Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.     

Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.     

Protein synthesis in synaptosomes: a proteomics analysis

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two-dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver-stained gels that were run in parallel. In-gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear-encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre-synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.     

Anti-TNF-alpha antibody allows healing of joint damage in polyarthritic transgenic mice

Anti-tumor-necrosis-factor-alpha (TNF-alpha) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-alpha (hTNF-alpha) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-alpha antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-alpha treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-alpha prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-alpha and local expression of various proinflammatory mediators were all diminished by anti-TNF-alpha treatment, confirming a critical role of hTNF-alpha in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-alpha treatment was observed in young mice but not in aged mice.     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.     

Identification of an endocrine disrupting agent from corn with mitogenic activity

A mitogenic agent in corncob bedding and fresh corn products disrupts sexual behavior and estrous cyclicity in rats. The mitogenic activity resides in an isomeric mixture of linoleic acid derivatives with a tetrahydrofuran ring and two hydroxyl groups (THF-diols) that include 9, (12)-oxy-10,13-dihydroxystearic acid and 10, (13)-oxy-9,12-dihydroxystearic acid. Synthetic THF-diols stimulated breast cancer cell proliferation in vitro and disrupted the estrous cycle in female rats at oral doses of approximately 0.30 mg/kg body weight/day. Exposure to THF-diols may disrupt endocrine function in experimental animals at doses approximately 200 times lower than classical phytoestrogens, promote proliferation of breast or prostate cancer, and adversely affect human health.     

Training common marmosets (Callithrix jacchus) to cooperate during routine laboratory procedures: ease of training and time investment

The first author trained 12 laboratory-housed common marmosets (Callithrix jacchus) in pairs to assess the practicality of positive reinforcement training as a technique in the management of these nonhuman animals. Behaviors taught were (a) target training to allow in homecage weighing and (b) providing urine samples. Between 2 to 13, 10-minute training sessions established desired behaviors. Training aggressive animals only after they had been fed eliminated aggression during training. Trained animals proved extremely reliable, and data collection using trained animals was considerably faster than collection using current laboratory techniques. The results suggest that positive reinforcement training is a practical option in the management of laboratory-housed marmosets.     

Abdominal cysticercosis in a rhesus macaque (Macaca mulatta)

A mid-abdominal mass was discovered during routine physical examination of a rhesus macaque (Macaca mulatta). Further diagnostics and exploratory laparotomy were performed, revealing a fluid-filled cyst attached to the caudal free margin of the greater omentum. Formation and pulsatile movement of white-colored circumferential bands within the wall of the cyst were observed during surgery. The cyst was removed and later was dissected. The discovery of a single invaginated scolex identified the cyst as a cysticercus. The location and characteristics of the cysticercus were consistent with the larval form of Taenia hydatigena.     

Ethanol ingestion via glutathione depletion impairs alveolar epithelial barrier function in rats

We determined that rats fed a liquid diet containing ethanol (36% of calories) for 6 wk had decreased (P < 0.05) net vectorial fluid transport and increased (P < 0.05) bidirectional protein permeability across the alveolar epithelium in vivo compared with rats fed a control diet. However, both groups increased (P < 0.05) fluid transport in response to epinephrine (10(-5) M) stimulation, indicating that transcellular sodium transport was intact. In parallel, type II cells isolated from ethanol-fed rats and cultured for 8 days formed a more permeable monolayer as reflected by increased (P < 0.05) leak of [(14)C]inulin. However, type II cells from ethanol-fed rats had more sodium-permeant channels in their apical membranes than type II cells isolated from control-fed rats, consistent with the preserved response to epinephrine in vivo. Finally, the alveolar epithelium of ethanol-fed rats supplemented with L-2-oxothiaxolidine-4-carboxylate (Procysteine), a glutathione precursor, had the same (P < 0.05) net vectorial fluid transport and bidirectional protein permeability in vivo and permeability to [(14)C]inulin in vitro as control-fed rats. We conclude that chronic ethanol ingestion via glutathione deficiency increases alveolar epithelial intercellular permeability and, despite preserved or even enhanced transcellular sodium transport, renders the alveolar epithelium susceptible to acute edematous injury.