Monoclonal antibody against human T cell leukemia virus p19 defines a human thymic epithelial antigen acquired during ontogeny.

Using monoclonal antibody 12/1-2 against a 19,000-dalton human T cell leukemia virus (HTLV) protein (anti-p19), previously demonstrated to be reactive with HTLV-infected human cells, but not in numerous other uninfected cells, we found a reactive antigen to be expressed on the neuroendocrine component of human thymic epithelial cells but not on any other normal epithelial or neuroendocrine human tissues. Moreover, this reactive antigen is acquired on neuroendocrine thymic epithelium during thymic ontogeny--first appearing on fetal thymic epithelial cells between 8 and 15 wk gestation. While only a portion of thymic epithelial cells in the subcapsular cortical region of 15- and 24-wk fetal thymuses contained anti-p19+ epithelial cells, the entire subcapsular cortical region of newborn thymus epithelium was anti-p19+. By age 3 yr, normal subjects' entire subcapsular cortical and medullary thymic epithelium was anti-p19+. Using antibody against HTLV core protein, p24, and c-DNA probes for HTLV DNA, neither HTLV-specific p24 protein nor proviral DNA could be demonstrated in anti-p19+ thymic epithelial tissue. However, thymic epithelial extracts, disrupted HTLV extracts, as well as purified HTLV p19 antigen all inhibited the binding of anti-p19 antibody to thymic epithelium. Thus, anti-p19 may recognize a determinant on an HTLV-encoded 19,000-dalton structural protein that is shared by human thymic epithelium. Alternatively, anti-p19 defines a host encoded protein that is selectively expressed by normal thymic epithelium, and is induced to be expressed in HTLV-infected malignant T cells.

A LMNA splicing mutation in two sisters with severe Dunnigan-type familial partial lipodystrophy type 2.

CONTEXT: To date, all cases of familial partial lipodystrophy type 2 (FPLD2; Mendelian Inheritance in Man 151660) result from missense mutations in LMNA, which encodes nuclear lamin A/C (Mendelian Inheritance in Man 150330). OBJECTIVE: The objective of the study was to carry out mutational analysis of LMNA in two sisters with a particularly severe FPLD2 phenotype. DESIGN: This was a descriptive case report with molecular studies. SETTING: The study was conducted at a referral center. PATIENTS: We report two sisters of South Asian origin. The first presented with acanthosis nigricans at age 5 yr, diabetes with insulin resistance, hypertension and hypertriglyceridemia at age 13 yr, and partial lipodystrophy starting at puberty. Her sister and their mother had a similar metabolic profile and physical features, and their mother died of vascular disease at age 32 yr. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES AND RESULTS: LMNA sequencing showed that the sisters were each heterozygous for a novel G>C mutation at the intron 8 consensus splice donor site, which was absent from the genomes of 300 healthy individuals. The retention of intron 8 in mRNA predicted a prematurely truncated lamin A isoform (516 instead of 664 amino acids) with 20 nonsense 3'-terminal residues. The mutant lamin A isoform failed to interact normally with emerin and failed to localize to the nuclear envelope. CONCLUSIONS: This is the first LMNA splicing mutation to be associated with FPLD2, and it causes a severe clinical and metabolic phenotype.

Dermal fibroblasts in Hutchinson-Gilford progeria syndrome with the lamin A G608G mutation have dysmorphic nuclei and are hypersensitive to heat stress.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare sporadic disorder with an incidence of approximately 1 per 8 million live births. The phenotypic appearance consists of short stature, sculptured nose, alopecia, prominent scalp veins, small face, loss of subcutaneous fat, faint mid-facial cyanosis, and dystrophic nails. HGPS is caused by mutations in LMNA, the gene that encodes nuclear lamins A and C. The most common mutation in subjects with HGPS is a de novo single-base pair substitution, G608G (GGC>GGT), within exon 11 of LMNA. This creates an abnormal splice donor site, leading to expression of a truncated protein. RESULTS: We studied a new case of a 5 year-old girl with HGPS and found a heterozygous point mutation, G608G, in LMNA. Complementary DNA sequencing of RNA showed that this mutation resulted in the deletion of 50 amino acids in the carboxyl-terminal tail domain of prelamin A. We characterized a primary dermal fibroblast cell line derived from the subject's skin. These cells expressed the mutant protein and exhibited a normal growth rate at early passage in primary culture but showed alterations in nuclear morphology. Expression levels and overall distributions of nuclear lamins and emerin, an integral protein of the inner nuclear membrane, were not dramatically altered. Ultrastructural analysis of the nuclear envelope using electron microscopy showed that chromatin is in close association to the nuclear lamina, even in areas with abnormal nuclear envelope morphology. The fibroblasts were hypersensitive to heat shock, and demonstrated a delayed response to heat stress. CONCLUSION: Dermal fibroblasts from a subject with HGPS expressing a mutant truncated lamin A have dysmorphic nuclei, hypersensitivity to heat shock, and delayed response to heat stress. This suggests that the mutant protein, even when expressed at low levels, causes defective cell stability, which may be responsible for phenotypic abnormalities in the disease.

Functional consequences of an LMNA mutation associated with a new cardiac and non-cardiac phenotype.

Heritable dilated cardiomyopathy is a genetically highly heterogeneous disease. To date 17 different chromosomal loci have been described for autosomal dominant forms of dilated cardiomyopathy with or without additional clinical manifestations. Among the 10 mutated genes associated with dilated cardiomyopathy, the lamin A/C (LMNA) gene has been reported in forms associated with conduction-system disease with or without skeletal muscle myopathy. For the first time, we report here a French family affected with a new phenotype composed of an autosomal dominant severe dilated cardiomyopathy with conduction defects or atrial/ventricular arrhythmias, and a specific quadriceps muscle myopathy. In all previously reported cases with both cardiac and neuromuscular involvement, neuromuscular disorders preceded cardiac abnormalities. The screening of the coding sequence of the LMNA gene on all family members was performed and we identified a missense mutation (R377H) in the lamin A/C gene that cosegregated with the disease in the family. Cell transfection experiments showed that the R377H mutation leads to mislocalization of both lamin and emerin. These results were obtained in both muscular (C2C12) and non-muscular cells (COS-7). This new phenotype points out the wide spectrum of neuromuscular and cardiac manifestations associated with lamin A/C mutations, with the functional consequence of this mutation seemingly associated with a disorganization of the lamina.

Thymopoietin-like substance in human skin.

A heterologous antithymopoietin (anti-TP) antibody was used to determine whether a TP-like molecule is present in the epidermis, since such factors have been postulated to play a part in known T cell-epidermal cell interaction. Examination of cytocentrifuge smears of freshly separated human epidermal cells stained by indirect immunofluorescence revealed that 8-14% of these cells possessed cytoplasmic reactivity with the anti-TP antibody. Similarly, 2-5% of human epidermal cells, maintained in tissue culture for 2-8 weeks, showed cytoplasmic staining with the anti-TP antibody. Double-labeling immunofluorescence studies, with the anti-TP antibody and a monoclonal antibody specifically reactive with Langerhans cells (OKT6), demonstrated that cells possessing this TP-like substance were not Langerhans cells. In situ studies of 4-microns frozen sections of normal human skin indicated that the cell population which possesses the TP-like substance is the basal layer of keratincoytes in the epidermis.

Radioimmunotherapy with alpha-particle emitters: microdosimetry of cells with a heterogeneous antigen expression and with various diameters of cells and nuclei.

Intercellular variations in the level of antigen expression and in cellular and nuclear radii were taken into account in a model used to estimate cell survival for an in vitro experiment with antibodies containing alpha-particle emitters that target the cell surface. Using measured variations in these characteristics for cells of two human cancer cell lines, the model gave results for cell survival and the fundamental parameter of radiation sensitivity, z(0), that differ substantially from those obtained using only mean values. The cell survival may be underestimated by a factor of 100 if only mean values of these cellular parameters are used, and calculated values of z(0) may be overestimated by a factor of 2. Most of this effect stems from the variation in antigen expression. The magnitudes of the differences were found to be a function of the fractions of mean specific energy delivered by surrounding activity and by activity bound to the cells.

Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone.

Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.

Immunoreactive thymopoietin in the mouse central nervous system.

A thymopoietin-immunoreactive substance (TP-IRS) has been detected in homogenates of mouse spinal cord and brain using a radioimmunoassay; levels were maximal at birth. TP-IRS was also detected in supernatants of mouse neuroblastoma (NIE-115) and primary spinal cord cultures but not human astrocytic and meningeal tumors or mouse primary astrocyte cultures. With affinity purified rabbit anti-TP globulin, immunofluorescent staining was seen in mouse spinal cord cultures in association with nuclear membranes of neurons and, to a lesser degree, flat background cells. From supernatants of NIE-115 cells grown in tritiated leucine and lysine, proteins of approximately 8000 and 4500 Da were isolated by TP affinity chromatography (compared with 5562 Da for thymic thymopoietin). When injected into mice, these neural proteins partially blocked neuromuscular transmission in a manner similar to thymic thymopoietin.

Detection of DNA fragmentation of myonuclei in myotonic dystrophy by double staining with anti-emerin antibody and by nick end-labeling.

To clarify the occurrence of apoptosis in skeletal muscle in pathological conditions, we studied 44 muscle biopsy specimens by immunohistochemical staining with monoclonal antibody against emerin, which is localized in muscle nuclear membrane, and by ApopTag Plus to detect DNA fragmentation. Five of six patients with myotonic dystrophy (DM) showed three to 35 myonuclei stained with anti-emerin antibody and ApopTag Plus in 1500 muscle fibers. Four of the 18 patients with polymyositis, one of those with thyroid myopathy and one with neurogenic atrophy showed a few myonuclei stained positively by these methods. Our study revealed that DNA fragmentation in myonuclei occurred in skeletal muscle fibers regardless of the type of disease, although the frequency was rather low in all of these diseases except DM. The DNA fragmentation detected in most of the patients with DM suggested a significant role of apoptosis in the pathomechanism of this disease.

Emericella venezuelensis, a new species with stellate ascospores producing sterigmatocystin and aflatoxin B1.

Emericella venezuelensis is a new species, differing from two other species with stellate ascospores, E. variecolor and E. pluriseminata, by triangular flaps on the convex sides of the ascospores, and further from E. variecolor by producing an Aspergillus anamorph only on unconventional growth media. The three species also differ in their profiles of extrolites (secondary metabolites). Emericella venezuelensis produces aflatoxin B1, sterigmatocystin, and terrein and compounds with chromophores of the shamixanthone, emerin and desertorin type of compounds. E. variecolor produces asteltoxin, shamixanthone, asperthecin, and terrein, in addition to metabolites unequivocally recorded in the literature or tentatively identified here as astellolide A & B, andibenin A, B, C, andilesin A, B, C, anditomin, astellatol, stellatic acid, stellatin, tajixanthone, radixanthone, najamxanthone, ajamxanthone, variecoxanthone A, B, C, isoemericellin, kojic acid, varitriol, varioxiran, dihydroterrein, 7-hydroxyemodin, avariquinone and stromemycin. E. pluriseminata produces several unknown specific extrolites. E. venezuelensis is the first organism of marine origin reported to produce aflatoxin. Aflatoxin production by E. venezuelensis makes this species an attractive model organism for the study of the regulation of this important type of carcinogenic mycotoxins in combination with the knowledge on sterigmatocystin production by E. nidulans, soon to be whole genome sequenced. The isolates were also analyzed cladistically using partial sequences of the beta-tubulin gene. Since three species of Emericella have stellate ascospores, and the type material of E. variecolor is equivocal, this species is epitypified with CBS 598.65. Emericella species normally do not appear to cause problems for food safety, as they are most often found in litter and soil.

1H-NMR studies on thymopoietin-type oligopeptides--assignment of the proton resonances and investigation of conformational preferences.

The thymopoietin-type tripeptides TP3 (HArg-Lys-AspOH), TP(D-Asp)3(HArg-Lys-D-AspOH) and tetrapeptide TP4 (HArg-Lys-Asp-ValOH) were studied by one- and two-dimensional, 500 MHz 1H-NMR spectroscopy in H2O and D2O solutions at four different pH values. All proton resonances of the three oligopeptides were assigned by two-dimensional phase-sensitive TOCSY experiments at pH 12.2, 9.1, 5.9 and 3.6. At these pH-values well-defined stages of protonation and concomitant molecular charges exist, allowing different possibilities for intra-molecular and inter-residual orientations. Conformation-sensitive rotating frame nuclear Overhauser enhancement (ROESY) two-dimensional experiments were also performed at the above pH values. These experiments indicated no definite solution conformation of any of the molecules at any pH. Standard one-dimensional experiments were also carried out and three-bond coupling constants were measured for the NH--CH and the Asp CH--CH moieties. The coupling constants provided evidence that non-statistical orientations of the functional groups exist which are changed upon protonation of the basic sites.

[Primary structure and species specificity of immunopoietins]. / Pervichnaia struktura i vidovaia spetsifichnost' immunopoétinov.

A model involving quasicyclization of protein molecules following the stepwise reactions of limited proteolysis was developed. On the basis of the model new active sites were detected in immunoglobulins of various species as well as chemical synthesis of the sites was carried out. The new group of the substances was designated as immunopoietins. The primary structure of various species immunoglobulins was analyzed and evolutional stability of the immunopoietin structure was shown.

Mutation Glu82Lys in lamin A/C gene is associated with cardiomyopathy and conduction defect.

Dilated cardiomyopathy is a form of heart muscle disease characterized by impaired systolic function and ventricular dilation. The mutations in lamin A/C gene have been linked to dilated cardiomyopathy. We screened genetic mutations in a large Chinese family of 50 members including members with dilated cardiomyopathy and found a Glu82Lys substitution mutation in the rod domain of the lamin A/C protein in eight family members, three of them have been diagnosed as dilated cardiomyopathy, one presented with heart dilation. The pathogenic mechanism of lamin A/C gene defect is poorly understood. Glu82Lys mutated lamin A/C and wild type protein was transfected into HEK293 cells. The mutated protein was not properly localized at the inner nuclear membrane and the emerin protein, which interacts with lamin A/C, was also aberrantly distributed. The nuclear membrane structure was disrupted and heterochromatin was aggregated aberrantly in the nucleus of the HEK293 cells stably transfected with mutated lamin A/C gene as determined by transmission electron microscopy.

Graphical determination of specific activity, binding constants, and antibody-site concentrations for radioimmunoassays, with application to thymopoietin.

A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.

[Effect of splenin and its fractions on the functional state of the adrenal cortex]. / Vliianie splenina i ego fraktsii na funktsional'noe sostoianie kory nadpochechnikov.

Effect of splenin (1 ml) and its fractions (0.5 ml) on adrenocortical function was studied after a single intramuscular injection of the preparations into rats. Splenin and its fractions containing water-soluble substances reduced the content of ascorbic acid in the rat adrenals thereby indicating the increased synthesis of adrenocortical hormones. Adenosine-5-monophosphate is one of the basic biological substances contained by splenin and its water-soluble fractions. It produces a stimulant effect on the synthesis of adrenocortical hormones.

Use of monoclonal antibodies to identify thymopoietin in cultured human thymic epithelial cells.

Murine monoclonal antibodies (mAbs) were developed to discriminate thymopoietin, a human thymic hormone, and thysplenin, a closely related molecule found in spleen. Three of these recognized both native and synthetic thymopoietin as well as thysplenin. Together they define two non-overlapping epitopes which withstand sodium dodecyl sulfate denaturation and can be detected by western blotting. We used these three mAbs to demonstrate the production of thymopoietin by cultured thymic epithelial cells for up to several weeks. Three additional mAbs were selective for thysplenin. Highly specific mAbs will be useful for characterizing further these physiologically distinct polypeptides.

Immunolocalization of thymosin alpha 1, thymopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: a light and electron microscopic study.

The secretory evolution of the thymic hormones (thymulin, thymosin alpha 1 and thymopoietin) in cultured thymic reticuloepithelial cells (TREC) was studied by immunocytochemical techniques using monoclonal anti-thymulin or anti-thymosin alpha 1 and polyclonal anti-thymopoietin antibodies (Ab). The culture of TREC was performed with a medium where L-valine was replaced by D-valine, thus ensuring rapid and selective development of these cells. The number of thymulin, thymosin alpha 1 or thymopoietin-containing cells increased progressively from Day 6 to Day 12 of the culture. The localization of the three thymic hormones within the TREC also varied according to the age of the culture. By light microscopy the staining of the three hormones was localized in some cytoplasmic granules at the beginning of the culture and at Day 90, while at Day 12 it was throughout the cytoplasm. In electron microscopy these localizations corresponded respectively to vacuoles of different sizes and to cytosol. All these results show that the synthesis and excretion of thymulin, thymosin alpha 1 and thymopoietin evolve during the development of TREC in culture.

Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins.

Lamina-associated polypeptide 2 (LAP2) and the thymopoietins (TPs) are a family of proteins described in somatic cells of mammals, which are derived by alternative splicing from a single gene. For one of the members of the family (LAP2 = TPbeta) it has been shown that this integral membrane protein locates to the inner membrane of the nuclear envelope, and that it binds to chromatin and B-type lamins. In the present study, we observed that during the third phase of spermatogenesis (i.e. spermiogenesis), TP-labelling shifted progressively to one half of the nuclear periphery in round spermatids. In the elongating spermatid the signal then becomes restricted to one spot located at the posterior (centriolar) pole of the nucleus. Changes in localization are accompanied by the disappearance, first of TPgamma, and later on of LAP2/TPbeta. TPalpha is the only member of the family detectable in the mature sperm. Concomitantly, lamin B1, the only nuclear lamina protein known to be expressed in mammalian spermatids, showed a similar behaviour, i.e. shifted progressively to the centriolar pole of spermatid nuclei before it became undetectable in fully differentiated mature sperms. These results are the first demonstration that expression and localization patterns of TPs are coordinately and differentially regulated with lamins during a differentiation process.

Thymopentin structure and relation to thymic factors.

High-performance liquid chromatography (HPLC) was used to evaluate the purity and batch-to-batch consistency of four commercially available thymic extracts and thymopentin, the synthetic pentapeptide corresponding to the biologically active region of the thymic hormone thymopoietin. The thymus extracts were all extremely heterogeneous, differing one from the other. Additionally, they showed high batch-to-batch variation. In contrast, thymopentin was homogeneous and this homogeneity was consistent in different batches.

Thymopoietin radioreceptor assay utilizing lectin-purified glycoprotein from a biologically responsive T cell line.

Radiolabeled thymopoietin that was biologically active and of high specific activity was prepared by a novel technique involving protection of free amino groups, selective excision of the protected N-terminal prolyl group with post-proline cleaving enzyme, reaction of the newly exposed alpha-amino group with a highly radioiodinated compound, and deprotection and purification of the polypeptide. Binding of this radiolabeled thymopoietin was not demonstrable by conventional techniques with cells, cell membranes, or solubilized cell membranes, apparently due to the presence of active proteases in these preparations. A glycoprotein with thymopoietin binding properties was prepared by lectin purification from the detergent-solubilized membranes of CEM cells, a human T cell line that responds to thymopoietin in vitro with increases in intracellular cyclic GMP. Presumably this procedure separated the thymopoietin binding protein from membrane proteases, thus permitting the development of a radioreceptor assay. Evidence is presented that the thymopoietin binding protein represents a thymopoietin receptor that is probably related to the mediation of immunoregulatory actions of thymopoietin on a subset of peripheral T cells.