The ßLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.

It has been proposed that introducing tyrosine residues into human hemoglobin (e.g. ßPhe41Tyr) may be able to reduce the toxicity of the ferryl heme species in extracellular hemoglobin-based oxygen carriers (HBOC) by facilitating long-range electron transfer from endogenous and exogenous antioxidants. Surface-exposed residues lying close to the solvent exposed heme edge may be good candidates for mutations. We therefore studied the properties of the ßLys66Tyr mutation. Hydrogen peroxide (H2O2) was added to generate the ferryl protein. The ferryl state in ßLys66Tyr was more rapidly reduced to ferric (met) by ascorbate than recombinant wild type (rwt) or ßPhe41Tyr. However, ßLys66Tyr suffered more heme and globin damage following H2O2 addition as measured by UV/visible spectroscopy and HPLC analysis. ßLys66Tyr differed notably from the rwt protein in other ways. In the ferrous state the ßLys66Tyr forms oxy, CO, and NO bound heme complexes similar to rwt. However, the kinetics of CO binding to the mutant was faster than rwt, suggesting a more open heme crevice. In the ferric (met) form the typical met Hb acid-alkaline transition (H2O to -OH) appeared absent in the mutant protein. A biphasicity of cyanide binding was also evident. Expression in E. coli of the ßLys66Tyr mutant was lower than the rwt protein, and purification included significant protein heterogeneity. Whilst, ßLys66Tyr and rwt autoxidised (oxy to met) at similar rates, the oxygen p50 for ßLys66Tyr was very low. Therefore, despite the apparent introduction of a new electron transfer pathway in the ßLys66Tyr mutant, the heterogeneity, and susceptibility to oxidative damage argue against this mutant as a suitable starting material for a HBOC.

A functional alternative splicing mutation in human tryptophan hydroxylase-2.

The brain serotonergic system has an essential role in the physiological functions of the central nervous system and dysregulation of serotonin (5-HT) homeostasis has been implicated in many neuropsychiatric disorders. The tryptophan hydroxylase-2 (TPH2) gene is the rate-limiting enzyme in brain 5-HT synthesis, and thus is an ideal candidate gene for understanding the role of dysregulation of brain serotonergic homeostasis. Here, we characterized a common, but functional single-nucleotide polymorphism (SNP rs1386493) in the TPH2 gene, which decreases efficiency of normal RNA splicing, resulting in a truncated TPH2 protein (TPH2-TR) by alternative splicing. TPH2-TR, which lacks TPH2 enzyme activity, dominant-negatively affects full-length TPH2 function, causing reduced 5-HT production. The predicted mRNA for TPH2-TR is present in postmortem brain of rs1386493 carriers. The rs13864923 variant does not appear to be overrepresented in either global or multiplex depression cohorts. However, in combination with other gene variants linked to 5-HT homeostasis, this variant may exhibit important epistatic influences.

A pilot randomized controlled trial to examine the feasibility and efficacy of an educational nursing intervention to improve self-management practices in patients with mild-moderate psoriasis.

BACKGROUND: Large numbers of people are expected to self-manage their skin condition, but limited attention has been given to studies of self-management in psoriasis, neither clearly highlighting the challenge nor seeking to develop interventions to support its effectiveness. OBJECTIVES: 1. To test the feasibility of a new educational intervention to enable people with psoriasis to self-manage more effectively an adequately powered multi-centred trial design through piloting. METHOD: Pilot randomized controlled trial with adults (n = 64) with mild-moderate psoriasis in Primary Care in the United Kingdom. Both groups continued with usual treatment. A theory-based educational intervention was designed. The primary outcome measure was the Dermatology Life Quality Index (DLQI). Secondary measures included the Psoriasis Area and Severity Index (PASI) and qualitative feedback from participants. Assessment of the feasibility of the intervention included recruitment and acceptability to participants. RESULTS: Delivery of the intervention was feasible and positively evaluated. Recruitment strategies and the intervention need minor modification. As a pilot study there was insufficient power to detect significant score changes. Sub group analysis of participants with a PASI or DLQI of >6 indicated a modest reduction in PASI in the intervention group which demonstrates a trend that may indicate that this intervention has potential value for people with moderate psoriasis when combined with qualitative data. CONCLUSION: This study highlights the feasibility of delivering a self-efficacy based educational intervention for people with mild-moderate psoriasis in primary care establishing the numbers and design required for an adequately powered multi-centred trial.

The pathology of 'scale drop syndrome' in Asian seabass, Lates calcarifer Bloch, a first description.

This is the first pathological description of 'scale drop syndrome' (SDS) in Asian seabass, Lates calcarifer Bloch. Cumulative mortality was estimated at 40-50%. The vasculitis in all major organs including the skin and associated tissue necrosis was distinctive. The dermis overlying scale beds was often necrotic and associated with scale loss. Necrosis of splenic ellipsoids, renal glomeruli and choroid rete glands of eye were further hallmarks of a disease with systemic vascular involvement. The brain was not spared vascular damage, and the resulting multifocal encephalomalacia probably accounts for the spiral swimming behaviour in some affected fish. Other lesions included accentuated hepatic lobulation and gastric gland necrosis. Nuclear chromatin margination and karyolysis in hepatocytes, renal tubular epithelium and gastric and intestinal epithelium suggest specific targeting of cells. Basophilic cytoplasmic inclusions were present in spleen, kidney, liver, heart and choroid rete, but they were not prominent. Using transmission electron microscopy, two morphological forms of virions were observed: single- and double-enveloped hexagonal virions. Based on size and morphology, these virions resemble iridovirus or herpesvirus. The cause of SDS is unknown, but the pathological changes, especially the vasculitis, suggest an infectious aetiology, possibly viral.

Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

Remission of histiocytic ulcerative colitis in Boxer dogs correlates with eradication of invasive intramucosal Escherichia coli.

BACKGROUND: Historically, histiocytic ulcerative (HUC) (or granulomatous) colitis of Boxer dogs was considered an idiopathic immune-mediated disease with a poor prognosis. Recent reports of dramatic responses to enrofloxacin and the discovery of invasive Escherichia coli within the colonic mucosa of affected Boxer dogs support an infectious etiology. HYPOTHESIS: Invasive E. coli is associated with colonic inflammation in Boxer dogs with HUC, and eradication of intramucosal E. coli correlates with clinical and histologic remission. ANIMALS: Seven Boxer dogs with HUC. METHODS: Prospective case series. Colonic biopsies were obtained at initial evaluation in 7 dogs, and in 5 dogs after treatment with enrofloxacin. Biopsies were evaluated by standardized histopathology, and fluorescence in situ hybridization (FISH) with probes to eubacteria and E. coli. RESULTS: Intramucosal E. coli was present in colonic biopsies of 7/7 Boxers with HUC. Clinical response was noted in all dogs within 2 weeks of enrofloxacin (7 + or - 3.06 mg/kg q24 h, for 9.5 + or - 3.98 weeks) and was sustained in 6 dogs (median disease-free interval to date of 47 months, range 17-62). FISH was negative for E. coli in 4/5 dogs after enrofloxacin. E. coli resistant to enrofloxacin were present in the FISH-positive dog that relapsed. CONCLUSIONS AND CLINICAL RELEVANCE: The correlation between clinical remission and the eradication of mucosally invasive E. coli during treatment with enrofloxacin supports the causal involvement of E. coli in the development of HUC in susceptible Boxer dogs. A poor response to enrofloxacin treatment might be due to colonization with enrofloxacin-resistant E. coli.

A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers.

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778).

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.

Spore ornamentation of Haplosporidium hinei n. sp. (Haplosporidia) in pearl oysters Pinctada maxima (Jameson, 1901).

An infection of pearl oysters, Pinctada maxima, attributed to a Haplosporidium sp. by Hine and Thorne (1998) has been detected on 3 occasions and is considered to represent a serious concern to the pearling industry in Australia. The spore ornamentation of the parasite was determined by scanning electron microscopy and transmission electron microscopy. Spores of the parasite were pleomorphic, or elongated 3.5-4 microm x 2.5-3.0 microm in size. Two filaments were wound around the spore and originated from 2 'knob-like' posterior thickenings. Both filaments passed up one side of the spore together until just below the operculum whereupon each split and passed obliquely under the lip of the opercula lid. Each filament wrapped around the spore 4 times. The posterior thickenings seem to appear late in the development of the spore and were composed of spore wall material. A second set of branching tubular filaments composed of a different material was observed on the spore body although not on mature spores possessing a 'knob-like' posterior thickening. The ornamentation on the spores of the pearl oyster parasite was unique amongst described haplosporidian species where spore ornamentation is known. The parasite is named in this manuscript as Haplosporidium hinei n. sp.

Acute necrotising pulmonary vasculitis and pulmonary hypertension in a juvenile dog.

A five-month-old female Jack Russell terrier was presented for investigation of acute lethargy, anorexia, coughing, respiratory distress and weakness. Examination findings included cyanosis, a grade 3 of 6 systolic heart murmur and prolonged capillary refill time. Radiography and echocardiography revealed severe pulmonary hypertension, cor pulmonale and right-sided heart failure. Indirect measurement of the systolic pulmonary artery pressure estimated pressures over 100 mmHg. Despite treatment the patient died. Post-mortem examination did not identify a congenital cardiovascular anomaly. Histopathology confirmed acute necrotising pulmonary arteritis and immunohistochemistry failed to identify any immune complex or complement deposition.

Response to ciclosporin treatment in Ethiopian and Nepali patients with severe leprosy Type 1 reactions.

Leprosy type 1 reactions (T1R) are immune-mediated events with inflammation of peripheral nerves and skin. We report the clinical outcomes of a closely monitored open prospective trial in which eight Nepali and 33 Ethiopian patients with T1Rs were treated with an Indian generic formulation of ciclosporin (Cn; 5-7.5 mg/kg/day) for 12 weeks and followed up for 24 weeks after starting treatment. Outcomes were measured using a clinical severity score. Among the Nepalis, 75-100% improved in all acute clinical parameters; 67-100% patients maintained improvement, except for those with acute sensory nerve impairment among whom 67% relapsed after stopping treatment. The skin lesions of all Ethiopians on 5 mg/kg/day of Cn improved and 50-60% had peripheral nerve function improvement. Most Ethiopians needed a higher dose of Cn to improve nerve impairment and neuritis, and 50-78% of them developed worse clinical severity scores when Cn was stopped. Four Ethiopians and two Nepalis developed elevated serum creatinine levels on 7.5 mg/kg/day Cn, and three (9%) Ethiopians developed treatable hypertension. This suggests that Cn monotherapy is an effective treatment for severe T1R with few adverse effects. A dose of 5 mg/kg/day seems efficacious in Nepalis, but a higher dose may be required in Ethiopian patients.

Corticosteroids for treating nerve damage in leprosy.

BACKGROUND: Leprosy causes nerve damage which can result in nerve function impairment and disability. Corticosteroids are commonly used for treating nerve damage, although the long-term effect is uncertain. OBJECTIVES: To assess the effects of corticosteroids on nerve damage in leprosy. SEARCH STRATEGY: We searched the Cochrane Neuromuscular Disease Group Register, the Cochrane Central Register of Controlled Trials (Issue 4), MEDLINE (from 1966), EMBASE (from 1980), CINAHL (from 1980), LILACS (from 1982) in January 2006. We checked reference lists of the studies identified, the Current Controlled Trials Register (www.controlled-trials.com), conference proceedings and contacted trial authors. SELECTION CRITERIA: Randomised and quasi-randomised controlled trials of corticosteroids for nerve damage in leprosy. DATA COLLECTION AND ANALYSIS: The primary outcome was improvement in sensory and motor nerve function after one year. Secondary outcomes were improvement in nerve function after two years, change in nerve pain and tenderness, and adverse events. Two authors independently extracted data and assessed trial quality. We contacted trial authors for additional information. We collected adverse effects and cost effectiveness information from the trials and non-randomised studies. MAIN RESULTS: We included three randomised controlled trials involving 513 people. Two trials compared prednisolone with placebo. One trial treated mild sensory impairment of less than six months duration and the other trial treated nerve function impairment of 6 to 24 months duration. Both trials examined an effect twelve months from the start of treatment. There was no significant difference in nerve function improvement between people treated with prednisolone or with placebo. The third trial compared three corticosteroid regimens for severe type 1 reactions. This trial did not report the prespecified outcomes. However, after 12 months, a significantly higher proportion of individuals on a 3-month course of prednisolone required extra corticosteroids compared to the groups with a high-dose and low-dose regimen of five months duration. Diabetes and peptic or infected ulcer were sometimes reported as serious adverse events in the placebo-controlled trials, but not significantly more often in the corticosteroid than placebo groups. AUTHORS' CONCLUSIONS: Corticosteroids are used for treating acute nerve damage in leprosy, but evidence from randomised controlled trials does not show a significant long-term effect. Randomised controlled trials are needed to establish their effectiveness, the optimal regimens and to examine new therapies.

Klossiella quimrensis (Apicomplexa: Klossiellidae) causes renal coccidiosis in western barred bandicoots Perameles bougainville (Marsupialia: Peramelidae) in Western Australia.

Previous studies have described a range of Klossiella species parasitic in marsupial hosts. Klossiella quimrensis is the etiologic agent of renal coccidiosis in the peramelid marsupial hosts Isoodon obesulus and Perameles gunnii in Eastern Australia, but there is no previous report of klossiellosis in Western Australian peramelids. This study describes klossiellosis diagnosed by histology of renal tissue sections collected during necropsy of 20 Perameles bougainville between 2000 and 2005. Sporonts, sporoblasts, and macrogametes were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. The prevalence of renal coccidiosis in P. bougainville diagnosed by renal histology is estimated at 30%. Only a single unsporulated sporocyst was detected by examination of cystocentesis-collected urine, indicating that microscopic evaluation of urine samples is an insensitive diagnostic test for detection of K. quimrensis in P. bougainville. This infection in P. bougainville is indirectly associated with mild multifocal interstitial lymphohistiocytic nephritis and is likely to be only minimally pathogenic in otherwise healthy individuals. Our study also extends the host and geographic range of K. quimrensis to include P. bougainville and Western Australia.

Gall bladder torsion and rupture in a dog.

A 6-year-old desexed female German Shepherd dog was referred to the Murdoch University Veterinary Hospital for assessment and management of acute onset vomiting, diarrhoea, polydipsia and lethargy of 2 days duration. Surgical, microbiological and histological findings were consistent with necrotising cholecystitis secondary to gall bladder torsion, resulting in gall bladder rupture and secondary non-septic bile peritonitis. A chronic peritoneopleural perforation resulting from an abdominal cavity foreign body and congenital peritoneopericardial hernia were also present. The dog made a full recovery following cholecystectomy, foreign body removal, repair of the peritoneopleural perforation and peritoneopericardial herniorrhaphy. This is the first recorded case of gall bladder torsion in the dog.

Cellobiose dehydrogenase--a flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi.

Cellobiose dehydrogenase, the only currently known extracellular flavocytochrome, is formed not only by a number of wood-degrading but also by various phytopathogenic fungi. This inducible enzyme participates in early events of lignocellulose degradation, as investigated in several basidiomycete fungi at the transcriptional and translational level. However, its role in the ascomycete fungi is not yet obvious. Comprehensive sequence analysis of CDH-encoding genes and their translational products reveals significant sequence similarities along the entire sequences and also a common domain architecture. All known cellobiose dehydrogenases fall into two related subgroups. Class-I members are represented by sequences from basidiomycetes whereas class-II comprises longer, more complex sequences from ascomycete fungi. Cellobiose dehydrogenase is typically a monomeric protein consisting of two domains joined by a protease-sensitive linker region. Each larger (dehydrogenase) domain is flavin-associated while the smaller (cytochrome) domains are haem-binding. The latter shorter domains are unique sequence motifs for all currently known flavocytochromes. Each cytochrome domain of CDH can bind a single haem b as prosthetic group. The larger dehydrogenase domain belongs to the glucose-methanol-choline (GMC) oxidoreductase superfamily - a widespread flavoprotein evolutionary line. The larger domains can be further divided into a flavin-binding subdomain and a substrate-binding subdomain. In addition, the class-II (but not class-I) proteins can possess a short cellulose-binding module of type 1 at their C-termini. All the cellobiose dehydrogenases oxidise cellobiose, cellodextrins, and lactose to the corresponding lactones using a wide spectrum of different electron acceptors. Their flexible specificity serves as a base for the development of possible biotechnological applications.

Promoting early detection in leprosy--a literature review to identify proven and potential interventions addressing patient-related delay.

OBJECTIVES: The objective of the literature review was to identify proven and potential interventions to promote early diagnosis and start of treatment in leprosy, specifically, forms of intervention addressing needs at the local or primary level. DESIGN: Using a structured search procedure, we identified recent leprosy-related publications describing proven interventions. To identify potential interventions the search was extended to publications assessing knowledge and attitudes towards leprosy and extended again to identify publications relating to patient-related delay in the context of other infectious diseases. RESULTS: The review identified just 19 publications reporting leprosy-related interventions that included a form of evaluation of which only 10 directly addressed patient-related delay. These included health education interventions focussed on people directly affected by leprosy, their family members and other key individuals or groups within the local community. We identified no reports of interventions focussed specifically on the needs of women. CONCLUSIONS: Our conclusion is that the evidence base available to inform the choice of small-scale interventions to promote early detection at the primary level is extremely limited. There is an urgent need to develop and extend the range of proven interventions, specifically those that address the needs of women, those that explore and develop the health promotion potential of people previously affected by,leprosy and those that exploit the potential of individuals with leadership roles within the community. This will require careful attention to planning, implementation, evaluation and reporting of interventions.

A new eimeria species parasitic in western barred bandicoots, Perameles bougainville (Marsupialia: Peramelidae), in western Australia.

Feces from western barred bandicoots, Perameles bougainville, examined during routine monitoring of captive breeding colonies and wild populations were frequently found to contain oocysts. Fecal oocysts from 1 individual housed at Kanyana Wildlife Rehabilitation Centre were allowed to sporulate in 2% potassium dichromate (K2Cr2O7) at room temperature. Sporulated oocysts are subspheroidal 18.8 X 17.9 (16.9-21.0 x 16.0-19.9) microm, with length/width (L/W) ratio of 1.05 (1.00-1.15), lack a micropyle and oocyst residuum, but they usually have a polar granule within a smooth trilaminate oocyst wall 1.0 (0.7-1.3) microm thick. Sporocysts are ovoid, 9.1 x 7.0 (8.1-10.8 x 6.1-8.6) microm, with L/W ratio of 1.32 (1.04-1.51), have a Stieda body, sporocyst residuum, and 2 comma-shaped sporozoites, each containing 2 spheroidal refractile bodies. Sporulation takes 2-5 days at room temperature. This is the first formal description of an Eimeria species parasitic in the order Peramelemorphia.

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain.

1. Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa3. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species. 2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome aa3 (a3 + a33+) and in the half-reduced species (a2 + a33+). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the aa3 spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of a32+ minus a33+, free of the difficulties with cyanide (which induces marked high leads to low spin spectral shifts in cytochrome a33+) and azide (which induces peak shifts of cytochrome a2+ towards the blue in both alpha- and Soret regions). 3. The rate of formate dissociation from cytochrome a2+ a33+ -HCOOH is faster than its rate of dissociation from a3+ a33+ -HCOOH, especially in the presence of cytochrome c. The Ki for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 degrees C. 4. Succinate-cytochrome c reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2. 5. Formate inhibition of ascorbate plus N, N, N', N'-tetramethyl-p-phenylenediamine oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in 'on' or 'off' inhibition rates were observed when intact mitochondria were compared with submitochondrial particles. 6. NADH-cytochrome c reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome aa3 level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.

The effect of sulphide on cytochrome aa3. Isosteric and allosteric shifts of the reduced alpha-peak.

1. Sulphide, like cyanide, is a slow-binding inhibitor of cytochrome aa3 with a high affinity (KD smaller than 0.1 MUM). 2. Unlike cyanide binding, the binding of sulphide is apparently independent of the redox state of components of the oxidase other than cytochrome a3 and shows no anomalous kinetics during complex formation. 3. Sulphide binding to cytochrome a3-3+ is accompanied by a blue-shift in the alpha-peak of the reduced enzyme (a-2+ a3-3+ H2S), similar to but smaller than that induced by azide. 4. The reduced sulphide-inhibited system shows a much higher Soret peak at 445 nm than the corresponding cyanide and azide complexes, suggesting that partial electron transfer from sulphide to haem may occur in the complex. No evidence was obtained for the formation of any sulfhaem derivatives of cytochrome a3. 5. The influence of energization on the spectrum of mitochondrial cytochrome oxidase, and the effects of calcium on the alpha-peak of isolated cytochrome aa3 (Wikström, M.K.F. (1974) Ann. N.Y. Acad. Sci. 227, 146-158) are distinct from the action of the cytochrome a3 ligands. 6. A classification of peak shifts in the alpha-region in terms of isosteric and allosteric ligands is proposed.

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles.

1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.