Comparison of the respiratory chain of Neurospora crassa wild type and the mi-mutants mi-1 and mi-3.
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Antibodies to Mi, an antigen in calf thymus extract, have been demonstrated by complement fixation inhibition in polymyositis (PM) and dermatomyositis (DM) sera but not in the sera of individuals without myositis. The original Mi reference serum defined 2 precipitating antibodies, using immunodiffusion (ID). Anti-Mi-1 was not active in complement fixation. We have now studied in further detail anti-Mi-2, which appears to be the antibody in Mi serum that fixes complement. Mi-2 antigen was purified by immuno-affinity chromatography. An enzyme-linked immunosorbent assay (ELISA) to measure Mi-2 antibody, using this antigen, was used to test the sera of 139 myositis patients: 52 had DM and 87 had PM. Control sera from 35 normal subjects and 93 patients with other connective tissue diseases were also tested. Only 13 sera were considered definitely positive for anti-Mi-2. All were from patients who had myositis, 11 of whom had DM. Only DM sera had anti-Mi-2 by ID, and all sera with anti-Mi-2 by ID were positive by ELISA. A number of other sera, including many from patients with other connective tissue diseases and 2 from normal subjects (all without precipitating antibodies) had lower elevations which were of uncertain significance. Detection of anti-Mi-2 by ID as well as by ELISA was significantly more frequent in DM than in PM. Anti-Mi-2 appears to be closely linked to DM, and is the first specific serologic marker for this form of myositis.
The rat somatosensory (SI) cortex contains a precise map of the cutaneous periphery, yet its rostromedial edge, which includes part of the fore- and hind paw representation, has been reported to functionally overlap with the electrically excitable primary motor (MI) cortex. Thus, the MI cortex in the rat contains two subregions: (i) rostrally, the "MI-agranular" cortex (i.e., "typical" MI cortex), and (ii) caudally, the "MI-SI-granular" cortex (i.e., the MI-SI overlap). The aim of this study was to assess the degree of overlap in the physiologic properties of single neurons recorded across the MI-SI boundary zone in awake, freely moving rats. Computer techniques were used to characterize both somatosensory receptive fields (cutaneous or "passive joint-manipulation") and discharge correlates of active limb movement in these MI-agranular and MI-SI-granular subregions of the MI. "Active-movement" units were defined as those which discharged strongly in correlation with specific limb movements, but in a manner which could not be predicted by their observable somatosensory properties. Of 92 completely analyzed cells in the MI-SI-granular forepaw region, 86 exhibited specific cutaneous receptive fields on the palmar surface of the forepaw. By contrast with the similarity of these neurons' responses to passive stimulation, they varied markedly in their discharge during active limb movements. For example, many did not respond when their forepaw receptive fields touched the ground during stepping. Furthermore, 31 (of 92) neurons in this region were identified as active-movement, firing in correlation with reaching movements of the forelimb. Seven of these were completely unresponsive to any sensory stimuli, but 24 exhibited an apparent convergence of cutaneous and active-movement properties. Of 86 units recorded in the MI-agranular subregion, 46 responded strongly to passive joint manipulation, but only three responded exclusively to cutaneous stimulation. Twenty-eight (of 86) cells were defined as active-movement, discharging mainly in correlation with forelimb reaching movements. Thus, the active-movement properties of neurons in these two subregions were quite similar, whereas the somatosensory properties were markedly different.
This study reports on the Mi Mark technique as a primary method of endometrial sampling in routine practice. The experience gained during the first year was evaluated with regard to diagnostic accuracy and acceptability. In total, 736 patients were examined using either the Mi Mark technique or dilatation and curettage (D&C). In the pre-menopausal and post-menopausal women, in the study group, 11% and 30%, respectively, of the Mi Mark specimens proved too scanty for evaluation. In women aged over 60 the proportion of specimens that were inadequate for diagnosis was around 60% and the Mi Mark technique was not therefore considered to be suitable for use in this age group. If the latter group is excluded, the Mi Mark procedure could replace D&C in 47% of cases. Where the sampling material obtained was inadequate a secondary D&C was performed, following which an atrophic endometrium was usually found, although endometrial cancers were detected in 2 cases. Thus, an inadequate specimen obtained by means of the Mi Mark helix did not exclude the possibility of endometrial malignancy. When questioned, 8% of the patients stated that they considered the pain associated with the examination to be severe. On the other hand, 93% were prepared to undergo another Mi Mark procedure if necessary. The Mi Mark technique was considered to be adequate in terms of both acceptability and accuracy provided a D&C was performed in cases where the Mi Mark specimen was inadequate for evaluation.
The possibility of an amino acid substitution (cysteine for tryptophan) in a membrane protein of the [mi-1] strain of Neurospora crassa has been investigated in detail by using a double radioactive labeling procedure. Auxotrophic strains of Neurospora having wild-type [+] or [mi-1] cytoplasm have been grown under conditions which result in the specific labeling of protein tryptophan with (3)H and protein cysteine with (35)S. Although the least soluble 1 to 20% of the [mi-1] mitochondrial membrane protein was usually found to have a higher Cys/Trp ratio (ratio of cysteine plus half-cystine to tryptophan) than the corresponding [+] fraction, it has been shown that these differences were due mainly to the presence of differential amounts of a very insoluble, cysteine-rich (Cys-rich) material. The same Cys-rich material was found in variable amounts in both [+] and [mi-1] cultures, but the concentration was usually higher in the [mi-1] cultures. The Cys-rich material is clearly distinct from "structural protein" on the basis of amino acid composition and appears to have no direct relationship to the [mi-1] phenotype. In the absence of the Cys-rich material, no difference between the Cys/Trp ratios of corresponding [+] and [mi-1] membrane proteins could be detected. We conclude, therefore, that the previously postulated amino acid substitution of cysteine for tryptophan in [mi-1] membrane protein is incorrect.
We have determined the DNA sequence of the oxi-3 gene and its 5' flanking region in the extranuclear [mi-3] mutant of Neurospora crassa. The oxi-3 gene encodes subunit 1 of cytochrome c oxidase, a protein known to be altered in the [mi-3] mutant (Bertrand, H., and Werner, S. (1979) Eur. J. Biochem. 98, 9-18). When the sequence from [mi-3] was compared to previously published sequences of the same region of mtDNA from wild-type N. crassa, a total of five differences was found. Four of these differences can be accounted for as either genetic polymorphisms or previous errors in DNA sequence determination. The remaining difference is a G/C to T/A transversion that changes a codon specifying an aspartic acid residue (GAC) to one that would specify tyrosine (TAC) at amino acid 448 of the 555 amino acid mature subunit 1 protein. This alteration was also found in the mtDNA of two separate heterokaryotic strains that had acquired the [mi-3] phenotype after repeated subculturing of heterokaryons forced between an [mi-3] strain and a strain containing a wild-type cytoplasm. The particular aspartic acid residue that would be affected by the mutation observed in [mi-3] is conserved in a diversity of species as either aspartic acid or glutamic acid, suggesting that an acidic residue at this position is important for the correct function of the subunit 1 protein. For these reasons, we consider it likely that the observed missense mutation is responsible for the [mi-3] phenotype.
Mutual information (MI)-based approaches have increasingly been applied to cancer genomics; however, their use for genome-wide prognostic biomarker discovery remains relatively underexplored. The present article summarizes the conceptual workflow of Mutual Information-based Prognostic Omics Gene (MI-POG) based on previously published applications in breast cancer, lower-grade glioma, and other cancer datasets. The framework consists of clinical endpoint discretization, genome-wide MI-based screening, candidate ranking, and downstream validation using conventional survival-analysis approaches. Previous MI-POG applications identified solute carrier family 20 member 1 (SLC20A1) as a prognostic biomarker in hormone receptor-positive breast cancer. Elevated SLC20A1 expression was associated with unfavorable survival outcomes and was independently validated in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort. Methodological analyses demonstrated how survival endpoints can be integrated into an information-theoretic framework through fixed-time outcome discretization, enabling model-independent assessment of molecular-clinical dependencies. Applications across multiple cancer datasets suggested the potential applicability of the framework across biologically distinct tumor types, although further validation will be required to establish its robustness and generalizability. In conclusion, MI-POG can be formalized as an information-theoretic framework for genome-wide identification of prognostic biomarkers by quantifying molecular-clinical dependencies using mutual information. Representative applications from previously published studies suggest that MI-POG may complement conventional survival-analysis approaches and provide a useful strategy for biomarker discovery, although additional benchmarking and prospective validation will be required.
Outcome after myocardial infarction (MI) is commonly conceptualized as exercise tolerance, recurrent infarction and return to work. In the present study a relative measure of return to former leisure and social activities (LSA) was tested in 366 myocardial infarction (MI) patients. Next, the hypothesis that psychosocial factors are more important predictors for return to LSA than medical ones, was tested. It appeared that at baseline (3 weeks after MI) 36%, at post-test (3 months after MI) 51% and at follow-up (1 year after MI) 54%, reached former levels of LSA. It could be shown, both in multiple regression analysis and path analysis, that psychological vulnerability at baseline is the most important predictor of return to former LSA. Females, older patients, single patients and patients with angina pectoris showed an unfavorable psychological profile at baseline. It is suggested that more attention should be given to the improvement of the psychological status soon after MI for the categories of patients mentioned.
In the Miltenberger class V (Mi. V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossing-over between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-GPA antibodies specific for discrete domains of GPA, it was found that the Mi. V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus. As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in lambda EMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-1-A-7) and five exons (B-1-B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi. V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi. V glycoprotein gene was composed of exon B-1 of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossing-over has occurred and it was deduced that the Mi. V glycoprotein is a hybrid molecule composed of amino acid residues 1-58 from GPA fused to amino acid residues 27-72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from GPB suggests that the genetic background of the Mi. V variant is rather complex and may involve a cascade of recombination or gene conversion events.
Impairment of transport and metabolism of retinal pigment epithelium (RPE) has been recognized to play a role in the development of diabetic macular edema. To understand the mechanism(s) of action of high glucose levels in alteration of RPE metabolism, primary cultures of RPE cells were used as an in vitro model of diabetic retinopathy/maculopathy. RPE cells were grown with 5 mM (control) or 40 mM glucose (a monosaccharide that enters the cells), or 40 mM sucrose (a disaccharide that does not enter the cells), and the extent of Na(+)-dependent active transport of an osmolyte ([3H]-myo-inositol, MI, 10 microM) into cells was determined. While 40 mM glucose down-regulated 3H-MI transport, 40 mM sucrose stimulated it, compared to 5 mM glucose feeding. Addition of 1 mM amiloride, an inhibitor of Na+/H+ exchanger, in the incubation media, significantly inhibited MI transport. Cells treated with high sucrose or high glucose were more sensitive toward amiloride inhibition, compared to controls. Inhibition of either pump or leak pathway alone was not sufficient to completely inhibit MI transport, but simultaneous inhibition of both pathways, by amiloride and ouabain (1 mM each), strongly inhibited osmolyte accumulation. The strongest inhibition of uptake occurred when 150 mM NaCl in the incubation media was replaced by 150 mM choline-Cl, and the percent inhibition of uptake, with choline-Cl, was highest with sucrose-fed cells, compared to normal or high glucose-fed cells. Imposition of a pH gradient [pHi (6.1) less than pH0 (8.0)] across the cell membrane, a condition that stimulates Na+/H+ exchange activity, also reduced MI accumulation. Cellular water content, measured by the extent of [3H]-3-O-methyl glucose uptake, in the presence of balanced salt solution (BSS), BSS containing half the ionic strength (hypotonic solution), or BSS containing 20 mM K+, for induction of cell swelling, varied when cells were fed with various sugars. Cells fed with high glucose were less sensitive toward media tonicity compared to normal. These results suggested that in cultured RPE cells, changes in Na+/H+ exchanger activity (intracellularly or extracellularly), through its inhibition by amiloride, its activation via intracellular acidification, or perhaps by chronic feeding with high sucrose or high glucose, affected the Na(+)-dependent active accumulation of MI. A metabolic factor involved in the development of diabetic macular edema is perhaps associated with glucose-induced alterations in Na+ fluxes (e.g., changes in Na+/H+ exchanger activity), which can secondarily influence osmolyte accumulation, impairment of pump-leak balance, and/or intracellular pH.(ABSTRACT TRUNCATED AT 400 WORDS)
A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.
Oxidation of exogenous NADH in mitochondria isolated from wild type and mi-1 mutant of Neurospora crassa decreases rapidly in vitro. In mi-1 mutant mitochondria the inactivation concerns the alternate pathway of oxidation whereas in the wild type it involves an unknown component of the respiratory chain. The activity of the primary NADH dehydrogenase is constant within the time of the experiments (2-4 h). NADH oxidase is not inactivated if oxygen is removed from the incubation medium by nitrogen bubbling. Succinate oxidase does not show any remarkable changes in activity within 2-3 h. In fresh mitochondria of the mi-1 mutant reduced ubiquinone is completely reoxidized by cytochrome oxidase but only 80% reoxidized by the alternate oxidase. In aged mitochondria of the mi-1 mutant in the presence of cyanide, ubiquinone is reduced to the level characteristic for fresh mitochondria in which respiration is completely inhibited by cyanide plus salicylhydroxamic acid. In these mitochondria the reoxidation of the reduced ubiquinone proceeds only via the cytochrome pathway. It is supposed that a labile component(s) of the respiratory chain present in the mi-1 mutant and the wild type mitochondria may, in mi-1 mutant, act as an alternate oxidase.
Psychosocial adjustment following myocardial infarction (MI) has received significant research attention during the past 20 years. This article highlights research addressing the relationship of anxiety, depression, and denial in influencing specific outcomes following MI. Additional research describing patterns of emotional response to MI is also included. Based on reported research, specific nursing interventions, in a care plan format, are suggested to foster positive psychosocial outcomes in the post-MI patient. Even though a significant amount of research has been completed on this subject, definitive conclusions regarding patient management supportive of positive outcomes are not possible. Suggestions for future research focus on this topic area are identified.
Continuous subcutaneous insulin infusion (CSII) and multiple injections (MI) have been shown to have metabolic advantages in highly-selected insulin-dependent diabetics (IDDs), but there have been few comparative studies in self-selected IDDs. With MI, the optimal insulin preparation for overnight insulin delivery has not been defined. We compared conventional 2-3 injection therapy (CT), CSII and MI with human isophane insulin (MI/human isophane) and human ultralente insulin (MI/human ultralente), respectively, at bedtime in self-selected IDDs. Of 275 IDDs who were invited to participate, 52 individuals (18.9%) entered the study. Most indices of glycaemic control showed better values on CSII and also on MI compared to CT, but the differences were small. Fasting blood glucose was higher on MI/human ultralente than on MI/human isophane. Only one subcutaneous abscess and one case of ketoacidosis requiring hospitalization occurred on CSII. Serious hypoglycaemic episodes were non-significantly increased on intensified therapy. Most patients clearly preferred intensified insulin therapy; approximately one half CSII.
The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.
Oxidative phosphorylation has been demonstrated with mitochondria of the mi-1 respiratory mutant of Neurospora crassa. The P/O ratios observed with these mitochondria were approximately 0.8 with citrate and 0.4 with either externally added reduced nicotinamide adenine dinucleotide (NADH), succinate, or ascorbate-tetramethyl-p-phenylenediamine (TPD). These P/O ratios suggest that there are only two sites of phosphorylation in mitochondria isolated from young (20 to 24 h) cultures of the mi-1 mutant. The energy-dependent reduction of NAD(+) with succinate and the phosphorylation associated with ascorbate-TPD oxidation indicate that the first and the third sites of energy coupling are present in this mutant. Difference spectra of mitochondria from young cultures of the mi-1 mutant revealed the presence of cytochrome c. Cytochromes b and a + a(3) were not detected. However, in the presence of antimycin A, a small peak in the Soret region at 430 nm was observed. A carbon monoxide difference spectrum revealed the presence of a component of the respiratory chain with a spectrum similar to that of cytochrome o. It is of interest that respiratory inhibitors such as antimycin A, 2-n-nonylhydroxyquinoline N-oxide, and cyanide abolished phosphorylation but only partially inhibited oxidation. It is postulated that the mi-1 respiratory system contains two pathways of electron transport-the first is associated with a phosphorylating pathway, whereas the second is a non-phosphorylating electron transport pathway.
Mutations at three loci in the mouse--W, Steel Sl), and microphthalmia (mi)--can lead to a deficiency in melanocytes and mast cells. As well, W and Sl mutants can be anemic and sterile, whereas mi mice are osteopetrotic due to a monocyte/macrophage defect. Recent data have shown that the c-kit receptor tyrosine kinase is the gene product of the W locus, whereas Sl encodes the ligand for this growth factor receptor. We show here that ectopic expression of c-fms, a gene that encodes a macrophage growth factor receptor that is closely related to the c-kit receptor, complements mutations at the W locus in an in vitro mast cell/fibroblast coculture system but is unable to reverse the inability of mi/mi mast cells to survive under these conditions. Furthermore, mast cells expressing the c-fms receptor survive on a monolayer of fibroblasts homozygous for the Sl mutation. These results suggest that ligand binding to the c-kit or c-fms receptor activates identical or overlapping signal transduction pathways. Furthermore, they suggest that mi encodes a protein necessary for transducing signals mediated by way of either the c-kit or c-fms receptor.
We have examined the behavior of the [mi-3] mitochondrial mutation and two physical mtDNA markers in heterokaryotic cultures of Neurospora crassa. Previous workers showed that a 1.2-kilobase insertion in the larger polymorphic form of EcoRI-5 restriction fragment is a site of high frequency and rapid unidirectional gene conversion. We have confirmed this observation and determined by DNA sequence analysis that the insertion in the EcoRI-5 fragment corresponds precisely to an optional intron that contains a long open reading frame in the ND1 gene. Thus, the conversion of the short, intron-lacking, form of EcoRI-5 to the longer, intron-containing, form may be analogous to the unidirectional gene conversion events catalyzed by intron-encoded proteins in other organisms. The resolution of two polymorphic forms of the mtDNA EcoRI-9 restriction fragment in our heterokaryons differs from that observed previously and suggests that this locus is not a site of gene conversion in our heterokaryon pair. The size polymorphism of the EcoRI-9 fragments is due to a tandemly reiterated 78-base-pair sequence which occurs two times in the short form and three times in the long form. One copy of the repeat unit and 66 base pairs following it have been duplicated from the ND2 gene which is located about 30 kilobases distant on the mtDNA. In contrast to the [poky] mitochondrial mutant, which was completely dominant over wild-type mitochondria in heterokaryons, the [mi-3] mutant was recovered in only seven of twenty heterokaryons after ten cycles of conidiation and subculturing. The resolution of the [mi-3] or wild-type phenotype in heterokaryons may depend solely on random factors such as allele input frequency, drift, and segregation rather than specific dominant or suppressive effects.