Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination.

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Isolation of the pituitary gonadotrophic α-subunit hormone of the giant amazonian fish: pirarucu (Arapaima gigas).

The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.

Novel series of (177)Lu-labeled bombesin derivatives with amino acidic spacers for selective targeting of human PC-3 prostate tumor cells.

AIM: Bombesin (BBN) has demonstrated the ability to bind with high affinity and specificity to GRP receptor, overexpressed on human prostate cancer. A large number of BBN derivatives have been synthesized for this purpose but most of them exhibit high abdominal accumulation, which may represent a problem in their clinical use due to serious side effects to patients. In this study we describe the results of radiolabeling with lutetium-177, stability and in vivo studies of novel phenyl-glycine-extended bombesin derivatives. The spacers were inserted to improve bombesin in vivo properties and to reduce its target to non-tumor sites. METHODS: Preliminary studies were done to establish the ideal conditions for labeling bombesin derivatives. Chromatography systems were applied to determine free lutetium and the stability of the preparations was evaluated either after storing at 2-8 ºC or incubation in human serum at 37 ºC. In vivo experiments included biodistribution, pharmacokinetics and SPECT images and were performed in Balb-c and Nude mice bearing PC-3 xenografts. RESULTS: The derivatives were labeled with high yield and kept stable at 2-8 ºC and are metabolized by human serum enzymes. In vivo studies showed fast blood clearance of labeled peptides and rapid excretion, performed mainly by renal pathway. In addition, biodistribution and imaging studies showed low abdominal accumulation and significant and specific tumor uptake of (177)Lu-labeled derivatives. CONCLUSIONS: The derivative with longer spacer holds a higher potential as radiopharmaceutical for prostate tumor diagnosis and the derivatives with shorter spacers are potential radiopharmaceuticals for prostate tumor treatment.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells.

Human prolactin (hPRL) is a 199 aminoacid protein hormone with a wide spectrum of biological activities which is best known for its stimulation of lactation and development of mammary gland. This protein contains only one potential asparagine-linked glycosylation site, which is partially (10-30%) occupied when the protein is synthesized in eukaryotic cells. Although the biological activity of glycosylated hPRL (G-hPRL) has been found to be approximately 4-fold lower than that of hPRL, its physiological function is not yet well defined. In order to better characterize and study this hormone variant, we carried out its laboratory scale purification from conditioned medium of genetically modified CHO cells that had been supplemented with cycloheximide. Addition of cycloheximide increased the absolute concentration of G-hPRL approximately 4-fold and the glycosylated versus non-glycosylated hPRL concentration ratio by approximately 7-fold. G-hPRL purification was carried out via a two-step process based on a cationic exchanger and a size-exclusion HPLC (HPSEC) column. Characterization was carried out by HPSEC, Western blotting, MALDI-TOF-MS and in vitro bioassay based on Nb2 and Ba/F3-LLP cells, the biological activity being of the same order (11-15 IU mg(-1)) in the two assays. Our results show that addition of cycloheximide can be an important strategy for increasing glycosylated protein production, facilitating the purification and characterization of these isoforms.

Lymphocytic prolactin does not contribute to systemic lupus erythematosus hyperprolactinemia.

OBJECTIVES: Lymphocytic prolactin (PRL) gene expression is detected in the majority of the immune cells and it is not known if this source contributes to hyperprolactinemia in systemic lupus erythematosus (SLE). We have therefore evaluated lymphocytic PRL secretion and gene expression in SLE and healthy controls. METHODS: Thirty SLE patients (ACR criteria) and 10 controls were selected for the study. Serum levels of PRL and macroprolactin were detected by immunofluorometric assay and gel filtration chromatography, respectively. The lymphocytic biological activity was determined by Nb2 cells bioassays. Lymphocytic PRL gene expression was evaluated by RT-PCR assay. RESULTS: The median serum PRL levels of the 30 SLE patients was higher than the control group (9.65 (1.9-38.9) vs. 6.40 (2.4-10.3) ng/mL, p=0.03). A significant difference was detected between median serum PRL levels of active SLE, inactive SLE and controls (10.85 (5-38.9) vs. 7.65 (1.9-15.5) vs. 6.40 (2.4-10.3) ng/mL), p=0.01). The higher frequency of mild hyperprolactinemia was detected among active SLE in comparison with inactive SLE and controls (7 (38.9%) vs. 1 (8.3%) vs. 0 (0%)), with statistical significance (p=0.02). Nb2 cells assay revealed uniformly low levels of lymphocytic PRL in active, inactive and control groups without statistical significance among them (24.2 (8-63) vs. 27 (13.6-82) vs. 29.5 (8-72) ng/mL), p=0.84). Furthermore, median lymphocytic PRL gene expression evaluated by RT-PCR assay was comparable in both active and inactive SLE groups (p=0.12). CONCLUSIONS: This is the first study to exclude a lymphocytic source of PRL, pointing out a pituitary etiology for hyperprolactinemia in SLE. However, other sources from the immune system cannot be ruled out.

Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields.

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).

Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in Chinese hamster ovary (CHO) cells.

The synthesis, purification and characterization of G129R-hPRL and S179D-hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the first time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 microg/10(6) cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the specific denaturation, refolding and purification conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step purification process and included SDS-PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-flight mass spectral (MALDI-TOF-MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). This last technique revealed a considerable difference in hydrophobicity due to a single amino acid substitution, with S179D-hPRL less (t(RR) = 0.85 +/- 0.010) and G129R-hPRL more (t(RR) = 1.10 +/- 0.013) hydrophobic than hPRL, where t(RR) is the relative retention time. The biological characterization was based on further refinement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 x 10(-3) for S179D-hPRL and 70 x 10(-5) for G129R-hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting effects of hPRL in vivo in animal models of breast and prostate cancer.