Summary A proteolysis-inducing factor (PIF) isolated from a cachexia-inducing murne tumour (MAC16) produced a decrease in body weight (1.6 g, P. 0.01 compared with control subjects) within 24 h after i.v. administration to non-tumour-bearing mice. Weight loss was associated with significant decreases in the weight of the spleen and soleus and gastrocnemius muscles. with no effect on the weight of the heart or kidney and with an increase in weight of the liver. Protein degradation in isolated soleus muscle was signfficantly increased in mice bearing the MAC16 tumour. To define which proteolytic pathways contribute to this increase, soleus muscles from mice bearing the MAC16 tumour and non-tumour-bearing animals administered PIF were incubated under conditions that modify different proteolytic systems. In mice bearing the MAC16 tumour, there were increases in both cathepsin B and L, and the Ca2--dependent lysosomal and ATP-dependent pathways were found to contribute to the increased proteolysis; whereas, in PIF-injected animals, there was activation only of the ATP-dependent pathway. Further studies in mice bearing the MAC16 tumour have provided evidence for increased levels of ubiquitin-conjugated proteins and increased mRNA levels for the 14 kDa ubiquitin carrier protein E2 and the C9 proteasome subunit in gastrocnemius muscle, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. A monoclonal antibody to PIF attenuated the enhanced protein degradation in soleus muscle from mice bearing the MAC16 tumour, confirming that PIF is responsible for the loss of skeletal muscle in cachectic mice.

No abstract

Summary Urine from cancer patients with weight loss showed the presence of an antigen of Mr 24 000 detected with a monoclonal antibody formed by fusion of splenocytes from mice with cancer cachexia. The antigen was not present in the urine of normal subjects, patients with weight loss from conditions other than cancer or from cancer patients who were weight stable or with low weight loss (1 kg month-1). The antigen was present in the urine from subjects with carcinomas of the pancreas, breast, lung and ovary. The antigen was purified from urine using a combination of affinity chromatography with the mouse monoclonal antibody and reversed-phase high-performance liquid chromotography (HPLC). This procedure gave a 200 000-fold purification of the protein over that in the original urine extract and the material isolated was homogeneous, as determined by silver staining of gels. The N-terminal amino acid sequence showed no homology with any of the recognized cytokines. Administration of this material to mice caused a significant (P<0.005) reduction in body weight when compared with a control group receiving material purified in the same way from the urine of a normal subject. Weight loss occurred without a reduction in food and water intake and was prevented by prior administration of the mouse monoclonal antibody. Body composition analysis showed a decrease in both fat and non-fat carcass mass without a change in water content. The effects on body composition were reversed in mice treated with the monoclonal antibody. There was a decrease in protein synthesis and an increase in degradation in skeletal muscle. Protein degradation was associated with an increased prostaglandin E2 (PGE2) release. Both protein degradation and PGE2 release were significantly reduced in mice pretreated with the monoclonal antibody. These results show that the material of Mr 24 000 present in the urine of cachectic cancer patients is capable of producing a syndrome of cachexia in mice.

Loss of skeletal muscle is a major factor in the poor survival of patients with cancer cachexia. This study examines the mechanism of catabolism of skeletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intravenous administration of PIF to normal mice produced a rapid decrease in body weight (1.55 ± 0.12 g in 24 h) that was accompanied by increased mRNA levels for ubiquitin, the Mr 14 000 ubiquitin carrier-protein, E2, and the C9 proteasome subunit in gastrocnemius muscle. There was also increased protein levels of the 20S proteasome core and 19S regulatory subunit, detectable by immunoblotting, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. An increased protein catabolism was also seen in C2C12 myoblasts within 24 h of PIF addition with a bell-shaped dose–response curve and a maximal effect at 2–4 nM. The enhanced protein degradation was attenuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lactacystin. Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis showed a dose-related increase in expression of MSSI, an ATPase that is a regulatory subunit of the proteasome, with a dose–response curve similar to that for protein degradation. These results confirm that PIF acts directly to stimulate the proteasome pathway in muscle cells and may play a pivotal role in protein catabolism in cancer cachexia. © 2001 Cancer Research Campaign http://www.bjcancer.com

Summary An antigen of apparent molecular weight of 24000, reactive with a murine monoclonal antibody, has been isolated from a cachexia-inducing tumour (MAC 16) and has been shown to initiate muscle protein degradation in vitro using isolated soleus muscle. Administration of this material to female NMRI mice (20 g) produced a pronounced depression in body weight (2.72 ± 0.14 g; P<0.005 from control) over a 24 h period. This weight loss was attenuated in mice pretreated with the monoclonal antibody (0.06 + 0.26 g over 24 h) and occurred without a reduction in food and water intake. There was no change in body water composition, and the major contribution to the decrease in body weight was a decrease in the non-fat carcass dry weight (mainly lean body mass). The plasma levels of glucose and most amino acids were also significantly depressed. The decrease in lean body mass was accounted for by an increase (by 50%) in protein degradation and a decrease (by 50%) in protein synthesis in gastrocnemius muscle. Protein degradation was significantly decreased and protein synthesis increased to control values in mice pretreated with the monoclonal antibody. Protein degradation initiated in vitro with the proteolysis-inducing factor was abolished in mice pretreated with eicosapentaenoic acid (EPA), which had been shown to prevent muscle wastage in mice bearing the MAC16 tumour. Protein degradation was associated with a significant elevation of prostaglandin E2 production by isolated soleus muscle, which was inhibited by both the monoclonal antibody and EPA. These results suggest that this material may be the humoral factor mediating changes in skeletal muscle protein homeostasis during the process of cancer cachexia in animals bearing the MAC16 tumour, and could potentially be involved in other cases of cachexia.

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1–5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign http://www.bjcancer.com

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anionexchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. Carbon monoxide (CO) dehydrogenases (CODHs) are key enzymes in the CO metabolism of physiologically and phylogenetically diverse microbes. Carboxidotrophic bacteria are aerobic chemolithoautotrophs characterized by the utilization of CO as a sole source of carbon and energy. They are taxonomically diverse bacteria, encompassing more than 15 described species in eight genera (5, 27). The CODHs of Oligotropha carboxidovorans (37) and Pseudomonas thermocarboxydovorans (32) have been cloned and sequenced. The CODH from O. carboxidovorans is the best characterized (21-23, 30). The enzyme is an O 2 -stable, molybdenum-iron-sulfurflavin hydroxylase that catalyzes the oxidation of CO to CO 2 according to the equation CO ϩ H 2 O 3 CO 2 ϩ 2e Ϫ ϩ 2H ϩ . It contains the molybdopterin cytosine dinucleotide-type molybdenum cofactor (29) and [2Fe-2S] centers of type I and type II (2, 10). In anaerobic microorganisms, CODHs are nickeliron-sulfur proteins, usually O 2 labile, that function in a variety of energy-yielding pathways. The CODH from acetogenic Moorella thermoacetica reduces CO 2 to acetyl coenzyme A, providing acetate as the major end product (33), and that from acetotrophic Methanosarcina and Methanothrix obtains energy by fermenting acetate to CH 4 and CO 2 (17). A similar enzyme from phototrophic Rubrivivax gelatinosus and Rhodospirillum rubrum reduces CO and water to CO 2 and H 2 (40-42). Acetotrophic sulfate reducers also utilize CODH to cleave acetyl coenzyme A, whereas sulfate is reduced to sulfide (12, 36). CO oxidation and the fundamental role of CODHs in aerobic and anaerobic pathways of carbon metabolism have been reviewed previously (5,28,30).Bradyrhizobium japonicum species are gram-negative soil bacteria with the unique ability to establish N 2 -fixing symbiosis with soybeans (Glycine max). Although all rhizobia have been considered to be aerobic chemoorganotrophs that grow best on complex media (38, 43), some hydrogenase uptake-positive (Hup ϩ ) B. japonicum strains have been shown to grow chemolithoautotrophically utilizing H 2 and CO 2 as sole sources of energy and carbon, respectively (9, 19). With only very few exceptions, carboxidotrophs not only oxidize CO but can use H 2 plus CO 2 as well, indicating the presence of two different capacities for chemolithoautotrophic growth (25). Given the similarities observed between carboxidotrophic bacteria and hydroge...

We report the design and discovery of a 2-aminobenzimidazole-based series of potent and highly selective p38alphainhibitors. The lead compound 1 had low-nanomolar activity in both ATP competitive enzyme binding and inhibition of TNFalpha release in macrophages. Compound 18 showed excellent pharmacokinetics properties and oral activity in the rat collagen induced arthritis model compared with other p38 reference compounds. A SAR strategy to address CyP3A4 liability is also described.