From bench to bedside: initial experience with the Primus drug-coated balloon catheter.

AIM: Drug-coated balloon (DCB) technology has emerged as a promising therapy particularly in the treatment of coronary in-stent restenosis. Although a variety of devices are available for clinical use, clinical outcomes have been variable and scope for significant improvement exists. METHODS: In a preclinical study, a total of 10 juvenile healthy farm pigs underwent catheter-based DCB deployment in coronary arteries with angiographic and pathological follow-up at 7 or 28 days. Animals were randomly allocated to the PRIMUS or Dior® DCB (N.=10 per group) and evaluated by histopathology and morphometric analysis. In a first-in-man clinical study a total of 19 consecutive patients presenting with restenosis within drug-eluting stents were treated with the PRIMUS DCB. Clinical follow-up was performed out to 6 months. RESULTS: Neointimal thickness was similar between the PRIMUS and Dior® DCB groups, while fibrin deposition and inflammation were more sustained in the PRIMUS group at 28 days. In 19 consecutive patients presenting with in-stent restenosis of drug-eluting stents, treatment with the PRIMUS DCB catheter resulted in high procedural efficacy. There were no adverse clinical events observed out to 6 months. CONCLUSION: The PRIMUS DCB demonstrates high preclinical safety and excellent acute performance and safety. Further studies are needed to delineate the relative merits of this novel DCB compared to other devices.

Selective activation of the 20 S proteasome (multicatalytic proteinase complex) by histone h3.

Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment.

Heat shock protein-90 and the catalytic activities of the 20 S proteasome (multicatalytic proteinase complex).

The effect of heat shock protein 90 (Hsp-90) and several other proteins on the catalytic activities of the 20 S proteasome (MPC) was examined. The chymotrypsin-like (ChT-L) and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of the pituitary MPC were inhibited by Hsp-90 with IC50 values of 8 and 28 nM, respectively. Bovine serum albumin and two other proteins tested inhibited the same activities with much higher IC50 values. The trypsin-like and branched-chain amino-acid-preferring activities were not affected by any of the proteins. None of the activities of the bovine spleen MPC, an enzyme form in which the X, Y, and Z subunits are virtually completely replaced by the LMP2, LMP7, and LMP10 subunits, was affected by either Hsp-90 or the other proteins tested. Hsp-90 inhibited the degradation of the oxidized B-chain of insulin by the pituitary MPC but not by its spleen counterpart. The PA28 activator (11 S regulator; REG) of the proteasome abolished the inhibitory effect of Hsp-90 and other proteins on the ChT-L and PGPH activities of the pituitary MPC. It is suggested that Hsp-90 induces conformational changes that affect the ChT-L and PGPH activities expressed by the X and Y subunits, respectively, but does not affect the activities expressed by LMP subunits.

Evaluation of acid gastric secretion with 24-hour pH-metry in patients with acute and chronic hepatitis induced by HBV.

BACKGROUND: Acute and chronic diseases of the liver often coexist with functional disorders and pathological changes in the stomach. Lavish data indicate greatly higher incidence of ulcerations and gastric erosions in patients with various hepatopathies. Due to considerable discrepancies in the assessment of acid gastric secretion, an attempt has been made to evaluate this phenomenon with 24-hour pH-metry in patients with acute and chronic HBV. MATERIAL AND METHODS: The study material consisted of two equal groups of patients with dyspepsia--12 subjects with acute HBV and 12 subjects with chronic HBV, as well as 15 healthy volunteers forming a control group. At enrollment, the patients over 65 years of age were excluded from the study as well as those with chronic diseases, with the history of surgery in gastrointestinal tract and the subjects treated with the medicines affecting gastric secretion. All the patients underwent pH-metry with the use of DL70 pH-meter with a glass electrode. Median pH (Me) and mean pH (Arith. mean) obtained during 24 hours were used in the analysis. RESULTS: The following results were obtained: Me = 1.57, Arith. mean = 1.97 in control group, Me = 2.57, Arith. mean = 3.12 in patients with acute HBV, Me = 1.47, Arith. mean = 1.77 in patients with chronic HBV. On the basis of the statistical analysis, no statistically significant differences in gastric secretion determined with the help of 24-hour pH-metry were found between the analysed groups. CONCLUSION: Patients with acute and chronic HBV do not display statistically significant differences in gastric secretion determined with the help of 24-hour pH-metry.

Correlation between liver damage and degree of gastric mucose colonization by Helicobacter pylori in subjects with parenchymatous liver damage.

BACKGROUND: Helicobacter pylori (H. pylori) is an important factor responsible for chronic inflammatory conditions of the gastric mucosa. It has been demonstrated in numerous animal studies that some Helicobacter species may cause parenchymatous liver damage. The aim of the study was to investigate whether there is any correlation between the incidence of parenchymatous liver damage, and the incidence and degree of colonization of the gastric mucosa by H. pylori. MATERIAL AND METHODS: The study was carried out in the group of 30 patients (14 females, 16 males) whose mean age was 37 years, hospitalized because of parenchymatous liver damage without clinical symptoms of cirrhosis. All the patients had gastroscopy and urease tests performed, and mucosal biopsies were taken for immunomorphological investigations. The patients were divided into groups, group I comprising those with positive, and group II with negative urease test results. RESULTS: Positive urease tests were obtained in 26/30 patients (group I), 18/26 of whom demonstrated macroscopic changes of the gastric mucosa visible in gastroscopy. Group II with negative urease test results comprised 4/30 patients, 2/4 of whom had detectable changes in the gastric mucosa. The presence of H. pylori antigens was demonstrated by gastric mucosa immunomorphology in all 30 patients. The degree of invasion of H. pylori was visualized by immunofluorescence, which allowed to differentiate deep mucosal invasion of H. pylori (bacterial antigens present in lymph follicles and at the base of muciferous glands) observed in group I in 14/26 and in group II in 1/4 cases and superficial invasion (epithelium and mucosal surface) observed in group I in 12/26, in group II in 3/4. CONCLUSIONS: The obtained results may suggest more frequent H. pylori infections in subjects with parenchymatous liver damage than in the population without liver damage. Immunofluorescence seems to be a highly sensitive method allowing for detection of even small degrees of gastric mucosa colonization by H. pylori.

[Staphylococcus aureus sepsis--still life threatening disease]. / Posocznica gronkowcowa--choroba nadal zagrazajaca zyciu.

We report an analysis of clinical course of 18 patients presenting with Staphylococcus aureus sepsis. Community acquired infection was caused by Methicillin susceptible S. aureus (MSSA) in 11 patients. MSSA in 3 and Methicillin Resistant S. aureus strains (MRSA) in 4 patients, were the etiologic factor in 7 patients with nosocomial infection. From anamnestic data patients presented with: elevated body temperature--18/18, arthralgia and myalgia--9/18, headache--8/18, nausea--6/18, chills--2/18. Physical examination on admission revealed: meningismus--12/18, hepatomegaly--11/18, purulent and haemorrhagic skin lesions--7/18 and impaired neurological status (Glasgow Coma Scale < or = 12)--6/18. The mean APACHE III score, calculated from data collected at diagnosis of sepsis was 47 (7-114). Several complications had been observed: endocarditis--10, purulent meningitis--5, focal CNS lesions--5, pneumonia--8, pulmonary abscess--3, hydrothorax--1, abscesses of the spleen--5, renum--4, osteomyelitis--2. 11/18 patients required ICU treatment. Ventilator assistance of respiration was necessary in 7/18. Acute thrombocytopenia (< 100,000/ml) was diagnosed in 60%. In 5 patients suppurative meningitis had been diagnosed with a mean pleocytosis-837 (173-1898) microL. The results of treatment were satisfactory in 11 patients, 3 patients required further surgical treatment (2--cardiosurgery, 1--orthopedic surgery), 4 patients died. Infection caused by community acquired MSSA strains had been characterized by severe clinical course with increased incidence of endocarditis, organ failure and abscess forming. We conclude that Staphylococcus aureus sepsis is still a life-threatening disease, which should be treated at centers with immediate access to imaging techniques of CNS and circulatory system as well as intensive care and cardiosurgery. Community acquired S. aureus sepsis compared with nosocomial infection is characterized by more severe clinical course and higher mortality, despite of a great susceptibility to most antibiotics of causative S. aureus strains.

Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex.

The proteasome, a multisubunit, multicatalytic proteinase complex, is attracting growing attention as the main intracellular, extralysosomal, proteolytic system involved in ubiquitin-(Ub) dependent and Ub-independent intracellular proteolysis. Its involvement in the mitotic cycle, and control of the half-life of most cellular proteins, functions absolutely necessary for cell growth and viability, make it an attractive target for researchers of intracellular metabolism and an important target for pharmacological intervention. The proteasome belongs to a new mechanistic class of proteases, the N-terminal nucleophile hydrolases, where the N-terminal threonine residue functions as the nucleophile. This minireview focuses on the three classical catalytic activities of the proteasome, designated chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing in eukaryotes and also the activities of the more simple Archaebacteria and Eubacteria proteasomes. Other catalytic activities of the proteasome and their possible origin are also examined. The specificity of the catalytic components toward synthetic substrates, natural peptides, and proteins and their relationship to the catalytic centers are reviewed. Some unanswered questions and future research directions are suggested.

Ultra-high throughput screen of two-million-member combinatorial compound collection in a miniaturized, 1536-well assay format.

Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.

A statistical-based approach to assessing the fidelity of combinatorial libraries encoded with electrophoric molecular tags. Development and application of tag decode-assisted single bead LC/MS analysis.

A statistical sampling protocol is described to assess the fidelity of libraries encoded with molecular tags. The methodology, termed library QA, is based on the combined application of tag decode analysis and single bead LC/MS. The physical existence of library compounds eluted from beads is established by comparing the molecular weight predicted by tag decode with empirical measurement. The goal of sampling is to provide information on overall library fidelity and an indication of the performance of individual library synthons. The minimal sampling size n for library QA is l0 x the largest synthon set. Data are reported as proportion (p) +/- lower and upper boundary (lb-ub) computed at the 95% confidence level (alpha = 0.05). As a practical demonstration, library QA was performed on a 25,200-member library of statine amides (size = 40 x 63 x 10). Sampling was conducted three times at n approximately 630 beads per run for a total of 1902 beads. The overall proportions found for the three runs were consistent with one another: p = 84.4%, lb-ub = 81.5-87.2%; p = 83.1%, lb-ub = 80.2-85.95; and p = 84.5%, lb-ub = 81.8-87.3%, suggesting the true value of p is close to 84% compound confirmation. The performance pi of individual synthons was also computed. Corroboration of QA data with biological screening results obtained from assaying the library against cathepsin D and plasmepsin II is discussed.

[Gastric neurilemmoma complicated by gastroduodenal intussusception and with bleeding from the digestive tract]. / Nerwiak oslonkowy zoladka powiklany wglobieniem zoladkowo-dwunastniczym i krwotokiem z przewodu pokarmowego.

A case of Gastric Neurilemmoma with bleeding and gastroduodenal intussusception was described.

Components of the bovine pituitary multicatalytic proteinase complex (proteasome) cleaving bonds after hydrophobic residues.

Two catalytic components of the multicatalytic proteinase complex (MPC, proteasome) designated as chymotrypsin-like (ChT-L) and branched chain amino acid preferring (BrAAP) cleave bonds after hydrophobic amino acids. The possible involvement of the ChT-L and peptidylglutamyl-peptide hydrolyzing (PGPH) activities in the cleavage of bonds attributed to the BrAAP component was examined. Several inhibitors of the ChT-L activity containing a phenylalaninal group did not affect the BrAAP activity at concentrations that were more than 150 times higher than their K(i) values for the ChT-L activity. Concentrations of lactacystin that inactivated more than 90% of the ChT-L activity had no effect on the BrAAP activity. Concentrations of 3,4-dichloroisocoumarin (DCI) that inactivated the ChT-L activity activated by up to 10-fold the BrAAP activity toward synthetic substrates and by more than 2-fold the degradation of the insulin B chain in a reaction not inhibited by Z-LGF-CHO, a selective inhibitor of the ChT-L activity. These findings are incompatible with any significant involvement of the ChT-L activity in the cleavage of BrAAP substrates. Both the native and DCI-treated MPC cleaved the insulin B chain mainly after acidic residues in a reaction inhibited by Z-GPFL-CHO, an inhibitor of the BrAAP and PGPH activities. DCI exposure did not result in acylation of the N-terminal threonine in the active site of the Y subunit. These results suggest involvement of the PGPH activity in the cleavage of BrAAP substrates, but this conclusion is incompatible with DCI activation of the BrAAP activity and inactivation of the PGPH activity, and with the finding that proteins inhibiting the PGPH activity had no effect on the BrAAP activity. Rationalization of these contradictions is discussed.

Capecitabine: nursing implications of a new oral chemotherapeutic agent.

PURPOSE/OBJECTIVES: To describe the new oral chemotherapeutic agent capecitabine (Xeloda, Roche Laboratories Inc., Nutley, NJ) and key concepts driving its development and to delineate the nursing impact of patient-administered, home-based chemotherapies. DATA SOURCES: Published papers, investigational materials, package inserts, and clinical experience. DATA SYNTHESIS: Capecitabine recently was approved to treat metastatic breast cancer refractory to paclitaxel and anthracycline-containing regimens. Efficacy has been demonstrated. However, although the current regimen is well-tolerated, > or = 40% of patients require dose modification because of grade 2 or greater toxicity, usually hand-foot syndrome or gastrointestinal symptoms. CONCLUSIONS: Capecitabine frees nurses from infusion-related workload, allowing and demanding a new type and level of patient education. Such education emphasizes compliance with the treatment plan and prevention, timely recognition, and management of toxicities. These practice changes also challenge nurses to advocate for reimbursement of educational practices. IMPLICATIONS FOR NURSING PRACTICE: Oncology nurses will play more of a central role as use of patient-administered, home-based therapies increases. Nurses must enhance their patient-education and telephone symptom-management skills and help to secure reimbursement for such activities.

Tumor growth inhibition induced in a murine model of human Burkitt's lymphoma by a proteasome inhibitor.

Cell growth and viability are dependent on the function of the multicatalytic proteinase complex (proteasome), a multisubunit particle that affects progression through the mitotic cycle by degradation of cyclins. Exposure of rodent fibroblasts and human lymphoblasts in culture to benzyloxycarbonyl-leucyl-leucyl-phenylalaninal (Z-LLF-CHO), a cell-permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome, resulted in the induction of apoptosis in a rapid, dose-dependent fashion. Fibroblasts transformed with ras and myc, lymphoblasts transformed by c-myc alone, and a Burkitt's lymphoma (BL) cell line that overexpresses c-Myc were up to 40-fold more susceptible to apoptosis than were either primary rodent fibroblasts or immortalized nontransformed human lymphoblasts, respectively. To determine whether such preferential apoptosis could impact upon tumor growth in vivo, toxicological studies were performed in mice with severe combined immunodeficiency and showed that mice tolerated single interscapular doses of Z-LLF-CHO without unacceptable toxicity. Severe combined immunodeficient mice bearing s.c. BL tumors in the flank were treated interscapularly with Z-LLF-CHO or a comparable dose of the peptidyl alcohol (Z-LLF-OH), which does not induce proteasome inhibition or apoptosis. Single doses of Z-LLF-CHO induced statistically significant (P < 0.0001) early tumor regression and a significant (P < 0.0001) delay in tumor progression. Analysis of tumor specimens revealed increased apoptosis in BL tumors from mice treated with Z-LLF-CHO. These results, showing a 42% tumor growth delay, indicate that proteasome inhibitors have the potential of curbing the growth of a c-myc-related tumor.

Dissociation of proteasomal degradation of biosynthesized viral proteins from generation of MHC class I-associated antigenic peptides.

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.

Screening aspartyl proteases with combinatorial libraries.

Large numbers of pharmaceutically relevant low-molecular weight compounds can now be synthesized using combinatorial methods. Screening these large libraries of compounds requires high throughput assays. These methods are utilized to search for inhibitors of the aspartyl proteases, plasmepsin II and cathepsin D. Plasmepsin II, a protease found in the malaria parasite, hydrolyzes human hemoglobin, the nutrient source for the parasite and is a new target for anti-malaria therapy. Cathepsin D may be involved in many biological processes and inhibitors would help to clarify the role of cathepsin D in these processes. Plasmepsin II and cathepsin D are approximately 35% identical in amino acid sequence. Therefore, a comparison of the screening results of these two enzymes will be very useful in determining each enzyme's specificity and demonstrating the power of utilizing encoded combinatorial libraries.

CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

Evaluation of a structure-based statine cyclic diamino amide encoded combinatorial library against plasmepsin II and cathepsin D.

A structure-based 18,900-member combinatorial library was synthesized containing a statine template and three cyclic diamino acids as potential P1, P2-P4 surrogates. Evaluation of this encoded library against two aspartyl proteases, plasmepsin II and cathepsin D, led to the identification of selective inhibitors for each enzyme.

Identification of potent inhibitors of Plasmodium falciparum plasmepsin II from an encoded statine combinatorial library.

An encoded 13,020-member combinatorial library was synthesized containing a statine core. Evaluation of this library with plasmepsin II, an aspartyl protease required for hemoglobin metabolism in the malaria parasite, led to the identification of potent and selective inhibitors as well as novel structure-activity relationships.

Reactions of [14C]-3,4-dichloroisocoumarin with subunits of pituitary and spleen multicatalytic proteinase complexes (proteasomes).

Exposure to [14C]-3,4-dichloroisocoumarin (DCI) of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary and spleen leads to label incorporation into several beta-type subunits, to rapid inactivation of the chymotrypsin-like (ChT-L) activity, and to a slower inactivation of other activities of the MPC. The pituitary and spleen MPCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde inhibitors of the ChT-L activity protected the X subunit in the pituitary MPC and unexpectedly the LMP2 subunit in the spleen MPC from label incorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit. Losses in the yield of amino acids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reaction of DCI with the threonine OH group. Brief exposure to [14C]-DCI led to preferential incorporation of label into the LMP2 and X subunits, consistent with the high inactivation rate constants of the ChT-L activity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO, an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating differences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spleen MPC, respectively, and suggest the catalytic functions of two other beta-subunits.