocation of p47-phox and p67-phox to the plasma membrane. Thus, Rac plays a central role in the assembly of the neutrophil NADPH oxidase.
Asunto(s)
Proteínas de Unión al GTP/fisiología , NADH NADPH Oxidorreductasas/fisiología , NADPH Deshidrogenasa/fisiología , NADPH Oxidasas , Fosfoproteínas/fisiología , Secuencia de Aminoácidos , Membrana Celular/enzimología , Sistema Libre de Células , Grupo Citocromo b/metabolismo , Activación Enzimática , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Neutrófilos/enzimología , Péptidos/química , Relación Estructura-Actividad , Superóxidos/metabolismo , Proteínas de Unión al GTP racRESUMEN
We recently characterized a "semirecombinant" cell-free NADPH-oxidase system, comprised of plasma membrane plus the recombinant cytosolic proteins p47-phox and p67-phox, wherein superoxide generation was activated by an anionic amphiphile plus guanosine 5'-O-(2-thiotriphosphate) (GTP gamma S) (Uhlinger, D. J., Inge, K. L., Kreck, M. L., Tyagi, S. R., Neckelmann, N., and Lambeth, J. D. (1992) Biochem. Biophys. Res. Commun. 186, 509-516). Based on preincubation with guanine nucleotides, we show that plasma membrane contains G protein(s) that support oxidase activation at submaximal rates. By varying p47-phox and p67-phox concentrations, kinetic parameters (EC50 and Vmax) for each were determined. For both, GTP gamma S increased the Vmax and decreased the EC50, whereas guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) produced the opposite effect, consistent with the participation of a G protein in an activation complex containing p47-phox and p67-phox. Using [35S]methionine-labeled p47-phox and p67-phox, we investigated the association of these components with both normal plasma membranes and chronic granulomatous disease membranes lacking cytochrome b558. p47-phox translocation was stimulated by arachidonate but not GTP gamma S, was about 50% cytochrome-dependent, and occurred independently of p67-phox. Arachidonate-stimulated translocation of p67-phox required both cytochrome and p47-phox and was enhanced by GTP gamma S. The mass of p47-phox and p67-phox which assembled with cytochrome b558 indicated a ternary complex with a 1:1:1 stoichiometry.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Nucleótidos de Guanina/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Neutrófilos/enzimología , Estallido Respiratorio , Adulto , Secuencia de Bases , Membrana Celular/metabolismo , Sistema Libre de Células , ADN , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Cinética , Datos de Secuencia Molecular , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Fosfoproteínas/metabolismo , Superóxidos/metabolismoRESUMEN
Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.
Asunto(s)
NADH NADPH Oxidorreductasas/sangre , NADPH Oxidasas , Neutrófilos/enzimología , Animales , Baculoviridae/genética , Línea Celular , Membrana Celular/enzimología , Sistema Libre de Células , Humanos , Insectos , Cinética , Sustancias Macromoleculares , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Superóxidos/sangre , TransfecciónRESUMEN
Eleven morphologic criteria were studied from videotapes of 323 fresh and 103 thawed embryos. The proportion of thawed embryos (30/39; 77%) with more than one abnormality was (P = 0.03) higher than that of fresh embryos (5/13; 38%), despite similar implantation rates (18% and 15%, respectively). The best predictor of thawed embryo implantation was cell-cell adherence: when scored "positive," 11 of 17 women (65%) became pregnant, whereas none became pregnant (0/15; P = 0.0002) when blastomeres did not adhere. The total number of abnormalities for thawed embryos was an important prognostic. An increased percentage variation of zona pellucida thickness was the most important prognostic for fresh embryos. When the "best" embryo had a zona pellucida that varied more than 25%, 24 of 60 (40%) resulted in pregnancy; pregnancies were not induced (0/21) when the "best" embryo had less than 10% variation (P = 0.0003).
Asunto(s)
Transferencia de Embrión , Embrión de Mamíferos/ultraestructura , Congelación , Grabación de Cinta de Video , Blastómeros/ultraestructura , Humanos , Estudios Retrospectivos , Preservación de Semen , Zona Pelúcida/ultraestructuraRESUMEN
The incidence of cell injury, embryo survival, and implantation following cryopreservation of zygotes and two- to five-cell embryos was studied in 100 patients in order to evaluate the effect of duration of storage. The incidence of individual cell survival was 58% regardless of the length of time kept in liquid nitrogen or the stage of the embryo at freezing. There were 104 of 208 (50%) thawed embryos that survived completely intact, and of those, 24 implanted successfully. Twenty-one (21%) patients had a clinical pregnancy; two of them miscarried Neither the survival of zygotes or cleaved embryos upon thawing nor the incidence of implantation was affected by the duration of cryostorage.
Asunto(s)
Embrión de Mamíferos , Preservación Biológica , Fase de Segmentación del Huevo/fisiología , Transferencia de Embrión , Femenino , Congelación , Humanos , Hormona Luteinizante/metabolismo , Inducción de la Ovulación , EmbarazoRESUMEN
Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.