Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide.

Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.

The ornithine transcarbamylase leader peptide directs mitochondrial import through both its midportion structure and net positive charge.

The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is targeted to mitochondria by its NH2-terminal leader peptide. We previously established through mutational analysis that the midportion of the OTC leader peptide is functionally required. In this article, we report that study of additional OTC precursors, altered in either a site-directed or random manner, reveals that (a) the midportion, but not the NH2-terminal half, is sufficient by itself to direct import, (b) the functional structure in the midportion is unlikely to be an amphiphilic alpha-helix, (c) the four arginines in the leader peptide contribute collectively to import function by conferring net positive charge, and (d) surprisingly, proteolytic processing of the leader peptide does not require the presence of a specific primary structure at the site of cleavage, in order to produce the mature OTC subunit.

Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor.

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.

Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Antisense down regulation of connexin31.1 reduces apoptosis and increases thickness of human and animal corneal epithelia.

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells.

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.

Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.

A multidisciplinary approach was taken to investigate the intracellular locations of the 26-kDa integral membrane protein encoded by the bcl-2 gene. Subcellular fractionation analysis of a t(14;18)-containing lymphoma cell line revealed the presence of Bcl-2 protein in nuclear, heavy-membrane, and light-membrane fractions but not in cytosol. Sedimentation of heavy-membrane fractions in Nycodenz and Percoll continuous gradients demonstrated comigration of p26-Bcl-2 with mitochondrial but not other organelle-associated proteins. Fractionation of light-membrane fractions using discontinuous sucrose-gradients revealed association of Bcl-2 protein primarily with lighter-density microsomes (smooth endoplasmic reticulum) as opposed to heavy-density microsomes (rough endoplasmic reticulum). Immune microscopy studies using laser-scanning microscopy, pre- and postembedding electron microscopic methods, and six different anti-Bcl-2 antibodies demonstrated Bcl-2 immunoreactivity in the nuclear envelope and outer mitochondrial membrane in a patchy distribution. Furthermore, anti-Bcl-2 antibody immunoreactivity generally appeared to directly overlie the nuclear envelope in high magnification electron microscopic studies, reminiscent of nuclear pore complexes. Addition of in vitro translated p26-Bcl-2 to isolated translocation-competent mitochondria revealed transmembrane domain-dependent association of Bcl-2 protein with mitochondria but provided no evidence for import into a protease-resistant compartment, consistent with immunomicroscopic localization to the outer mitochondrial membrane. Taken together, the findings demonstrate that p26-Bcl-2 resides primarily in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane in a nonuniform distribution suggestive of participation in protein complexes perhaps involved in some aspect of transport.

Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy.

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

Putting a lid on protein folding: structure and function of the co-chaperonin, GroES.

The co-chaperonin GroES is an essential partner in protein folding mediated by the chaperonin, GroEL. Two recent crystal structures of GroES provide a structural basis to understand how GroES forms the lid on the folding-active cis ternary complex, and how the GroEL-GroES complex enhances folding.

Testing systems for assessment of negative symptoms in schizophrenia.

To compare methods of measuring negative symptoms, eight rating scales were employed to retrospectively assess and subtype 187 patients with schizophrenia from the Chestnut Lodge Follow-up Study. These included Andreasen's Schedule for Assessment of Negative Symptoms, Carpenter's Criteria for the Deficit Syndrome, Kay and Opler's Positive and Negative Symptom Scale, the scales developed by Krawiecka et al and Crow's modification of them, the Negative Symptom Scale developed by Lewine et al, Pogue-Geile and Harrow's Negative Symptom Scale, and Abrams and Taylor's Emotional Blunting Scale. The overlap and concordance, temporal stability, and predictive validity of these instruments are described. When rated from detailed medical records, the reliability of all scales was fair to good. Despite their inclusion of different items, there were high positive correlations between the scales when used to rate negative symptoms dimensionally. When used to classify individual patients as having the negative or deficit syndrome, however, concordance among criteria was low. Using the broadest criteria (Pogue-Geile and Harrow), 75 (40%) patients were diagnosed as having negative syndrome; the narrowest criteria (Andreasen and Olsen) yielded 11 (6%) diagnoses of negative syndrome. Narrower definitions tended to be subsets of broader ones. Carpenter's Criteria for the Deficit Syndrome focus on primary enduring negative symptoms and show the greatest temporal stability. Broader criteria, which diagnose the deficit or negative syndrome independent of severity of positive symptoms, had the greatest predictive validity.

The positive-negative distinction in schizophrenia. Review of natural history validators.

A review of the interaction between the positive-negative symptom distinction in schizophrenia and multiple measures of illness natural history reveals some redundant and compelling patterns. Negative or deficit symptoms are often associated with inferior social/instrumental functioning premorbidly, more abnormal voluntary/involuntary movements at illness presentation, and poorer long-term outcome when present beyond the early phase of illness. Negative symptoms are semi-independent of positive symptoms. They are variable early in the illness but accrue in severity, stability, and prognostic weight with time. The nature of the processes that generate negative symptoms and their specificity to schizophrenia remain to be elucidated. Nevertheless, it is clear that negative symptoms are a common and valid component of schizophrenia and deserve recognition as such in our nosology.

Natural history of schizophrenia subtypes. I. Longitudinal study of paranoid, hebephrenic, and undifferentiated schizophrenia.

To explore the validity of different approaches for subtyping schizophrenia, the conditions of 187 schizophrenic patients from the Chestnut Lodge follow-up study were rediagnosed with the use of classic subtype criteria. Independently collected data allowed construction of a longitudinal profile of the natural history of illness for patients who met operational criteria for paranoid (n = 78), hebephrenic (n = 26), and undifferentiated (n = 83) schizophrenia. Paranoid schizophrenia had an older age at onset, often developed rapidly in individuals with good premorbid functioning, tended to be intermittent during the first 5 years of illness, and was most associated with good outcome or recovery. Hebephrenia had an earlier age at onset, often developed insidiously, and was associated with a greater family history of psychopathology, poor premorbid functioning, and, frequently, a continuous illness with a poor long-term prognosis. While also early and insidious in onset, unlike hebephrenia, undifferentiated schizophrenia was poorly distinguished from the patients' premorbid state, associated with an early history of behavioral difficulties, and often resulted in a continuous but stable disability. We discuss implications for nosology. Although distinctive patterns were discernible, the considerable heterogeneity within subtypes calls for continued efforts to develop and explore alternate classification schemes.

Natural history of schizophrenia subtypes. II. Positive and negative symptoms and long-term course.

The natural history and long-term course of schizophrenia divided by pervasiveness of positive and negative symptoms was explored among 187 schizophrenic patients from the Chestnut Lodge follow-up study. Schizophrenia with many negative symptoms was associated with poor premorbid functioning, insidious onset, partial or no remissions during the first several years of illness, and in most cases a progressive course leading to permanent disability. Schizophrenia with few negative symptoms was associated with good premorbid functioning, acute onset, intermittent early course, and a better prognosis. Positive symptoms predicted future hospitalizations but were less powerful and specific as indicators of differential illness history, course, and long-term functional incapacity. As predictors of long-term outcome, negative symptoms were of greater value measured at index admission several years after illness onset than at first hospital admission. Multivariate analyses indicated that two negative symptoms (anhedonia and affective flattening) contribute significantly to outcome variance independent of their association with premorbid functioning or positive symptoms. Patients with the poorest long-term outcome tended to show an increase in negative symptoms during the early years of their illness. Progressive negative symptoms early in the course of schizophrenia may thus reflect or signal a process leading to long-term functional disability.

Risk factors for spontaneous dyskinesia in schizophrenia.

OBJECTIVE: We describe the prevalence, clinical correlates, and prognostic significance of spontaneous dyskinesias among 100 patients with schizophrenia from the Chestnut Lodge Follow-up Study who had never received treatment with neuroleptic agents up to and including the baseline assessment. DESIGN: Extensive case records were screened and descriptions of abnormal movements were recorded verbatim for blind rating. Neuroleptic-naive patients with and without abnormal oral-facial movements were compared across sign and symptom, schizophrenia subtype, and illness natural history variables. RESULTS: Excluding three patients with motor symptoms who had a history of neurologic illness or injury and three who had received prochlorperazine maleate therapy (Compazine), 23% of patient records documented some form of movement disorder; 15% documented oral-facial dyskinesias with sufficient detail so that their presence was considered nearly certain. Compared with patients with schizophrenia without oral-facial movements, patients with oral-facial dyskinesias were more likely to demonstrate a lower IQ score, had more negative symptoms at index admission, and were more symptomatic at follow-up an average of 23 years later. Both the classic hebephrenic schizophrenia subtype and Carpenter's Criteria for the Deficit Syndrome defined high-risk groups for spontaneous oral-facial dyskinesia. CONCLUSIONS: In previous studies, intellectual impairment and negative symptoms have been described as risk factors for neuroleptic-induced tardive dyskinesia. The present data, however, suggest that in many cases oral-facial dyskinesias in patients with intellectual impairment and negative symptoms may actually represent spontaneous movement disorders associated with hebephrenic or deficit forms of schizophrenia.

Training and career activity. The experience of the Yale Advanced Track Program.

The Advanced Track Program of the Yale psychiatric residency is examined by a study of the career patterns of 132 graduates of the program over a 9-year period (1973 to 1982). The findings demonstrate that specific track training sequences are associated with subsequent career activities and that track graduates differ significantly from each other along dimensions of professional activities, work settings, treatment modalities, and patient characteristics. These differences are discussed from the vantage of the relationship between training and professional activity.

GroEL-mediated protein folding.

I. Architecture of GroEL and GroES and the reaction pathway A. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding II. Polypeptide binding A. A parallel network of chaperones binding polypeptides in vivo B. Polypeptide binding in vitro 1. Role of hydrophobicity in recognition 2. Homologous proteins with differing recognition-differences in primary structure versus effects on folding pathway 3. Conformations recognized by GroEL a. Refolding studies b. Binding of metastable intermediates c. Conformations while stably bound at GroEL 4. Binding constants and rates of association 5. Conformational changes in the substrate protein associated with binding by GroEL a. Observations b. Kinetic versus thermodynamic action of GroEL in mediating unfolding c. Crossing the energy landscape in the presence of GroEL III. ATP binding and hydrolysis-driving the reaction cycle IV. GroEL-GroES-polypeptide ternary complexes-the folding-active cis complex A. Cis and trans ternary complexes B. Symmetric complexes C. The folding-active intermediate of a chaperonin reaction-cis ternary complex D. The role of the cis space in the folding reaction E. Folding governed by a "timer" mechanism F. Release of nonnative polypeptides during the GroEL-GroES reaction G. Release of both native and nonnative forms under physiologic conditions H. A role for ATP binding, as well as hydrolysis, in the folding cycle V. Concluding remarks.

Essential fatty acids, lipid membrane abnormalities, and the diagnosis and treatment of schizophrenia.

Recent research suggests that deficient uptake or excessive breakdown of membrane phospholipids may be associated with schizophrenia. We review available clinical research on abnormalities in membrane fatty acid composition and metabolism in schizophrenia, and therapeutic trials of fatty acid in this disorder. All potentially relevant English-language articles were identified from the medical and psychiatric literature with the aid of computer searches using key words such as lipids, phospholipids, prostaglandins and schizophrenia. All studies which include human subjects are reviewed. Empirical studies related to membrane hypotheses of schizophrenia focus on: 1) assessment of prostaglandins (PG) and their essential fatty acid (EFA) precursors in the tissues of patients with schizophrenia; 2) evaluation of the niacin flush test as a possible diagnostic marker; 3) evaluation of phospholipase enzyme activity; 4) NMR spectroscopy studies of brain phospholipid metabolism; and 5) therapeutic trials of PG precursors for the treatment of schizophrenia. The most consistent clinical findings include red blood cell fatty acid membrane abnormalities, NMR spectroscopy evidence of increased phospholipid turnover and a therapeutic effect of omega-3 fatty acid supplementation of neuroleptic treatment in some schizophrenia patients. Studies of EFA metabolism have proved fruitful for generating and testing novel etiologic hypotheses and new therapeutic agents for schizophrenia. Greater attention to factors that influence tissue EFA levels such as diet, tobacco and alcohol are required to reconcile inconsistent findings. Treatment studies, although promising, require independent replication.

Sustained remission in drug-free schizophrenic patients.

The inability to determine which schizophrenic patients do not require maintenance medication is a significant gap in current knowledge. This report describes 23 largely chronic DSM-III schizophrenic patients who, after a period of inpatient treatment, sustained good outcome without maintenance antipsychotic medication over an average of 15 years. Retrospective study of these patients revealed that their distinguishing characteristics at admission included better premorbid social and occupational adjustment, higher levels of accrued psychosocial competence and acquired skills, fewer hebephrenic traits, and the preservation of affect (depressed mood). Hence, even within a largely chronic patient sample, classic predictors of good outcome may also be useful in predicting sustained remission without medication.

The prognostic significance of obsessive-compulsive symptoms in schizophrenia.

Obsessive-compulsive symptomatology has been described in schizophrenia for more than 60 years, but its clinical significance has yet to be explored systematically. This report details the clinical characteristics and long-term course of a group of 21 schizophrenic patients with prominent obsessive-compulsive symptoms from the Chestnut Lodge Follow-Up Study. While this group differed on admission only minimally from schizophrenic patients without obsessive-compulsive symptoms, their long-term outcome in the areas of social relations, employment, psychopathology, and global functioning was significantly, and almost uniformly, poorer. Persistent obsessive-compulsive symptoms thus appear to be a powerful predictor of poor prognosis in schizophrenia.

Risk of schizophrenia in character disordered patients.

Inpatients from the Chestnut Lodge follow-up study diagnosed with character disorder were studied to predict future schizophrenic decompensation. Individually, three DSM-III criteria for schizotypal personality disorder predicted schizophrenia at long-term follow-up: magical thinking, suspiciousness or paranoid ideation, and social isolation. Additionally, lower IQ, poorer premorbid quality of work, and transient delusional experiences were predictive. No borderline personality disorder criterion was predictive. This suggests that schizotypal but not borderline personality disorder belongs in the schizophrenic spectrum. Within schizotypal personality disorder, criteria from both familial and clinical traditions appear to be dimensions of vulnerability to psychosis.