Performance Gel Filtration Chromatography Research Articles

Structural characteristics, immune-activating mechanisms in vitro, and immunomodulatory effects in vivo of the exopolysaccharide EPS53 from Streptococcus thermophilus XJ53

Our previous investigations have successfully identified the repeating structural units of EPS53, an exopolysaccharide derived from Streptococcus thermophilus XJ53 fermented milk, and substantiated its potential immunomodulatory properties. The present study further elucidated the structural characteristics of EPS53 and investigated the underlying mechanisms governing its in vitro immunoreactivity as well as its in vivo immunoreactivity. The results obtained from multi-detector high performance gel filtration chromatography revealed that EPS53 adopted a rigid rod conformation in aqueous solution, with the weight-average molecular weight of 1464 kDa, the number-average molecular weight of 694 kDa, and the polydispersity index of 2.11. Congo red experiment confirmed the absence of a triple helix conformation. Scanning electron microscopy showed that EPS53 displayed a three-dimensional fibrous structure covered with flakes. The in vitro findings indicated that EPS53 enhanced phagocytosis ability, reactive oxygen species (ROS) production, and cytokine levels of macrophages via the TLR4-mediated NF-κB/MAPK signaling pathways as confirmed by immunofluorescence staining experiments, inhibition blocking experiments, and Western blot assay. Additionally, the in vivo experiments demonstrated that EPS53 significantly increased macrophage and neutrophil number while enhancing NO and ROS levels in zebrafish larvae; thus, providing further evidence for the immunomodulatory efficacy of EPS53.

Biophysical Characterization of the C-Terminal Tail of T. rubrum PacC Reveals an Inherent Intrinsically Disordered Structure with pH-Induced Structural Plasticity.

PacC is a key transcriptional regulator of human pathogenic fungus Trichophyton rubrum with pivotal roles in pH homeostasis and virulence. We report the first biophysical characterization of the C-terminal inhibitory tail of PacC, pertinent to its physiological role in maintaining the inactive state of PacC at acidic pH which undergoes conformational changes for its proteolytic removal and activation, at alkaline pH. To gain insights into the structural features of PacC that enable the required conformational flexibility, we performed gel filtration chromatography, dynamic light scattering, circular dichroism, and 1-anilino-8-naphthalenesulfonate binding and showed that the tail exhibits properties similar to intrinsically disordered proteins, as also predicted by bioinformatics tools. We demonstrate that the C-terminal tail is conformationally flexible and attains a molten globule-like state at extremely acidic pH and undergoes biphasic GdmCl-induced unfolding in a noncooperative manner with an intermediate X state. We hypothesize that the conformational plasticity of the C-terminal tail of PacC may play a significant role in modulating its pH-dependent transcriptional activation.

Analysis of the polymerized impurities in cefmetazole sodium based on novel separation principle by liquid chromatography tandem ion trap/time-of-flight mass spectrometry

To effectively control the polymerized impurities in cefmetazole sodium, novel high performance gel filtration chromatography (HPSEC) with TSK-gel G2000SWxl column and RP-HPLC method with C18 column were used in replace of classical gel filtration chromatography with Sephadex G-10 gel. By studying the chromatographic behavior of polymerized impurities in both chromatographic systems with different chromatographic separation principles, the polymerized impurities in cefmetazole sodium were separated and detected effectively. The two-dimensional liquid chromatography tandem ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) was applied to characterize the structures of polymerized impurities eluted from HPSEC method, and liquid chromatography tandem ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and other unknown impurities eluted from RP-HPLC method. The structures of fourteen unknown impurities in cefmetazole sodium were deduced based on the MS n data, nine of which were polymerized impurities. The corresponding relationship between impurities in the HPSEC method and RP-HPLC method was established, and the specificity of the two methods was evaluated. The RP-HPLC method for analysis of the polymerized impurities has higher column efficiency and specificity than the HPSEC method. The RP-HPLC method is suitable for quality control of the polymerized impurities in cefmetazole sodium. The forming mechanisms of degradation impurities in cefmetazole sodium were also studied.

High-Efficiency Production of Auricularia polytricha Polysaccharides Through Yellow Slurry Water Fermentation and Its Structure and Antioxidant Properties.

Yellow slurry water is a kind of nutrient-rich wastewater of tofu. Firstly, the medium of yellow slurry was optimized. Then, APP40, APP60, and APP80 were obtained by sedimentation with different concentration of ethanol (40, 60, and 80%). The physicochemical properties and primary structures of the three polysaccharides were studied by high performance anion exchange chromatography (HPAEC), high performance gel filtration chromatography (HPGFC), scanning electron microscope (SEM), atomic force microscope (AFM), and Fourier transform infrared (FT-IR) spectrometer. Finally, the effects of three polysaccharides on antioxidation activity were studied. According to the experimental optimization the results, the biomass and the production of Auricularia polytricha Polysaccharides (APPS) reached the peak, and they were 13.5 ± .655 and 9.42 ± .253 g/L (p < .05). The SEM and the AFM showed that the height of APP80 gradually increased from 31.1 to 46.7 nm and from APP40 to APP80. The particle size of APP80 increased, the pores decrease or even disappear, and the molecules begin to aggregate. The FT-IR spectrum analysis showed that the three polysaccharides possessed key functional groups. The carbohydrate content of APP40, APP60, and APP80 was 20.2, 34.25, and 31.73%. The molecular weights of APP40, APP60, and APP80 are 9.462 × 104, 8.742 × 104, and 8.091 × 104 Da, respectively. The three polysaccharides were composed of rhamnose, galactose, glucose, mannose, and xylose but with different molar ratio. APP80 showed strong reducing ability and scavenging activity of 2,2–diphenyl–1–picrylhydrazyl (DPPH) and hydroxyl radicals through antioxidant activities evaluated in vitro. This study introduces a way for the effective use of yellow slurry water.

Multiple-fingerprint analysis of Poria cocos polysaccharide by HPLC combined with chemometrics methods

In this study, the multiple fingerprints, which were integrated with HPGFC-ELSD (high performance gel filtration chromatography - evaporative light scattering detector) fingerprint, PMP-HPLC-DAD (1-phenyl-3-methyl-5-pyrazolone-high performance liquid chromatography - diode array detector) fingerprint of complete acid hydrolysates and HILIC-HPLC-ELSD (hydrophilic interaction - high performance liquid chromatography - evaporative light scattering detector) fingerprint of enzyme hydrolysates, were established to evaluate the quality of polysaccharides from Poria cocos (PCPs). The similarity evaluation showed that 16 batches of PCPs from different origins had high similarity in structural characteristics based on the multiple fingerprints. The chromatographic data of multiple fingerprints of PCPs were fused, processed and analyzed by chemometric methods including HCA (hierarchical cluster analysis), PCA (principal component analysis) and PLS-DA (partial least squares discriminant analysis). The 16 batches of PCPs were divided into 3 categories in PCA, indicating a certain relationship between the structural characteristics and the origins. PLS-DA analysis indicated that Man, Glc, Gal, Fuc, the components with m/z of 2.22 × 104∼1.53 × 104 Da and 3.46 × 103∼2.69 × 103 Da, oligosaccharides with DPs of 6 and 7, respectively, could be regarded as potential chemical markers for the classification of PCPs from different origins. According to the multiple fingerprints and chemometric analysis, the two commercial samples were proved to be adulterants.

Structural determination of group A Streptococcal surface dehydrogenase and characterization of its interaction with urokinase-type plasminogen activator receptor

Streptococcus pyogenes (group A Streptococcus, GAS) has caused a wide variety of human diseases. Its multifunctional surface dehydrogenase (SDH) is crucial for GAS life cycle. Furthermore, GAS infection into human pharyngeal cells has been previously shown to be mediated by the interaction between SDH and host urokinase-type plasminogen activator receptor (uPAR). However, the structural information of SDH remains to be elucidated and there are few detailed studies to characterize its interaction with uPAR. In-depth research on these issues will provide potential targets and strategies for combating GAS. Here, we prepared recombinant SDH tetramer in Escherichia coli BL21 (DE3) cells. After purification and crystallization, we determined its crystal structure at 1.74 Å. The unique characteristics might be potentially explored as drug targets or vaccine immunogen. We subsequently performed gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and in vitro pull-down analyses. The results showed that their interaction was too weak to form stable complexes and the role of uPAR involved in GAS infection needs further demonstration. Altogether the current work provides the first view of SDH and deepens the knowledge of GAS infection.

Structure Characterization of Honey-Processed Astragalus Polysaccharides and Its Anti-Inflammatory Activity In Vitro.

Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which strengthens the tonic effect. Astragalus polysaccharide (APS), perform its immunomodulatory effects by relying on the tonic effect of Radix Astragalus, therefore, the improved pharmacological activity of honey-processed Astragalus polysaccharide (HAPS) might be due to structural changes during processing. The molecular weights of HAPS and APS were 1,695,788 Da, 2,047,756 Da, respectively, as determined by high performance gel filtration chromatography combined with evaporative light scattering detection (HPGFC-ELSD). The monosaccharide composition was determined by ultra-performance liquid chromatogram quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) after pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). The results showed that the essential components were mannose, glucose, xylose, arabinose, glucuronic acid and rhamnose, is molar ratios of 0.06:28.34:0.58:0.24:0.33:0.21 and 0.27:12.83:1.63:0.71:1.04:0.56, respectively. FT-IR and NMR analysis of HAPS results showed the presence of uronic acid and acetyl groups. The anti-inflammatory activities of HAPS were more effective than those of APS according to the NO contents and the expression of IFN-γ, IL-1β, IL-22 and TNF-α in lipopolysaccharide (LPS)-induced RAW264.7 cells. This findings suggest that the anti-inflammatory and bioactivity improvement might be associated with molecular structure changes, bearing on the potential immunomodulatory action.

Relative molecular mass and monosaccharide composition of polysaccharide in Dendrobium officinale flowers

In order to provide new information on polysaccharide in Dendrobium officinale flowers,the monosaccharide composition and relative molecular mass distribution of 11 families of flowers were investigated and analyzed by high performance gel filtration chromatography (HPGFC) and pre-column derivatization ultra performance liquid chromatography (UPLC) in this study. Then cluster analysis was carried out for the monosaccharide peak areas by utilizing SPSS 19.0 software. The results showed that the polysaccharides of all the above 11 hybrid families of D. officinale flower were separated into three fractions (DOP-1, DOP-2 and DOP-3) with the average relative molecular mass of 5.53×105, 3.49×105 and 2.12×105. The polysaccharides in 11 different families were mainly composed of glucose, mannose, galactose, galacturonic acid and arabinose; mannose had the highest proportion among them, with mannose/glucose ratio of 0.302-3.335. Additionally, the relative contents of various monosaccharides in different families varied. 11 families of D. officinale flower could be classified into four categories according to their monosaccharide components and relative contents. In this study, the relative molecular mass distribution and monosaccharide composition of polysaccharides in D. officinale flowers were defined, which can provide foundations for its resource utilization．.

Discussion and Analysis of &lt;em&gt;Flammulina velutipes Polysaccharides&lt;/em&gt; Compositions, Molecular Weight and Monosaccharide Composition

The aim of the present work was to explore the composition, molecular weight and monosaccharide composition of ,Flammulina velutipes polysaccharides by DEAE Cellulose-52, High Performance Gel Filtration Chromatography (HPGFC) and HPLC with PMP derivative with the FVP isolated and purified from Flammulina velutipes. The results showed that Flammulina velutipes polysaccharides were composed of three kinds of polysaccharides; the molecular weight of FVP was composed of 4191338, 372779, 19002; the molecular weight of 372779, 19002, one of the biggest average molecular weight polysaccharides is neutral polysaccharide, the rest of the two kinds of polysaccharide composition of less average molecular weight is acidic polysaccharides and the proportion of two kind of acidic polysaccharides significantly greater than neutral polysaccharide, 44.31 and 37.76% respectively, only 17.93% of neutral polysaccharide. FVP was composed of Glc, Man, Gal, Xyl, Fuc; the highest content of Glc, followed by Gal and Man, Xyl and Fuc content is relatively low. The molar ratio was relatively 13.05:2.75:3.16:1.48:1.00. In conclusion, FVP contained the more Glc, the less Fuc and a small amount of galacturonic acid and glucuronic acid, which showed FVP may contain acidic polysaccharides.

Isolation, purification, characterization and immunostimulatory activity of polysaccharides derived from American ginseng

In this study, crude American ginseng polysaccharide (AGPS) was extracted with hot water and preliminarily purified by using resin S-8 and Polyamide columns. Then, it was further purified and separated by DEAE-Sepharose CL-6B and Sepharose CL-6B chromatography, respectively. Five main fractions were obtained, named WPS-1, WPS-2, SPS-1, SPS-2 and SPS-3. Their homogeneities and structural characteristics were elucidated based on UV–vis spectroscopy, High Performance Gel Filtration Chromatography (HPGFC), Gas Chromatography (GC), Scanning Electron Microscopy (SEM), Infrared Spectrum (IR), and NMR Spectroscopy methods. Furthermore, the immunostimulatory effects of these fractions upon splenic lymphocyte proliferation, macrophage phagocytosis and nitric oxide (NO) production, were investigated in vitro. The results indicated that their stimulations could be ordered as SPS-3>SPS-1>CPS (crude polysaccharides)>WPS-1>WPS-2>SPS-2. Among them, SPS-3 showed more potent immunomodulatory activity and could be explored as a potential immunopotentiating agent for use in functional food or medicine.

Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter

The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240g DCW/L of biomass level and 96IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53mg/L of recombinant bovine chymosin production after 120h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800IMCU/Total Abs280nm and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.

Role of levoglucosan physiochemistry in cellulose pyrolysis

The fate of levoglucosan after it forms during cellulose pyrolysis was investigated experimentally using time-resolved thermogravimetric analysis/differential scanning calorimetric combined with mass spectrometry measurements followed by high performance liquid chromatography and gel filtration chromatography of the pyrolysis residue. This study indicates that levoglucosan formed during cellulose pyrolysis is initially a liquid that undergoes two simultaneous, competing processes of evaporation and polymerization. Levoglucosan that evaporates escapes the high temperature pyrolysis zone while levoglucosan that polymerizes is trapped in the pyrolysis zone and dehydrates to low molecular weight volatile products and char. The oligosaccharides that form during polymerization are subject to two simultaneous reaction pathways: direct decomposition to low molecular weight products such as water, carbon dioxide, 5-hydroxymethylfurfural, furfural, furan and acetic acid, and formation of polysaccharides that eventually dehydrate to char and low molecular weight volatiles. Based on the experimental observations and quantitative measurements, a modified cellulose pyrolysis pathway involving levoglucosan as the major intermediate is proposed.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.

Oligomerization and DNA-Binding Capacity of Pmr, a Histone-Like Protein H1 (H-NS) Family Protein Encoded on IncP-7 Carbazole-Degradative Plasmid pCAR1

Pmr, a histone-like protein H1 (H-NS) family protein encoded on plasmid pCAR1, is a key factor in optimizing gene transcription on both pCAR1 and the host chromosome. To clarify the mode of function of Pmr, we performed gel filtration chromatography analysis and protein-protein cross-linking, and found that Pmr forms homo-oligomers, consisting of its homodimers. We also found, by atomic force microscopy, that Pmr has DNA-bridging capacity. From these results, Pmr was deduced to have features common to H-NS family proteins. Additionally, evaluating protein-DNA affinity is important to clarify the mode of function of Pmr, and hence we performed an electrophoretic mobility shift assay. Though Pmr formed high-order protein-DNA complexes and did not show preference for nucleic acid sequences, the C-terminal region of Pmr did, suggesting that the DNA-binding affinity of Pmr can be evaluated by using its C-terminal region.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3 mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen > human fibrinogen > dog fibrinogen > goat fibrinogen >> rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.

A high performance gel filtration chromatography method for γ-glutamyltransferase fraction analysis

The clinical relevance of serum γ-glutamyltransferase (GGT) activity, in areas other than hepatic function, has recently been increased by several epidemiological associations. Still, GGT remains a nonspecific test because of the influence of various pathophysiological factors. We devised a procedure based on gel filtration chromatography, followed by postcolumn injection of fluorescent GGT substrate (γ-glutamyl-7-amido-4-methylcoumarin), permitting the quantification of GGT fractions in serum or plasma. Plasma GGT molecular weight distribution was analyzed in healthy volunteers (20 males; mean ± SD age 38 ± 10 years; 20 females; age 44 ± 13; total GGT 21 ± 11 for males vs 13 ± 7 for females; P < 0.01). The method is highly sensitive (determination limit: 0.5 U GGT/L), with a linear dynamic range between 0.5 and 150 U/L for each fraction. Four GGT fractions of different molecular weight were detected in all subjects of both genders: b-GGT, m-GGT, s-GGT (likely lipoprotein-bound, molecular masses >2000, 940, and 140 kDa, respectively), and a free fraction (f-GGT, 70 kDa). f-GGT and s-GGT were the main fractions in subjects with lower and higher total GGT activity, respectively. Higher total GGT activity in males is related mainly to f-GGT ( P < 0.01). GGT fraction analysis may increase the sensitivity and specificity of the GGT activity test.

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.

Regulation of Function by Dimerization through the Amino-terminal Membrane-spanning Domain of Human ABCC1/MRP1

Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.

Critical Elements of Oligosaccharide Acceptor Substrates for the Pasteurella multocida Hyaluronan Synthase

Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating beta1,3-N-acetylglucosamine-beta1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses both GlcNAc-transferase and glucuronic acid (GlcUA)-transferase activities. The recombinant Escherichia coli-derived PmHAS enzyme will elongate exogenously supplied hyaluronan chains in vitro with either a single monosaccharide or a long chain depending on the UDP-sugar availability. Competition studies using pairs of acceptors with distinct termini (where one oligosaccharide is a substrate that may be elongated, whereas the other cannot) were performed here; the lack of competition suggests that PmHAS contains at least two distinct acceptor sites. We hypothesize that the size of the acceptor binding pockets of the enzyme corresponds to the size of the smallest high efficiency substrates; thus we tested the relative activity of a series of authentic hyaluronan oligosaccharides and related structural analogs. The GlcUA-transferase site readily elongates (GlcNAc-GlcUA)(2), whereas the GlcNAc-transferase elongates GlcUA-Glc-NAc-GlcUA. The minimally sized oligosaccharides, elongated with high efficiency, both contain a trisaccharide with two glucuronic acid residues that enabled the identification of a synthetic, artificial acceptor for the synthase. PmHAS behaves as a fusion of two complete glycosyltransferases, each containing a donor site and an acceptor site, in one polypeptide. Overall, this information advances the knowledge of glycosaminoglycan biosynthesis as well as assists the creation of various therapeutic sugars for medical applications in the future.

Prolactin Secretion in Mice with Thyrotropin-Releasing Hormone Deficiency

The physiological roles of TRH in pituitary lactotrophs, particularly during lactation, remain unclear. We studied the prolactin (PRL) status, including serum PRL and PRL mRNA levels in the pituitary, in nonlactating and lactating TRH-deficient (TRH(-/-)) mice with a rescue study with thyroid hormone and TRH. We found that, as reported previously for male TRH(-/-) mice, neither the morphology of the lactotrophs, PRL content in the pituitary, nor the serum PRL concentration was changed in nonlactating female TRH(-/-) mice. However, concurrent hypothyroidism induced a mild decrease in the PRL mRNA level. In contrast, during lactation, the serum PRL level in TRH(-/-) mice was significantly reduced to about 60% of the level in wild-type mice, and this was reversed by prolonged TRH administration, but not by thyroid hormone replacement. The PRL content and PRL mRNA level in the mutant pituitary during lactation were significantly lower than those in wild-type mice, and these reductions were reversed completely by TRH administration, but only partially by thyroid hormone replacement. Despite the low PRL levels, TRH(-/-) dams were fertile, and the nourished pups exhibited normal growth. Furthermore, the morphology of the pituitary was normal, and high performance gel filtration chromatography analysis of the PRL molecule revealed no apparent changes. We concluded that 1) TRH is not essential for pregnancy and lactation, but is required for full function of the lactotrophs, particularly during lactation; and 2) the PRL mRNA level in the pituitary is regulated by TRH, both directly and indirectly via thyroid hormone.