Functional Activity of Enantiomeric Oximes and Diastereomeric Amines and Cyano Substituents at C9 in 3-Hydroxy-N-phenethyl-5-phenylmorphans.

The synthesis of stereochemically pure oximes, amines, saturated and unsaturated cyanomethyl compounds, and methylaminomethyl compounds at the C9 position in 3-hydroxy-N-phenethyl-5-phenylmorphans provided µ-opioid receptor (MOR) agonists with varied efficacy and potency. One of the most interesting compounds, (2-((1S,5R,9R)-5-(3-hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-9-yl)acetonitrile), was found to be a potent partial MOR agonist (EC50 = 2.5 nM, %Emax = 89.6%), as determined in the forskolin-induced cAMP accumulation assay. Others ranged in potency and efficacy at the MOR, from nanomolar potency with a C9 cyanomethyl compound (EC50 = 0.85 nM) to its totally inactive diastereomer, and three compounds exhibited weak MOR antagonist activity (the primary amine 3, the secondary amine 8, and the cyanomethyl compound 41). Many of the compounds were fully efficacious; their efficacy and potency were affected by both the stereochemistry of the molecule and the specific C9 substituent. Most of the MOR agonists were selective in their receptor interactions, and only a few had δ-opioid receptor (DOR) or κ-opioid receptor (KOR) agonist activity. Only one compound, a C9-methylaminomethyl-substituted phenylmorphan, was moderately potent and fully efficacious as a KOR agonist (KOR EC50 = 18 nM (% Emax = 103%)).

Investigating a psychological model of mental conditions and coping during the COVID-19 pandemic driven by participatory methods.

BACKGROUND: There is evidence of increased mental health problems during the early stages of the COVID-19 pandemic. We aimed to identify the factors that put certain groups of people at greater risk of mental health problems. METHODS: We took a participatory approach, involving people with lived experience of mental health problems and/or carers, to generate a set of risk factors and potential moderators of the effects of COVID on mental health. An online cross-sectional survey was completed by 1464 United Kingdom residents between 24th April and 27th June 2020. The survey had questions on whether respondents were existing mental health service users and or carers, level of depression (PHQ9) and anxiety (GAD7), demographics, threat and coping appraisals, perceived resilience (BRS), and specific coping behaviours (validated as part of this study). The relationship between responses and coping strategies was measured using tetrachoric correlations. Structural equation modelling was used to test the model. RESULTS: A model significantly fit our data (rel χ2 = 2.05, RMSEA = 0.029 95%, CI (0.016, 0.042), CFI = 0.99, TLI = 0.98, SRMR = 0.014). Age and coping appraisal predicted anxiety and depression. Whereas, threat appraisal and ethnicity only predicted anxiety, and resilience only predicted depression. Additionally, specific coping behaviours predicted anxiety and depression, with overlap on distraction. CONCLUSIONS: Some, but not all, risk factors significantly predict anxiety and depression. While there is a relationship between anxiety and depression, different factors may put people at greater risk of one or the other during the pandemic.

Neuropathological and biochemical investigation of Hereditary Ferritinopathy cases with ferritin light chain mutation: Prominent protein aggregation in the absence of major mitochondrial or oxidative stress.

AIMS: Neuroferritinopathy (NF) or hereditary ferritinopathy (HF) is an autosomal dominant movement disorder due to mutation in the light chain of the iron storage protein ferritin (FTL). HF is the only late-onset neurodegeneration with brain iron accumulation disorder and study of HF offers a unique opportunity to understand the role of iron in more common neurodegenerative syndromes. METHODS: We carried out pathological and biochemical studies of six individuals with the same pathogenic FTL mutation. RESULTS: CNS pathological changes were most prominent in the basal ganglia and cerebellar dentate, echoing the normal pattern of brain iron accumulation. Accumulation of ferritin and iron was conspicuous in cells with a phenotype suggesting oligodendrocytes, with accompanying neuronal pathology and neuronal loss. Neurons still survived, however, despite extensive adjacent glial iron deposition, suggesting neuronal loss is a downstream event. Typical age-related neurodegenerative pathology was not normally present. Uniquely, the extensive aggregates of ubiquitinated ferritin identified indicate that abnormal FTL can aggregate, reflecting the intrinsic ability of FTL to self-assemble. Ferritin aggregates were seen in neuronal and glial nuclei showing parallels with Huntington's disease. There was neither evidence of oxidative stress activation nor any significant mitochondrial pathology in the affected basal ganglia. CONCLUSIONS: HF shows hallmarks of a protein aggregation disorder, in addition to iron accumulation. Degeneration in HF is not accompanied by age-related neurodegenerative pathology and the lack of evidence of oxidative stress and mitochondrial damage suggests that these are not key mediators of neurodegeneration in HF, casting light on other neurodegenerative diseases characterized by iron deposition.

Exploring doped or vacancy-modified graphene-based electrodes for applications in asymmetric supercapacitors.

We report here density functional theory calculations and molecular dynamics atomistic simulations to determine the total capacitance of graphene-modified supercapacitors. The contributions of quantum capacitance to the total capacitance for boron-, sulfur-, and fluorine-doped graphene electrodes, as well as vacancy-modified electrodes, were examined. All the doped electrodes presented significant variations in quantum capacitance (ranging from 0 to ∼200 µF cm-2) due to changes in the electronic structure of pristine graphene. The graphene-modified supercapacitors show any appreciable effect on double-layer capacitance being virtually the same for all the devices investigated. The total differential capacitance was found to be limited by the quantum capacitance, and for all the systems, it is lower than the quantum capacitance over the entire voltage window. We found that the total capacitance can be optimized by considering an adequate modification to each electrode in the supercapacitor. In addition, we found that an asymmetric supercapacitor assembled with different doped electrodes, i.e. an F doped negative electrode and an N doped positive electrode, is the best choice for a supercapacitor since this combination results in better capacitance over the entire potential window.

Quantifying sperm egg interaction to assess the breeding efficiency through artificial insemination in guinea fowls.

1. Guinea fowl hens were inseminated weekly once with two doses of spermatozoa (75 million and 100 million) in two different diluents, Beltsville poultry semen extender (BPSE), and Instruments for Veterinary Medicine (IMV), each with and without pre-insemination vaginal douching. Per cent fertility, hatchability, dead germ, dead in shells along with data on sperm egg interaction and vaginal microbial counts were recorded. 2. Artificial insemination had significantly improved the per cent fertility and hatchability compared to natural mating. Dose dependent improvement in fertility was noticed with both diluents. 3. There was a beneficial effect of vaginal douching, which was more pronounced at lower insemination doses. 4. For optimum fertility and hatchability in guinea fowl, insemination of 75 million spermatozoa diluted in BPSE once in 4 d and 100 million spermatozoa diluted in BPSE or IMV once in 5 d coupled with vaginal douching is recommended.

Acridinium-Based Photocatalysts: A Sustainable Option in Photoredox Catalysis.

The emergence of visible light photoredox catalysis has enabled the productive use of lower energy radiation, leading to highly selective reaction platforms. Polypyridyl complexes of iridium and ruthenium have served as popular photocatalysts in recent years due to their long excited state lifetimes and useful redox windows, leading to the development of diverse photoredox-catalyzed transformations. The low abundances of Ir and Ru in the earth's crust and, hence, cost make these catalysts nonsustainable and have limited their application in industrial-scale manufacturing. Herein, we report a series of novel acridinium salts as alternatives to iridium photoredox catalysts and show their comparability to the ubiquitous [Ir(dF-CF3-ppy)2(dtbpy)](PF6).

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis.

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.

Safety, efficacy, and tolerability of memantine for cognitive and adaptive outcome measures in adolescents and young adults with Down syndrome: a randomised, double-blind, placebo-controlled phase 2 trial.

BACKGROUND: Down syndrome is a chromosomal disorder with considerable neurodevelopmental impact and neurodegenerative morbidity. In a pilot trial in young adults with Down syndrome, memantine (a drug approved for Alzheimer's disease) showed a significant effect on a secondary measure of episodic memory. We aimed to test whether memantine would improve episodic memory in adolescents and young adults with Down syndrome. METHODS: We did a randomised, double-blind, placebo-controlled phase 2 trial with a parallel design, stratified by age and sex. Participants (aged 15-32 years) with either trisomy 21 or complete unbalanced translocation of chromosome 21 and in general good health were recruited from the community at one site in Brazil and another in the USA. Participants were randomly assigned (1:1) to receive either memantine (20 mg/day orally) or placebo for 16 weeks. Computer-generated randomisation tables for both sites (allocating a placebo or drug label to each member of a unique pair of participants) were centrally produced by an independent statistician and were shared only with investigational pharmacists at participating sites until unblinding of the study. Participants and investigators were masked to treatment assignments. Neuropsychological assessments were done at baseline (T1) and week 16 (T2). The primary outcome measure was change from baseline to week 16 in the California Verbal Learning Test-second edition short-form (CVLT-II-sf) total free recall score, assessed in the per-protocol population (ie, participants who completed 16 weeks of treatment and had neuropsychological assessments at T1 and T2). Linear mixed effect models were fit to data from the per-protocol population. Safety and tolerability were monitored and analysed in all participants who started treatment. Steady-state concentrations in plasma of memantine were measured at the end of the trial. This study is registered at ClinicalTrials.gov, number NCT02304302. FINDINGS: From May 13, 2015, to July 22, 2020, 185 participants with Down syndrome were assessed for eligibility and 160 (86%) were randomly assigned either memantine (n=81) or placebo (n=79). All participants received their allocated treatment. Linear mixed effect models were fit to data from 149 (81%) participants, 73 in the memantine group and 76 in the placebo group, after 11 people (eight in the memantine group and three in the placebo group) discontinued due to COVID-19 restrictions, illness of their caregiver, adverse events, or low compliance. The primary outcome measure did not differ between groups (CVLT-II-sf total free recall score, change from baseline 0·34 points [95% CI -0·98 to 1·67], p=0·61). Memantine was well tolerated, with infrequent mild-to-moderate adverse events, the most common being viral upper respiratory infection (nine [11%] participants in the memantine group and 12 [15%] in the placebo group) and transient dizziness (eight [10%] in the memantine group and six [8%] in the placebo group). No serious adverse events were observed. Amounts of memantine in plasma were substantially lower than those considered therapeutic for Alzheimer's disease. INTERPRETATION: Memantine was well tolerated, but cognition-enhancing effects were not recorded with a 20 mg/day dose in adolescents and young adults with Down syndrome. Exploratory analyses point to a need for future work. FUNDING: Alana Foundation. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Multi-system neurological disease is common in patients with OPA1 mutations.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

OPA1 increases the risk of normal but not high tension glaucoma.

BACKGROUND: Primary open angle glaucoma is a progressive optic neuropathy characterised by the selective loss of retinal ganglion cells, pathological optic disc cupping and visual field defects. The OPA1 gene encodes an inner mitochondrial membrane protein crucial for normal mitochondrial function, and pathogenic mutations cause autosomal dominant optic atrophy by specifically targeting retinal ganglion cells. This raises the distinct possibility that more subtle genetic variations in OPA1 could alter the risk of developing glaucoma. METHODS: 137 patients with primary open angle glaucoma (67 patients with high-tension glaucoma (HTG), 70 patients with normal-tension glaucoma (NTG)) and 75 controls from the North East of England were studied. Three single-nucleotide polymorphisms in intron 8 (IVS8+4c-->t and IVS8+32t-->c) and exon 4 (c.473A-->G) of the OPA1 gene were genotyped in the study group. In addition, the entire OPA1 coding region was sequenced in 24 individuals with the CT/TT compound genotype using standard BigDye chemistries. RESULTS: There was no difference in either allele or genotype frequency for the IVS8+32t-->c single-nucleotide polymorphisms between patients and controls, but there was a significant association between the T allele at IVS8+4c-->t and the risk of developing NTG (OR=2.04, 95% CI=1.10 to 3.81, p=0.004), but not HTG. Logistic regression analysis also confirmed a strong association between the CT/TT compound genotype at IVS8+4 and IVS8+32 with NTG (OR=29.75, 95% CI=3.83 to 231.21, p=0.001). CONCLUSIONS: The CT/TT compound genotype at IVS8+4 and IVS8+32 is a strong genetic risk determinant for NTG but not HTG.

Ask me in your own words: paraphrasing for multitask question answering.

Multitask learning has led to significant advances in Natural Language Processing, including the decaNLP benchmark where question answering is used to frame 10 natural language understanding tasks in a single model. In this work we show how models trained to solve decaNLP fail with simple paraphrasing of the question. We contribute a crowd-sourced corpus of paraphrased questions (PQ-decaNLP), annotated with paraphrase phenomena. This enables analysis of how transformations such as swapping the class labels and changing the sentence modality lead to a large performance degradation. Training both MQAN and the newer T5 model using PQ-decaNLP improves their robustness and for some tasks improves the performance on the original questions, demonstrating the benefits of a model which is more robust to paraphrasing. Additionally, we explore how paraphrasing knowledge is transferred between tasks, with the aim of exploiting the multitask property to improve the robustness of the models. We explore the addition of paraphrase detection and paraphrase generation tasks, and find that while both models are able to learn these new tasks, knowledge about paraphrasing does not transfer to other decaNLP tasks.

Inherited mitochondrial optic neuropathies.

Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA) are the two most common inherited optic neuropathies and they result in significant visual morbidity among young adults. Both disorders are the result of mitochondrial dysfunction: LHON from primary mitochondrial DNA (mtDNA) mutations affecting the respiratory chain complexes; and the majority of DOA families have mutations in the OPA1 gene, which codes for an inner mitochondrial membrane protein critical for mtDNA maintenance and oxidative phosphorylation. Additional genetic and environmental factors modulate the penetrance of LHON, and the same is likely to be the case for DOA which has a markedly variable clinical phenotype. The selective vulnerability of retinal ganglion cells (RGCs) is a key pathological feature and understanding the fundamental mechanisms that underlie RGC loss in these disorders is a prerequisite for the development of effective therapeutic strategies which are currently limited.

Novel POLG1 mutations associated with neuromuscular and liver phenotypes in adults and children.

BACKGROUND: The POLG1 gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA replication and repair. Mutations in POLG1 have been linked to a spectrum of clinical phenotypes, and may account for up to 25% of all adult presentations of mitochondrial disease. METHODS AND RESULTS: We present 14 patients, with characteristic features of mitochondrial disease including progressive external ophthalmoplegia (PEO) and Alpers-Huttenlocher syndrome and laboratory findings indicative of mitochondrial dysfunction, including cytochrome c oxidase (COX) deficiency and multiple deletions or depletion of the mitochondrial DNA. Four novel POLG1 missense substitutions (p.R597W, p.L605R, p.G746S, p.A862T), are described, together with the first adult patient with a recently described polymerase domain mutation (p.R1047W). All novel changes were rare in a control population and affected highly conserved amino acids. CONCLUSION: The addition of these substitutions-including the first report of a dinucleotide mutation (c.1814_1815TT>GC)-to the growing list of defects further confirms the importance of POLG1 mutations as the underlying abnormality in a range of neurological presentations.

Factors associated with academic performance of physical therapy students.

UNLABELLED: Understanding the pre-matriculation factors that influence academic success facilitates the recruitment and retention of students who are more likely to graduate on time. OBJECTIVE: To determine the factors associated with the academic performance of students enrolled in the physical therapy diploma programme. METHODS: Records of 250 students enrolled over a twenty-year period at the School of Physical Therapy were reviewed. Data were collected and organized using a data collection sheet. Data were analysed using the Statistical Package for the Social Sciences (SPSS). Relationships between the independent variables: age, gender marital status, work history and entry qualifications, and the dependent variables: academic performance (percentage of subjects passed at the first sitting of examinations and success in the Final Qualifying Examination), withdrawal and delayed graduation were examined using correlation coefficient, t-test, ANOVA and chi-square as appropriate. RESULTS: Results revealed that students gaining the minimum entry qualifications at one sitting of GCE O' Level/CXC examinations (p < 0.01) and who had GCE A' Level passes (p < 0.05) were less likely to withdraw from the programme. Students without prior work experience performed better during the course of study (p < 0.05) but the older and married students were more likely to withdraw (p < 0.01). Older students performed better on the Final Qualifying Practical and Theory Examinations taken at the end of the academic programme (p < 0.05) but had a lower percentage of subjects passed at the first sitting of course examinations (p < 0.05). CONCLUSION: Students with better academic preparation demonstrated better academic performance. These findings were consistent with prior studies in the field.

On the Design of Broad-Based Neuropsychological Test Batteries to Assess the Cognitive Abilities of Individuals with Down Syndrome in the Context of Clinical Trials.

Down syndrome (DS) is the most common genetically-defined cause of intellectual disability. Neurodevelopmental deficits displayed by individuals with DS are generally global, however, disproportionate deficits in cognitive processes that depend heavily on the hippocampus and prefrontal cortex are also well documented. Additionally, DS is associated with relative strengths in visual processing and visuospatial short-term memory, and weaknesses in the verbal domain. Although reports of pharmacological rescuing of learning and memory deficits in mouse models of DS abound in the literature, proving the principle that cognitive ability of persons with DS can be boosted through pharmacological means is still an elusive goal. The design of customized batteries of neuropsychological efficacy outcome measures is essential for the successful implementation of clinical trials of potential cognitive enhancing strategies. Here, we review the neurocognitive phenotype of individuals with DS and major broad-based test batteries designed to quantify specific cognitive domains in these individuals, including the one used in a pilot trial of the drug memantine. The main goal is to illustrate the essential considerations in planning trials to enhance cognitive functions in individuals with DS, which should also have implications for the design of similar studies in individuals with other forms of intellectual disability.

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

Acquisition of the lateral inconsistency in involuntary behaviour of upper limbs in 12-year-old children during walking at moderate speed.

The aim of this work was to investigate possible lateralisation in the behaviour of periodic motion of the human upper limb, during normal walking at a comfortable speed of locomotion. Ten healthy pre-adolescent, strongly right-handed, 12-year-old males participated in the experiment. Participants were walking on a treadmill with a standardised velocity of 1.1m/s (comfortable speed for all of them). A video analysis system with Silicon software was used to synchronically measure various angles of arms and forearms. The initial, final and interim angular positions of both arms and forearms in 10 cycles of each participant were compared in terms of variations (cycle to cycle) between both upper extremities at corresponding phases of each cycle for distal and proximal segments, respectively. We compared the coefficients of variation in relation to the spatial and temporal data of both limbs and their angular velocities. In addition we investigated the level of cycle-to-cycle regularity (constancy) of behaviour in relation to various positions, periods and velocities of movement of upper extremities (specifically arms and forearms) using the Eta non-linear method of correlation. All participants exhibited a lower level of regularity for the distal segments. The spatial and temporal variations in the dominant limb were also greater than the non-dominant limb for all participants. This may be due to a larger contribution from the right-sided muscles that are considered to be the main contributing factor to the motion of the dominant upper limb during walking, rather than simply gravity force acting alone. A possible practical application of this information may be useful in the objective clinical identification of the level of dominance of the upper extremity (arm plus forearm), in addition to 'traditional' handedness.

Exome sequencing in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is the second most common form of degenerative dementia. Siblings of affected individuals are at greater risk of developing DLB, but little is known about the underlying genetic basis of the disease. We set out to determine whether mutations in known highly penetrant neurodegenerative disease genes are found in patients with DLB. Whole-exome sequencing was performed on 91 neuropathologically confirmed cases of DLB, supplemented by independent APOE genotyping. Genetic variants were classified using established criteria, and additional neuropathological examination was performed for putative mutation carriers. Likely pathogenic variants previously described as causing monogenic forms of neurodegenerative disease were found in 4.4% of patients with DLB. The APOE ɛ4 allele increased the risk of disease (P=0.0001), conferred a shorter disease duration (P=0.043) and earlier age of death (P=0.0015). In conclusion, although known pathogenic mutations in neurodegenerative disease genes are uncommon in DLB, known genetic risk factors are present in >60% of cases. APOE ɛ4 not only modifies disease risk, but also modulates the rate of disease progression. The reduced penetrance of reported pathogenic alleles explains the lack of a family history in most patients, and the presence of variants previously described as causing frontotemporal dementia suggests a mechanistic overlap between DLB and other neurodegenerative diseases.

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]

The chloroplast CF0I subunit can replace the b-subunit of the F0F1-ATPase in a mutant strain of Escherichia coli K12.

The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E. coli F0F1-ATPase. However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function. This proposition was investigated directly. A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein. An E. coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene. The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control. Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E. coli b-subunit in the E. coli F0F1-ATPase.