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.

The lamina-associated polypeptide 2 (LAP2) isoforms beta, gamma and omega of zebrafish: developmental expression and behavior during the cell cycle.

Zebrafish lamina-associated polypeptides 2 (ZLAP2) beta, gamma and omega have in common an N-terminal region with a LEM domain, and in the C-terminal half of the molecule a lamina binding domain and a membrane spanning sequence. The maternally synthesized omega is the largest isoform and the only LAP2 present in the rapidly dividing embryonic cells up to the gastrula stage. ZLAP2omega levels decrease during development, concomitant with the increase of the somatic isoforms ZLAP2beta and gamma. In somatic zebrafish cells ZLAP2gamma is the predominant isoform, whereas only small amounts of ZLAP2beta are present. During early embryonic development, ZLAP2omega becomes associated with mitotic chromosomes before anaphase. The surface of these chromosomes is decorated with vesicles, and each chromosome assembles its own nuclear envelope at the end of mitosis (karyomere formation). Ectopically expressed ZLAP2omega-green fluorescent protein (GFP) fusion protein targets vesicles to mitotic chromosomes in Xenopus A6 cells, suggesting that ZLAP2omega is involved in karyomere formation during early zebrafish development. When ZLAP2beta and gamma were expressed as GFP fusion proteins in Xenopus A6 cells, the beta- but not the gamma-isoform was found in association with mitotic chromosomes, and ZLAP2beta-containing chromosomes were decorated with vesicles. Further analysis of ZLAP2-GFP fusion proteins containing only distinct domains of the ZLAP2 isoforms revealed that the common N-terminal region in conjunction with beta- or omega-specific sequences mediate binding to mitotic chromosomes in vivo.

Thymopentin and splenopentin as immunomodulators. Current status.

Splenopentin (SP-5, Arg-Lys-Glu-Val-Tyr) and thymopentin (TP-5, Arg-Lys-Asp-Val-Tyr) are synthetic immunomodulating peptides corresponding to the region 32-34 of a splenic product called splenin (SP) and the thymic hormone thymopoietin (TP), respectively. TP was originally isolated as a 5-kDa (49-amino acids) protein from bovine thymus while studying effects of the thymic extracts on neuromuscular transmission and was subsequently observed to affect T cell differentiation and function. TP I and II are two closely related polypeptides isolated from bovine thymus. A radioimmunoassay for TP revealed a crossreaction with a product found in spleen and lymph node. This product, named splenin, differs from TP only in position 34, aspartic acid for bovine TP and glutamic acid for bovine splenin and it was called TP III as well. Synthetic pentapeptides (TP-5) and (SP-5), reproduce the biological activities of TP and SP, respectively. It is now evident that various forms of TPs were created by proteolytic cleavage of larger proteins during isolation. cDNA clones have been isolated for three alternatively spliced mRNAs that encodes three distinct human T cell TPs. The immunomodulatory properties of TP, SP, TP-5, SP-5 and some of their synthetic analogs reported in the literature have been briefly reviewed.

[High performance liquid chromatography and capillary electrophoresis investigation of the neww immunostimulant tetrapeptide drug, thymocartin]. / Az új immunstimuláló tetrapeptid, a thymocartin nagyhatékonyságú folyadékkromatográfiás és kapillár elektroforetikus vizsgálata.

High-performance liquid chromatographic (HPLC) and capillary electrophoretic (CE) methods have been developed for the separation of 22 related peptides (potential and real impurities) from a new immunostimulant tetrapeptide derivative (thymocartin, Arg-Lys-Asp-Val) being under clinical examination. The described methods are suitable for the purity control of the bulk drug material. In the course of the reversed-phase ion-pair HPLC separations C-18 columns (Hypersil BDS and Ultrasphere IP) were used. In the case of using sodium hexanesulphonate as the ion-pairing reagent, the eluent contained phosphate buffer (pH = 3) and 32% v/v methanol, while in another method a gradient system with a sodium perchlorate solution (pH = 2 set by perchloric acid) and acetonitrile was applied. The optimum separation in the CE investigations was achieved at pH = 3 using triethylammonium phosphate buffer and 10% v/v acetonitrile as the organic modifier.

Expression of thymopoietin beta/lamina-associated polypeptide 2 (TP beta/LAP2) and its family proteins as revealed by specific antibody induced against recombinant human thymopoietin.

An expression vector was constructed to produce a common region of human thymopoietin family proteins. The recombinant protein was expressed in Escherichia coli as a fusion protein with a biotinylated tag region and purified by affinity chromatography on a monomeric avidin resin. The thymopoietin family-specific antibody was induced in rabbits by immunization with the recombinant fusion protein. Western blotting analysis using the antibody revealed that the expression of thymopoietin family proteins was remarkably tissue specific. Among those, thymopoietin beta/lamina-associated polypeptide 2 appears to be specifically expressed in tissues with high proliferative activity.

Three distinct human thymopoietins are derived from alternatively spliced mRNAs.

Thymopoietin (TP) was originally isolated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission and was subsequently observed to affect T-cell differentiation and function. We now report the isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, which we have named TP alpha (75 kDa), TP beta (51 kDa), and TP gamma (39 kDa), contain identical N-terminal regions, including sequences nearly identical to that of the originally isolated 49-aa protein, but divergent C-terminal regions. TP mRNAs are expressed in many tissues, most abundantly in adult thymus and fetal liver of the tissues so far examined. Distinct structural domains and functional motifs in TPs alpha, beta, and gamma suggest that the proteins have unique functions and may be directed to distinct subcellular compartments.

Effect of direct control of electroosmosis on peptide and protein separations in capillary electrophoresis.

The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.

Amino acid sequence of thymopoietin isolated from skin.

The isolation of thymopoietin-reactive material in fetal bovine skin was monitored by means of a radioimmunoassay to thymopoietin. The amino acid sequence of this material was determined to be identical with that of thymopoietin isolated from the thymus. Experimental evidence suggests that thymopoietin in the circulation derives from the thymus and not from the skin, suggesting that the thymopoietin in keratinocytes has a local function, either apocrine and/or immunoregulatory.

Isolation and complete amino acid sequence of human thymopoietin and splenin.

Human thymopoietin and splenin were isolated from human thymus and spleen, respectively, by monitoring tissue fractionation with a bovine thymopoietin RIA cross-reactive with human thymopoietin and splenin. Bovine thymopoietin and splenin are 49-amino acid polypeptides that differ by only 2 amino acids at positions 34 and 43; the change at position 34 in the active-site region changes the receptor specificities and biological activities. The complete amino acid sequences of purified human thymopoietin and splenin were determined and shown to be 48-amino acid polypeptides differing at four positions. Ten amino acids, constant within each species for thymopoietin and splenin, differ between the human and bovine polypeptides. The pentapeptide active site of thymopoietin (residues 32-36) is constant between the human and bovine thymopoietins, but position 34 in the active site of splenin has changed from glutamic acid in bovine splenin to alanine in human splenin, accounting for the biological activity of the human but not the bovine splenin on the human T-cell line MOLT-4.

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.

Complete amino acid sequences of bovine thymopoietins I, II, and III: closely homologous polypeptides.

Complete amino acid sequences were determined for thymopoietins I and II (revision), isolated from bovine thymus, and for thymopoietin III, a newly identified polypeptide isolated from bovine spleen. Thymopoietin III (TP-III) is a 49 amino acid monomeric peptide that shows minor microheterogeneity at residue 34. The three thymopoietins have largely identical sequences yet some distinct differences, suggesting very recent evolution from a common gene. The complete amino acid sequences are (Formula: see text).

[Modified method of isolating thymopoietin and its effect on neuromuscular transmission]. / Modifikatsiia metoda vydeleniia timopoétina i ego vliianie na nervno-myshechnuiu peredachu.

A modified method for isolation of the calf thymus hormone thymopoetin has been developed. For hormone purification high-speed multi-stage centrifugation and gel-filtration on sefadexes were used instead of filtration. The thymus hormonal factor was identified on the basis of its ability to block neuromuscular transmission.

Chromatographic isolation of thymic factors impairing neuromuscular transmission.

Thymopoietins I and II are chemically related peptides able to inhibit neuromuscular transmission. They were first isolated from bovine thymus by a protein-denaturating method, which may have destroyed other thymic factors displaying the same biological activity. The present investigation, based on a new gentle isolation procedure, suggests that thymopoietins are the only factors impairing neuromuscular transmission that are present in the thymus. The yield of the new procedure is at least twice as high as that of the original method.