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One of the most substantial complications in orthopedic surgeries is implant associated infection. While the implant interface can be a permissive surface for bacterial colonization, an appropriate coating on the implant can effectively hinder biofilm formation. To this aim, henna and thyme extract loaded into a chitosan (CS)/polyethylene oxide (PEO) based nanofibrous hydrogel. CS/PEO/henna and CS/PEO/thymus extract nanofibers were fabricated via two-nozzle electrospinning. The proper weight ratio of CS/PEO/herbal extract was optimized. The appropriate ratio of CS/PEO and the most suitable extract concentrations was obtained to be 7/3 (v/v) and 1 % (v/v), respectively. Contact angle measurements showed an appropriate degree of hydrophilicity in all groups including herbal loaded electrospun nanosheets. Tensile strength measurements showed that inclusion of henna and thymus made minor changes in tensile strength and strain of herbal loaded nanofiber, compared to control CS/PEO fibers. Then antibacterial tests were performed and the fibers either by themselves or when used as coating on orthopedic screws exhibited a good inhibition zone against both gram negative and gram positive bacteria. The coatings showed no adverse effect on the red blood cells in leading to hemolysis. Cytocompatibility assay indicated that the prepared nanofibrous hydrogel coating was absolutely biocompatible as tested with fibroblast cells. The results of this study indicated that our henna and thyme extracts loaded CS/PEO coatings can be used in various medical fields such as wound dressing and implantation coating to prevent bacterial adhesion, growth and generally, infection.

This study aimed to evaluate the supplementation (of drinking water) effects of L-Carnitine (LC) and/or calf thymus extract (TE) on immunity, antioxidant, duodenal histomorphometric, growth, and economic performance of Japanese quail. Six hundred, one day old unsexed quail were divided into four groups, the control group (G1) received plain drinking water, while G2, G3, and G4 were supplemented with LC and/or TE, respectively. The treated groups recorded a significant (p < 0.05) improvement in the final body weight, weight gain, feed conversion ratio, phagocytic activity %, phagocytic index, lymphocytes %, and plasma glutathione level compared to the G1. On the other hand, these supplementations significantly lower the mortality rate %, heterophil %, H/L ratio and plasma malondialdehyde level of the supplemented groups relative to G1. The supplemented groups recorded a non-significant increase in duodenal histomorphometric parameters (villous height, villous width, surface area, and crypt depth) relative to G1. Generally, LC and/or TE improve the values of economic profile (total returns, net profit, total return/total costs %, etc.) in treated groups compared to the control one. In conclusion, L-carnitine supplementation was more effective than TE or their combination with TE in the immunological, anti-oxidative, growth, and economic performance.

The polyphenolic composition and anti-inflammatory activity of selected Thymus species (T. longicaulis C. Presl, T. praecox Opiz subsp. polytrichus (A. Kerner Ex Borbas) Jalas, T. pulegioides L., T. serpyllum L. subsp. serpyllum and T. striatus Vahl) was studied in comparison with T. vulgaris, as an officinal medicinal plant, to evaluate the biomedical potential of these Croatian wild-growing species. This paper provides a first insight into the polyphenolic profiles of T. praecox subsp. polytrichus and T. striatus. Also, some flavone and hydroxycinnamic derivatives were at first identified in the other studied species by LC-DAD-ESI–MS/MS analysis. Rosmarinic acid and glycosides of scutellarein and luteolin were found to be the most abundant polyphenolic constituents. The Thymus extracts inhibited Src tyrosine kinase activity and they reduced the production of proinflammatory cytokine interleukin-6 in Balb/C mice splenocytes. The obtained results highlighted T. longicaulis as a potential source of herbal drugs with anti-inflammatory properties and/or novel functional food rich in flavonoids and phenolic acids.

THIS STUDY illustrates a simple method for the detection of the ability of Aspergillus parasiticus, isolated from peanut seeds, for the production of aflatoxins. The indicator of aflatoxins production ability is the appearance of colony reverse yellow color of the fungus on the agar media and the natural fluorescence of aflatoxin around the colony when observed under UV (black light 350BL) after three days of incubation at 28oC. The morphological identification of A. parasiticus was confirmed by molecular identification rDNA sequencing. The addition of β-Cyclodextrin to media enhanced the natural fluorescence of aflatoxin around the colony under UV. The extract of Thymus vulgaris had an inhibitory effect on the growth of A. parasiticus. The minimum inhibitory concentration (MIC) of Thymus extract against A. parasiticus was 80μL of Thymus extract /10 ml of YSA medium. The effective concentration of Thymus extract causing a 50% inhibition of mycelial growth (EC50) was 20μL of Thymus extract /10mL of YSA medium. The chemical analysis of Thymus extract by Gas Chromatography-Mass Spectroscopy (GC-MS) illustrated that the thymol has a retention time (RT) (min) of 37.538 at the highest percent area (13.8717 Area %).

The Thymus and Origanum extracts were obtained at different temperature, separate and combined biological effects of them were investigated. There were shown the influence of this substances at the rye grain bioactivation taking into account their antioxidant and membrane-protective properties. The antioxidant activity of Thymus extracts, but not Origanum, directly depended on the temperature of extraction (30 or 80°C). When “hot” Thymus extract (80°C) used the reduction degree of chemiluminescence was 3…6 times higher than in the control and 2…3 times higher than the oregano extracts used. The results are in good agreement with the data on the assessment of extracts membrane-protective activity. Under the influence of individual preparations of Thymus, as well as the combined preparation of Thymus+Origanum (1:1), the osmotic resistance of the liposomal suspension increased. Under using combined phytoextracts of oregano and thyme (1: 2; 2: 1; 1: 1), the dynamics and energy of germination exceeded the control level significantly. The best result was observed at a 1:1 ratio, when the number of germinated seeds was more then in control by 4 times by the 12th hour of exposure, and the optimal length of germinated seeds was formed by the 10th hour. Thus, using of the combined preparation of thyme + oregano (1: 1) allows to halve the longest stage of plant raw materials preparation for obtaining a multi-grain flourless bakery product.

Water with modified isotopic composition (D/H = 40 ppm) as a solubilizing agent for biologically active substances extraction from immune organs Sus Scrofa increases protein yield in thymus to 20%, in spleen up to 38%, mesenteric lymph nodes up to 35% in comparison with distilled water (D/H = 150 ppm). It was found a significant amount of neurotransmitter amino acids, such as aspartic and glutamic acids, glycine (thymus - 10.5; 13.7; 7.6%; spleen - 12.2; 10.7; 7.8% lymph nodes - 11.0; 13.3; 11.1%, respectively) having the immunological and adaptogenic activity, in extracts of Sus scrofa immunocompetent organs (thymus, spleen, mesenteric lymph nodes). Application of water with modified isotopic composition as solubilizing agent (D / H = 40 ppm) for extraction of immunocompetent organs' biologically active compounds led to increased amino acid content, including hydrophilic amino acids, in thymus extracts - up to 22%, in spleen extracts - up to 15% , in lymph nodes exctracts - up to 8%, in comparison to distilled water (D / H = 150 ppm). Peptide profile analysis revealed positive effect of water (D/H = 40 ppm) on a quality protein content extracts in molecular weight range from 10 to 20 kDa and from 30 to 45 kDa and peptide composition (2 kDa), at the same time quantitative content of compounds adaptogenic and immunocorrective action increased. Reducing of deuterium content in the solubilizing agent enhances quantitative amino acid content, i.e. extraction in an aqueous- salt solution based on deuterium depleted water of animal tissues with a high content of amino acids with hydrophilic radicals proceeded more completely.

Microfluidics based systems could be useful for drug discovery as they allow for miniaturization and could potentially be run as multiple parallel cell based assays. Such miniaturization allows assays at single cell level and reduces the amount of test material needed, which, in the case of natural product extracts, simplifies the preparation. Thyme species extracts have been reported to show some promising anti-cancer effects. In the present work, we used a microfluidics based system to study the effects of Thymus kotschyanusm Boiss plant extract on two human breast cancer cells lines which are MDA-MB-231 and MCF-7. For better understanding a single cancer cell death mechanism and a flow control, a Polydimethylsiloxane (PDMS) microfluidic device has been fabricated. The morphology of single cancer cells in the microfluidic system showed that the higher concentration of plant Thymus extract (560 µg/ml) had a significant effect on the cell membrane compared to the lower concentration (15 µg/ml). In addition, the results showed that MDA-MB-231 cells were more sensitive to the cytotoxic effects of the extracts compared to the MCF-7 cells. These results concur with the MTT assay analysis that showed the IC50 values of the extract in MDA-MB-231 and MCF-7 cells to be 15 µg/ml and 60 µg/ml, respectively. This proof-of-principle study suggests the possibility of the use of microfluidics systems for natural product research. These systems could allow the development of miniaturized multiple parallel cell based assays which are measured with sensors and used for natural product drug discovery consistent with the needs of Industry 4.0.

Background The plant species Rosmarinus officinalis (RO), Thymus algeriensis (TA) and Thymus capitatus (TC) are widely used in traditional medicine in Tunisia. Their bioactivities have been reported before and particularly referred to their essential oils. The main objective of this work was to assess the phytochemical composition, the antioxidant activity, the antibacterial, antifungal, and cytotoxic potential of these 3 plants. Method The High Performance Liquid Chromatography (HPLC), chemical tests and spectrophotometric methods were used for screening, quantification of phytochemicals and for antioxidant activities. Extracts were evaluated for antibacterial potential by the microdilution method. Antifungal activities were tested using the Poisoned food method against: Aspergillus niger and Aspergillus flavus. The cytotoxic potential of the plant extracts was checked using HCT 116 cultures. Results Results revealed that aqueous extracts are not toxic compared to the methanolic extracts. Phenolic compounds were detected and these extracts showed excellent antioxidant activity presenting dose-dependent relationship. For antibacterial potential, all tested strains are more sensitive to Thymus extracts than Rosmarinus extracts. However, for antifungal activities, only Rosmarinus extracts inhibited mycelial growth. HPLC analysis allowed the identification of ten compounds with the abundance of gallic acid. Conclusion This study showed important bioactivities (antioxidant, antimicrobial, and safety potential) of the plant species RO, TA and TC used in traditional medicine.

The present study aimed to evaluate antioxidant and acetylcholinesterase (AChE) inhibitory activities of the ethanolic extracts of six selectedThymusspecies growing in Croatia (T. longicaulis,T. praecoxsubsp.polytrichus,T. pulegioides,T. serpyllumsubsp.serpyllum,T. striatus, andT. vulgaris). Antioxidant effectiveness was assessed using six different assays, in comparison with rosmarinic acid, luteolin, and reference antioxidants. All testedThymusextracts possessed DPPH (IC50= 3–6 μg/mL) and nitric oxide (IC50= 70–177 μg/mL) free radical scavenging activities, strong reducing properties (IC50= 11–15 μg/mL), ferrous ion chelating activity (IC50= 126–389 μg/mL), ability to inhibit lipid peroxidation (IC50= 34–80 μg/mL), and high total antioxidant capacities (238–294 mg AAE/g). AChE inhibitory activity was examined using Ellman's colorimetric method and all tested extracts showed anti-AChE activity in a dose dependent manner. The values of 10–28%, 23–39%, and 64–86% were obtained for tested concentrations of 0.25, 0.5, and 1 mg/mL, respectively. Additionally, the contents of total hydroxycinnamic derivatives, flavonoids, and tannins in dried plant samples were determined spectrophotometrically. Our results highlightedThymusspecies as a rich source of natural antioxidants and AChE inhibitors that could be useful in preventing and treating Alzheimer’s disease and other neurodegenerative disorders.

Aim. To study the efficiency of individual and combined use of thymus extract (tactivin) and vitamine E (tocopherol) for 2,4-dichlorophenoxyacetic acid amine salt hepatotoxicity correction. Methods. Experiments were carried out on 75 white non-imbred mature rats. Animals were distributed to 5 groups 15 animals each: controls, receiving 2,4-dichlorophenoxyacetic acid amine salt, receiving 2,4-dichlorophenoxyacetic acid amine salt and tactivin, receiving 2,4-dichlorophenoxyacetic acid amine salt and tocopherol, receiving 2,4-dichlorophenoxyacetic acid amine salt and both tactivin and tocopherol. 2,4-Dichlorophenoxyacetic acid amine salt (42 mg/kg) was introduced into the stomach during 28 days. Tocopherol (50 mg/ kg) and Tactivin (0,25 mg/kg) were injected from day 1 to day 6 after the discontinuation of toxin administration. Serum levels of alanine aminotransferase, aspartate transaminase, total and direct bilirubin were examined. The liver morphological state was assessed by microscopy of the liver tissue slices stained with hematoxylin and eosin, electron microscopy was also performed.The results were registered on the 7th day after the toxin discontinuation. Results. 2,4-Dichlorophenoxyacetic acid amine salt led to alanine aminotransferase, aspartate transaminase, total and direct bilirubin levels increase. Hemodynamic alterations, reduced number of mitoses, increased number of necrotic hepatocytes, fiber tissue scarring were observed morphologically. The use of tactivin reduced the severity of morphological changes, and decreased, but did not normalized the level of transaminases and bilirubin. The use of tocopherol normalized the level of transaminases, bilirubin, there were morphological signs of the regeneration processes and hepatocyte mitotic activity intensification. The combined use of tactivin and tocopherol normalized alanine aminotransferase, aspartate transaminase bilirubin levels. Morphologically, signs of regenerative processes and protein synthesis activation were observed in hepatocytes. Conclusion. Tocopherol and Tactivin effectively compensate the hepatotoxic effect of 2,4-dichlorophenoxyacetic acid amine salt, the best result was obtained when both of the drugs used in combination.

Two components of the thymic microenvironment have been separated, and their biological properties have been studied. The first component, soluble thymic factor (STF), after injection into syngeneic recipients, provoked a temporary decrease in thymic weight on day 7, concomitant with an increase in the level of T cells in lymph nodes (but not in the spleen), followed by a general hypertrophy of lymph nodes peaking on day 21. In contrast, after the injection of the second component, insoluble thymic fraction (ITF), there was an increase in the number of prothymocytes in the thymic subcapsulary cortex, followed by general thymic hypertrophy on days 14 and 21. STF is believed to trigger a selective migration of lymph-node-seeking T cells from the thymus, whereas ITF seems to play a role in the differentiation step preceding that imparted by STF.

A calf thymus extract was prepared and fractionated into lipid and non-lipid fractions. The non-lipid fraction was isolated from the calf thymus extract using the Folch method. The components isolated from the non-lipid fraction were characterized by IR, NMR, biuret and HPLC method. The results of the analyses indicated the presence of peptides. The lipid fraction contained phospholipids, glycolipids and neutral lipids. The biological activity of both the isolated lipid and peptide fractions was determined by the in vivo hemolytic plaques method in Wistar rats with an involuted thymus. The peptide and phospholipid fractions of the thymus extract showed a significant increase of hemolytic plaques. The glycolipid and neutral lipid fraction failed to express a significant immunological response.

The studies were carried out on Balb/c mice (5-6 weeks of age) exposed to immunosuppression by a single intraperitoneal dose (125 mg/kg) of hydrocortisone. Prior to hydrocortisone injection the mice were treated with diethyldithiocarbamate (DTC) intra-peritoneally at a dose of 20 mg/kg, five times at 48 h intervals or calf thymus extract (TFX) at a dose of 10 mg/kg, 10 times at 24 h intervals. The two drugs were used per se or in zinc ions interactions, by adding zinc ions (as sulphate salt) to drinking water at a dose of 72 microg/mouse per day. The results obtained in the study show that hydrocortisone injection drastically decreases the number of thymocytes and splenocytes, which is also accompanied by a decreasing weight ratio of the thymus and spleen. The decreasing number of thymic and spleen cells corresponds to a decreasing percentage of CD4+, CD8+ and CD19+ splenocytes and double positive CD4+CD8+ thymocytes. Changes in the number of thymic cells affect their activity, which is expressed in a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA). It has also been found that a single hydrocortisone dose decreases interleukin (IL)-1 production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from Escherichia coli. TFX or DTC counteract hydrocortisone-induced immunosuppression, which is expressed in partial normalization of the total number of thymic and spleen cells, accelerated regeneration of the two lymphatic organs, shorter suppressive action of hydrocortisone on the percentage of CD4+, CD8+ splenocytes and double positive (CD4+CD8+) and CD4+ thymocytes. Furthermore, total counteraction against the suppressive action of hydrocortisone to proliferative activity of thymocytes stimulated in vitro with Con A and PHA was observed. TFX administered prior to hydrocortisone injection partially prevented the suppressive action of the drug on IL-1 production by intraperitoneal macrophages, but such an effect was not observed with DTC. The immunorestorative effect of TFX and DTC was augmented by zinc supplementation. The results obtained in the study show that neither TFX nor DTC administration per se and in interaction with zinc supplementation were able to change the suppressive effect of hydrocortisone on the percentage of B splenocytes (CD19+ cells).

A highly active form of human recombinant deoxyguanosine kinase (dGK) phosphorylated purine nucleoside analogs active against cytomegalovirus, hepatitis B virus, and human immunodeficiency virus, such as penciclovir, 2′,3′-dideoxyguanosine and 3′-fluoro-2′,3′-dideoxyguanosine. The antiherpesvirus drug ganciclovir, which is also used in gene therapy, was a substrate for dGK, but with low efficiency. ATP and UTP were both good phosphate donors, with apparent K m values of 6 and 4 μM and V max values of 34 and 90 nmol of dGMP/mg of dGK/min, respectively. With a mixture of 5 mM ATP and 0.05 mM UTP, which represent physiologically relevant concentrations, the activities of dGK with ganciclovir and penciclovir was 1% and approximately 10%, respectively, of that with dGuo. The levels of dGK in different tissues were determined with a selective enzyme assay and the total activities per gram of tissues were similar in liver, brain, heart, and thymus extracts. The fact that the cellular dGK enzyme can phosphorylate antiviral guanosine analogs may help to explain the efficacies and side effects of several forms of chemotherapy.

The level of natural apoptosis in rat thymus on day 18 of embryo development attained 25%, while at subsequent terms it was about 5%. In the spleen, this parameter gradually decreased from 15 to 37% starting from day 18 of embryo development to postnatal day 30. Tactivin prevented the development of dexamethasone-induced apoptosis in thymocytes of 30-day-old rats, but had no effect on spontaneous apoptosis. Tactivin can be used as a modulator of apoptotic processes.

The influence of glucocorticoids on the composition and mineral/organic content of the mandible in tested animals after recovery and healing phase was investigated in this work. The results of FTIR analysis demonstrated that bone tissue composition was changed after glucocorticoid treatment. The increase of calcium, magnesium, phosphorus content and mineral part of bones was statistically significant in recovery phase and in treatment phase that included calcitonin and thymus extract. Some changes also happened in the organic part of the matrix, as indicated by intensity changes for already present IR bands and the appearance of new IR bands in the region 3500–1300 cm−1.

The oxidatively modified DNA base 8-oxo-7,8-dihydroguanine (8-oxoGua) is nontoxic and weakly mutagenic. Here we report on new data suggesting a potential for 8-oxoGua to affect the expression of several genes via epigenetic changes resulting in chromatin relaxation. Using pig thymus extract, we analyzed the distribution of 8-oxoGua among different nuclei fractions representative of transcriptionally active and silenced regions. The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) found in transcriptionally active euchromatin (4.37/10(6) nucleotides) and in the matrix fraction (4.16/10(6) nucleotides) were about 5 times higher than in transcriptionally silenced heterochromatin (0.91/10(6) nucleotides). Other experimental data are presented which suggest that 8-oxoGua present in specific DNA sequences may be widely used for transcription regulation. Like 8-oxoGua, 5-hydroxymethyluracil (5-hmUra) is another oxidatively modified DNA base (the derivative is formed by thymine oxidation). Recent experimental evidence supports the notion that 5-hmUra plays an important role in active DNA demethylation. This involves overexpression of activation-induced cytidine deaminase (AID) and ten-eleven translocation 1 (TET1) protein (the key proteins involved in active demethylation), which leads to global accumulation of 5-hmUra. Our preliminary data demonstrate a significant increase of the 5-hmUra levels in pig brain extract when compared with liver extract. The lack of 5-hmUra in Escherichia coli DNA also speaks for a role of this modification in the active demethylation process. It is concluded that 8-oxodG and 5-hmUra in DNA may be considered as epigenetic marks.

In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide.

Using phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] prepared from phosphatidylinositol 4,5-bisphosphate and inositolphospholipid 3-kinase, we identified in bovine thymus extracts the enzyme activity which catalyzed dephosphorylation of PtdIns(3,4,5)P3, to produce phosphatidylinositol biphosphate. Since bovine thymus exhibited the highest level of activity among tissues screened, we tried to purify this enzyme PtdINs(3,4,5)P3 phosphatase from bovine thymus. After sequential chromatographies using S-Sepharose, heparin-Sepharose, blue Sepharose, and Toyopearl HW55, the enzyme was purified 1875-fold with a yield of 10%. SDS/PAGE analysis revealed that a 120-kDA protein band copurified with the enzyme activity. The apparent molecular mass of the active protein was 120 kDa on size-exclusion chromatography, suggesting that the 120-kDa band on SDS/PAGE is the PtdIns(3,4,5)P3 phosphatase. Since PtdIns(3,4,5)P3 phosphatase seemed to be the only activity that metabolized PtdIns(3,4,5)P3, and the enzyme did not hydrolyze phosphatidylinositol 4,5-biphosphate, the enzyme may play a critical role in the inositolphospholipid 3-kinase signalling.

The human promyelocytic cell line HL-60 can be differentiated with retinoic acid (RA) along the granulocytic pathway. Numerous studies have identified many synergistic combinations of RA with cytostatics, cytokines and other inducers. A combination of RA with the crude thymus extract Thymex-L increased differentiation of HL-60 cells as confirmed by two functional assays and morphology, whereas the extract itself did not show any effect. The functional markers phagocytosis-associated chemiluminescence and nitroblue tetrazolium reduction were more enhanced (up to 4-fold with 1000 micrograms/ml Thymex-L) than morphology. The effect was found over a wide RA concentration range (10(-11) - 10(-6) M) and was dependent on extract concentration. The half-maximal induction of both functional markers was reached at 400 micrograms/ml. To achieve the same effect with the combination in comparison with RA alone, an RA dose reduction of about 100-fold was estimated. The effect was also seen when the cells were pretreated with the thymus extract for two days. The enhancement of RA action by Thymex-L was not correlated with an increase of extracellular or intracellular RA concentration. The active compound in Thymex-L is heat stable and bigger than 5 kDa as confirmed by gelfiltration. The defined thymus peptides thymosin alpha 1, prothymosin alpha 1 and thymopentin were unable to synergistically enhance HL-60 differentiation. These data suggest that the treatment with a thymus extract can increase the sensitivity of HL-60 cells for RA. This may have clinical implications.

胸腺抽出物の高分子画分(HM)および数十種類の酸性または中性のタンパク質で構成されているDEAE吸着画分(DEAE-HM)にはハイブリドーマに対する細胞増殖促進活性があり,抗体産生量も増加させることが明らかになった

Acute poisoning with alcohols and cholinotropic preparations carboxyphosphamide and atropine (0.8 LD(50)) was modeled on male outbred mice weighing 18-24 g. The decrease in activity of natural killer cells was most pronounced after injection of atropine, but insignificant after treatment with ethanol. The inhibitory effect of ethylene glycol, methanol, and methanol on functional activity of natural killer cells in vitro directly depended on their concentration. The effects of alcohols in equimolar concentrations of 10, 100, and 500 mM were similar. Therefore, immunotoxicity of alcohols was associated with the action of their metabolites. The ability of products formed after biotransformation of ethylene glycol, methanol, and ethanol in equimolar concentrations to cause damage to natural killer cells decreased in the following order: glyoxylic acid>formic acid>acetaldehyde>glycolaldehyde>glycolic acid. T-Activin injected subcutaneously in doses of 2.5 and 5.0 microg/kg for 3 days normalized activity of natural killer cells suppressed after acute poisoning with alcohols and cholinotropic preparations.

Poly(ADP-ribose) polymerase responds to DNA strand breaks in nuclei by producing ADP-ribose polymers covalently attached to proteins. Here we report two fast protein liquid chromatographic applications to aid investigations on poly(ADP-ribosyl)ation. The first rapidly purifies poly(ADP-ribose) polymerase from crude calf thymus extract. The purification protocol, involving successive fractionations over four columns, reduces the time for polymerase purification from four days to 14 h resulting in a > 50% increase in enzyme-specific activity. The second application employs a complex salt gradient to reproducibly separate ADP-ribose polymers into individual size classes.

Thymulin (formerly called "Facteur Thymique Sérique or FTS) is a metallopeptidic hormone selectively produced by thymic epithelial cells (TEC) and known to induce intra and extra-thymic T cell differentiation. It was initially isolated from porcine serum and shown to be present in calf thymus extract. Its amino-acid sequence was determined (

Sera from 64 patients with systemic lupus erythematosus (SLE) were tested for the presence of anti-Sm, anti-RNP, and anti-SSB antibodies using commercially available solid-phase enzyme-linked immunosorbent assays (ELISAs) and recombinant nuclear proteins as substrates. The results were compared to those obtained with counterimmunoelectrophoresis (CIE) and immunoblotting (IBT) using a rabbit thymus extract (RTE) as the substrate. The ELISAs detected antibodies to Sm, RNP, and SSB in, respectively, 25%, 36%, and 15% of the SLE sera. Neither IBT-positive/ELISA-negative nor CIE-positive/ELISA-negative sera were found, regardless of the specificity considered, suggesting that ELISAs using recombinant nuclear antigens are highly sensitive. Discrepancies were observed between the results obtained with these different techniques. In addition to sera positive by both IBT and ELISA but negative by CIE, a substantial number of sera had ELISA-detectable anti-Sm, anti-RNP, and anti-SSB antibodies which failed to react with the corresponding polypeptides by IBT. The reasons for ELISA/IBT discrepancies were explored; however, no single explanation was found. Instead, a higher sensitivity of the ELISA to detect antibodies directed against certain polypeptides, the possible inability of IBT using RTE as the substrate to detect antibodies reacting with conformational antigenic determinants, and false-positive reactions in the ELISAs were suggested. Thus, it is still advisable to perform both IBT and ELISAs simultaneously in well-defined autoimmune diseases to further analyze the potential advantages of ELISAs using recombinant antigens.

Exogenous antioxidants, e.g. tocopherol, prevent undesirable changes in the thymus and accelerate its recovery after intensive physical exercise. Four weeks after the end of training (swimming) the general structure of the thymus and content of LPO products in rats treated with tocopherol corresponded to the control values, in contrast to animals receiving no correction.

Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)

Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides, reported within the central nervous system, which have very high specificity and affinity for the mu-opioid receptor. We have used newly developed and well-characterised radioimmunoassays (RIAs) in combination with reversed-phase high-performance liquid chromatography (HPLC) to detect EM-1 and EM-2 immunoreactivity (ir) in rat immune tissues. Endomorphins were detectable in extracts of rat spleen (total EM-1-ir/spleen: 440+/-73 pg, mean+/-SEM, a=group of eight rats; EM-2-ir: 150+/-12 pg) and thymus (EM-1-ir: 152+/-18 pg, mean+/-SEM n=8; EM-2-ir: 156+/-28 pg). EM-2-ir was detectable in extracts of human spleen (338+/-196 pg/g tissue, n=3). Multiple peaks of EM-1-ir and EM-2-ir were observed in rat spleen and thymus extracts, and multiple peaks of EM-2-ir were observed in extracts of human spleen, following reversed-phase HPLC and RIAs. This is the first report of endomorphin immunoreactivity in tissues of the rat and human immune systems.

Apoptosis involves a cascade of biochemical events that is tightly regulated. Induction of apoptosis in tumor cells is a promising approach to curing cancer. Therefore, new and safe treatment methods that maximize cancer cells apoptosis are therefore highly sought after. In this study, we examined the effect of gross thymus extract (GTE) combined with freeze dried, heat-killed Lactobacillus kefiri P-IF in the induction of apoptosis in human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and evaluated the mechanism underlying its effect. L. kefiri P-IF is a specific strain of lactic acid bacteria that has a unique DNA sequence and PET scans show a 99.6% homology with regular kefiris. To identify any synergistic effect of these two products, tumor cells were cultured for three days with 1) GTE at a concentration of 1.25 mg/ml or 2) L.kefiri P-IF at a concentration of 1.25 mg/ml or 3) a combination of both products at the same concentrations. The apoptotic response was assessed by propidium iodide assay and expression of Bcl2, anti-apoptotic protein, was determined by flow cytometry. Results showed that GTE and L.kefiri P-IF induced apoptosis in HL60/AR cells; GTE caused 37% apoptosis while L.kefiri P-IF caused 17% apoptosis, as compared to 7% for control cancer cells without treatment. When cancer cells were cultured in the presence of both GTE and L.kefiri P-IF, 60% apoptosis of cancer cells was detected. This data was confirmed by giemsa stained cytospin preparation. The synergistic apoptotic effect was associated with decreased expression of Bcl2. MFC: 74, 68, 61 and 50 for control untreated cells, GTE, L.kefiri P-IF and GTE + kefiri P-IF respectively. Atomic Force Microscopy studies were performed and showed clear signs of apoptotic cancer cells including membrane blebbing. These results demonstrate that GTE + L.kefiri P-IF induces apoptosis on cancer cells that exceed the effect of either agent alone, and this agent may represent a new class of adjuvants for the treatment of leukemia. GTE (Thymax) was provided by the YS Nature Company, Tokyo, Japan; L.kefiri P-IF was provided by Paitos Co., Ltd. Yokohama, Kanagawa, Japan. Citation Format: Mamdooh H. Ghoneum, Mark Henary, Yoshikazu Seto. Apoptotic effect of thymus extract plus Lactobacillus kefiri P-IF on human myeloid leukemia (HL60/AR) cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3294. doi:10.1158/1538-7445.AM2013-3294 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Thymostimulin is a partially purified extract of calf thymus, consisting of a group of polypeptides with a total molecular weight of approximately 12,000 D. It is used as an immunologic response modifier in primary and secondary immunodeficiencies. We report a patient who had a severe anaphylactic reaction with the first injection of thymostimulin. Type I hypersensitivity to thymostimulin was suggested by an immediate skin test response, specific IgE determination by indirect ELISA and a positive histamine release test. This patient also reacted to bovine serum albumin (BSA), fetal calf serum (FCS) and cow hair and dander. The controls did not react to any of these tests. Clinicians need to be aware that the use of medical products of xenogeneic origin may lead to severe allergic reactions.

Thymopoietin, a polypeptide hormone of the thymus that has pleiotropic actions on the immune, endocrine, and nervous systems, potently interacts with the neuromuscular nicotinic acetylcholine receptor. Thymopoietin binds to the nicotinic alpha-bungarotoxin (alpha-BGT) receptor in muscle and, like alpha-BGT, inhibits cholinergic transmission at this site. Evidence is given that radiolabeled thymopoietin similarly binds to a nicotinic alpha-BGT-binding site within the brain and does so with the characteristics of a specific receptor ligand. Thus specific binding to neuronal membranes was saturable, of high affinity (Kd = 8 nM), linear with increased tissue concentration, and readily reversible; half-time was approximately 5 min for association and 10 min for dissociation. Binding of 125I-labeled thymopoietin was displaced not only by unlabeled thymopoietin but also by alpha-BGT and the nicotinic receptor ligands d-tubocurarine and nicotine; various other receptor ligands (muscarinic, adrenergic, and dopaminergic) did not affect binding of 125I-labeled thymopoietin. Thymopoietin was shown by ELISA to be present in brain extracts, displacement curves of thymus and brain extracts being parallel to the standard thymopoietin curve, and Western (immuno) blot identified in brain and thymus extracts a thymopoietin-immunoreactive polypeptide of the same molecular mass as purified thymopoietin polypeptide. We conclude that thymopoietin and thymopoietin-binding sites are present within the brain and that the receptor for thymopoietin is the previously identified nicotinic alpha-BGT-binding site of neuronal tissue.

Mutagenesis research has been focused for a long period on the interaction between mutagens (mostly X-rays) and the cell structure modified by them. Subsequently, the emphasis was shifted to mutation as a cellular process in which the primary lesion was extensively modified by enzymatic DNA-repair systems constitutively present in the cell (Auerbach 1976). Finally, over the past 15 years, considerable interest has been focused on inducible cellular functions (possibly DNA repair activities) occurring in the cell after exposure to very low doses of mutagens, probably through DNA lesions (for reviews, see Walker 1985; Elespuru 1987). DNA-damage-inducible processes are probably part of a set of general biological phenomena by means of which the cells and the organism respond to various types of stress (Luckey 1980; McClintock 1984; Lindquist 1986; Morgan et al. 1986). When stress is represented by a mutagenic agent one of the most interesting responses is the adaptive response (AR; Samson and Cairns 1977). The mechanisms underlying the AR are reasonably well known in prokaryotes in which they were discovered (Sekiguchi and Nakabeppu 1987; Rebeck et al. 1988). In plant (Rieger et al. 1982; Heindorff et al. 1987) and animal cells (Samson and Schwartz 1980, 1983) investigation is focused mainly on the reduction of the yield of induced chromatid aberrations by various pretreatment procedures (clastogenic adaptation).

Combined DEAE-Sephacel and hydroxyapatite chromatography resulted in a separation of free Sm antigen from the Sm/RNP complex in rabbit thymus extracts. In Western blots, the free Sm preparation contained an immunoreactive 14-kd (D) protein, whereas the Sm/RNP complex contained, in addition to the 14-kd protein, a 68-kd-reactive protein and its putative degradation fragments. Ethidium bromide staining of these preparations separated by agarose gel electrophoresis showed that the Sm/RNP preparation contained RNA, but the free Sm preparation did not. These preparations were used as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera characterized as anti-Sm only, anti-Sm/RNP, and anti-RNP only were assayed. A quotient (Q) was derived from the ELISA optical density obtained with Sm/RNP as antigen divided by the optical density obtained with free Sm as antigen. Q values less than 4.0 characterized sera with anti-Sm only, values of 4.0-12.0 were observed in sera with anti-Sm plus anti-RNP, and values greater than 12.0 in sera with anti-RNP only. Sera with antibodies to other nuclear antigens were not reactive in this system. It has traditionally been difficult to identify sera with anti-RNP when this is present simultaneously with, and in lower concentration than, anti-Sm. With this method, such sera can be identified by their Q value, which falls in an intermediate region between a lower Q for anti-Sm and a higher Q for anti-RNP.

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [3H]thymidine incorporation. The reliability of the method was tested with l -alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [3H]thymidine was stimulated with disks containing l -alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l -alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.

A novel DNA polymerase, which could use both poly(rA) · oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocusing ( p I = 6.5–7.0 ) or sucrose rate-zonal centrifugation (10–10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) · oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) · oligo(dT)-dependent activity differed from the previously described DNA polymerase γ from other sources in the following ways: 1. The activity was inhibited by 100–300 mM KCl and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26°C than at 37°C. 3. The K m value for dTTP was 2.6–3.0 · 10−4 M, which is several hundred-fold greater than that of DNA polymerase γ. 4. Mn2+ was essential for the reaction and could not be replaced by Mg2+. The activated DNA-dependent activity shared many properties with DNA polymerase α, except that it was less sensitive to N- ethylmaleimide and anti-α polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.

Serum anti-ribonucleoprotein (RNP) antibody levels were measured using an enzyme linked immunosorbent assay (ELISA) technique. Paper discs, activated by CNBr and coupled covalently with rabbit thymus extract (RTE), were used for the solid phase of ELISA.Twenty serum samples containing antibodies specific only to the RNP antigen, determined by double immunodiffusion, were examined by ELISA. The sera were obtained from patients with MCTD, SLE and PSS. The samples were measured for anti-RNP antibody levels in each immunoglobulin class. It was found that most of the anti-RNP antibodies were IgG class and the rest were IgA class. No IgM or IgE anti-RNP antibodies were detected in this study.To determine the absolute concentration of anti-RNP antibodies in sera, the IgG fraction of a sample from a patient with MCTD was labelled with N-succinimidyl [2, 3-3H] propionate ([3H] NSP). Then, Sepharose 4B coupled with RTE was reacted with the [3H] NSP-labelled IgG, and non-binding [3H] NSP-labelled IgG was removed by washing. The radioactivity of the Sepharose 4B obtained was measured and the absolute concentration of anti-RNP antibodies in serum was calculated from the binding ratio of the radioactivities of the entire [3H] NSP-labelled IgG to that bound with Sepharose 4B. Thus, the IgG anti-RNP anibody concentration of the IgG fraction was determined. The IgG fraction was sequentially diluted, and the anti-RNP antibody levels of the fractions were measured by ELISA. A dose response cure was obtaned from these values. The concentration of anti-RNP antibodies of twenty serum samples was determined from each antibody level by ELISA, using the dose response curve. The concentration of anti-RNP antibodies in sara tested ranged from 0.16 to 3.98mg/ml.

A factor in ultraftitrated (UM 10) extracts of calf thymus, liver and spleen stimulated the uptake of 3H-thymidine in DNA of cultured lymphocytes. The factor was purified by ion-exchange chromatography and gel chromatography and identified as alanine, which is lacking in the culture medium, RPMI 1640. An optimal amount of L-alanine increased the uptake of 3H-thymidine in DNA of lymphocytes from different species by about 100%. L-alanine should be regarded as a growth factor for lymphocytes in vitro and should be added to RPMI 1640, when this tissue culture medium is used for lymphocytes.

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in a variety of eukaryotic cells and shown to differ in the molecular weight of their largest subunit, designated IIo, IIa, and IIb, respectively. The objectives of this study were to establish the in vivo molecular structure of RNA polymerase II in mammalian cells and to examine conditions that influence the stability of RNA polymerase II subspecies. Subunit affinity-purified antibodies were used to determine the relative concentration of subunits IIo, IIa, and IIb in crude extracts of calf thymus tissue, cultured bovine kidney cells, and HeLa cells. HeLa cells contain exclusively RNA polymerase IIO whereas both cultured bovine kidney cells and calf thymus tissue contain RNA polymerases IIO and IIA. RNA polymerase IIB was not detected at significant levels in any of the cell extracts examined. Cell extracts were aged at either 4 degrees or 37 degrees C and the stability of RNA polymerases IIO and IIA determined by protein blotting. In the presence of buffer normally used for RNA polymerase purification, subunit IIo disappears from calf thymus extracts within 24 h at 4 degrees C or within 5 min at 37 degrees C. RNA polymerase IIO is partially stabilized by the inclusion of protease inhibitors and further stabilized by the presence of relatively high concentration of EDTA and EGTA. The prior fractionation of nuclei does not have an appreciable effect on RNA polymerase II stability. An increase in the amount of reducing agent causes a dramatic reduction in the stability of subunit IIo. The following manuscript (Bartholomew, B., Dahmus, M. E., and Meares, C. F. (1986) J. Biol. Chem. 14226-14231) examines the transcriptional activity of RNA polymerases IIO and IIA in reactions dependent on the major late promoter of adenovirus-2. Photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription extract, indicates that the transcriptional activity of RNA polymerase IIO is greater than 10 times that of IIA.

Thymus humoral factor (THF), a thymus hormone which participates in the processes leading to acquisition of immunocompetence of lymphoid cells has been isolated in our laboratory by a stepwise gel filtration through various Sephadex columns. THF so isolated appears to be a polypeptide of 3,000 mol wt which contains approximately 30 amino acid residues. Here we have tested the biological activity of THF fractions of successive degrees of purity upon lymphoid cells from both intact and neonatally thymectomized mice. The lymphoid cell populations were treated with the various THF fractions by in vitro incubation for a short time and by repeated injection in vivo. The treated cells evidenced increased ability to react in the graft-versus-host assay in vivo and in mixed lymphocyte cultures in vitro concomitantly with the rise of intracellular cAMP. On the other hand no activity whatsoever was shown by any of the control materials tested. These bioassays permitted isolation of fractions progressively more active than the original crude dialyzate of thymus extract tested. Thus the active peptide component of THF eluted from DEAE Sephadex A-25 column was estimated to be 2 X 10(4)-fold more active than the crude dialyzate of thymus extract which served as a starting material.

A peptidic effector from calf thymus causes a strong stabilization of DNA double-stranded molecule in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (less than 5000). The biological activity of the thymic factor cannot be attributed to a histone fragment. Melting data of the control DNA and of the DNA-active factor complex in various conditions of ionic strength and dielectric constant of the solution medium are recorded.

The major NaCl-stimulated protein-tyrosine kinase activity found in soluble thymus extracts, as measured by the phosphorylation of angiotensin I, is a 40-kDa enzyme known as p40 (Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986) J. Biol. Chem. 261, 15637-15643). Antibodies prepared against p40 cross-react with a 72-kDa protein-tyrosine kinase (p72) from spleen or thymus that is closely related to p40 by peptide-mapping experiments. The recovery of p40 from spleen homogenates is reduced while the recovery of p72 is enhanced by the addition of high concentrations of leupeptin or soybean trypsin inhibitor to the homogenization media. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with trypsin or papain. The p72 protein-tyrosine kinase is found at the highest levels in spleen, thymus, and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 following homogenization. p72 is also found in certain T and B cell-derived cell lines and in NIH3T3 cells.

Antibodies to the RNase-sensitive RNP and to the RNase-resistant Sm nuclear antigens were used to affinity purify these antigens from a saline extract of rabbit thymus acetone powder. Determination of the protein subunits recovered by either glycine-HCl, pH 2.8, or 2.5 M MgCl2 elution on gradient sodium dodecyl sulfate-polyacrylamide electrophoresis containing mercaptoethanol revealed that RNP was composed of five proteins with mol. wts from 10,000 to 15,000 whereas Sm contained the same or similar five chains plus six additional subunits with mol. wts from 21,000 to 42,000. RNase treatment of the thymus extract increased the recovery in Sm of the same bands compared to untreated extract. Thus, RNP and Sm appear to have different numbers of protein components and RNP may be a subset of Sm. Sucrose gradient centrifugation of the 125I-labeled, pH 2.8 eluted antigens gave peaks of 3 and 6S for RNP and Sm, respectively. Sucrose gradient centrifugation of the crude untreated thymus extract followed by quantitative single radial immunodiffusion analysis of each fraction produced a broad peak from 16S to the top of the gradient while pretreatment of the extract with RNase resulted in a discrete 6S peak. These results indicate that in rabbit thymus acetone powder native RNP and Sm exist as larger polydisperse complexes with additional material including RNA and that after acid elution or RNase treatment the antigens are found in a smaller monodisperse form.

In tierexperimentellen Untersuchungen wurde die Eignung von 2-Cyanathylharnstoff, Thymus-Extrakt und Lithiumkarbonat zur potentiellen Verminderung bzw. Verkurzung der Leukozytendepression nach Ganzkor

An HPLC system for quickly isolating individual peptides from thymus extract is described. A series of reversed phase separations followed by ion-exchange HPLC produces pure peptides with minimal chromatographic time. A 2.4 gram column load of crude extract was carried through to the isolation of 4 mg of pure thymosin α1 within 6–8 hours of chromatography.