The role of vitamins and amino acids on hybridoma growth and monoclonal antibody production

A balanced supplementation method was applied to develop a serum and protein- free medium supporting hybridoma cell batch culture. The aim was to improve systematically the initial formulation of the medium to prevent limitations due to unbalanced concentrations of vitamins and amino acids. In a first step, supplementation of the basal formulation with 13 amino acids, led to an increase of the specific IgA production rate from 0.60 to 1.07 pg cell⁻¹ h⁻¹. The specific growth rate remained unchanged, but the supplementation enabled maintenance of high cell viability during the stationary phase of batch cultures for some 70 h. Since IgA production was not growth- related, this resulted in an approximately4-fold increase in the final IgA concentration, from 26.6 to 100.2 mgl⁻¹. In a second step, the liposoluble vitamins E and K₃ were added to the medium formulation. Although this induced a slightly higher maximal cell concentration, it was followed by a sharp decline phase with the specific IgA production rate falling to 0.47 pg cell⁻¹ h⁻¹. However, by applying a second cycle of balanced supplementation with amino acids this decline phase could be reduced and a high cell viability maintained for over 300 h of culture. In this vitamin- and amino acid- supplemented medium, the specific IgA production rate reached a value of 1.10 pg cell⁻¹h⁻¹ with a final IgA concentration of 129.8 mgl⁻¹. The latter represents an increase of approximately5-fold compared to the non- supplemented basal medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Serious renal transplant rejection and adrenal hypofunction after gradual withdrawal of prednisolone two years after transplantation.

Ten patients with stable renal function two years after transplantation had their sole immunosuppressive treatment (oral prednisolone 10 mg daily) withdrawn by reducing the daily dose by 1 mg at monthly intervals. Plasma prednisolone concentration, cortisol concentration, creatinine clearance, and serum creatinine concentration were measured in all patients, and the adrenal response to corticotrophin was determined in five by measuring plasma cortisol concentrations before and after tetracosactrin injection. No episodes of rejection occurred in patients taking over 7 mg prednisolone daily. Although three patients apparently required only minimal immunosuppressive treatment (less than 5 mg daily) the remainder suffered episodes of rejection at daily doses below 7 mg. There was a tenuous association between rejection and low plasma cortisol concentration, but neither the pattern of plasma prednisolone concentrations nor the response to tetracosactrin were related to episodes of rejection. Reducing the daily dose of oral prednisolone to under 7 mg should not be attempted in patients with renal transplants unless there are extenuating circumstances.     

The role of CD154 in organ transplant rejection and acceptance

CD154 plays a critical role in determining the outcome of a transplanted organ. This simple statement is amply supported by experimental evidence demonstrating that anti-CD154 antibodies are potent inhibitors of allograft rejection in many rigorous transplant models. Unfortunately, despite intensive investigation over the past ten years, the precise mechanisms by which antibodies against CD154 exert their anti-rejection effects have remained less obvious. Though originally classified with reference to B-cell function, CD154-CD40 interactions have also been shown to be important in T cell-antigen-presenting cell interactions. Accordingly, CD154 has been classified as a T-cell co-stimulatory molecule. However, mounting data suggest that treatment with anti-CD154 antibodies does not simply block costimulatory signals, but rather that the antibodies appear to induce signalling in receptor-bearing T cells. Other data suggest that anti-CD154 effects may be mediated by endothelial cells and possibly even platelets. In fact, the current literature suggests that CD154 can either stimulate or attenuate an immune response, depending upon the model system under study. CD154 has secured a fundamental place in transplant biology and general immunology that will no doubt be the source of considerable investigation and therapeutic manipulation in the coming decade.     

Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment.

Chemokines are thought to contribute to the cellular infiltrate characteristic of renal transplant rejection. We show that Met-RANTES, a chemokine receptor antagonist, suppresses recruitment of inflammatory cells into renal allografts. In a renal transplant model (Fisher RT1(lvl) rat kidney into Lewis RT1(l) rat) where no additional immune suppressant was used, Met-RANTES-treated animals showed a significant reduction in vascular injury score (16.10 +/- 5.20 vs. 62.67 +/- 18.64) and tubular damage score (15.70 +/- 5.22 vs. 33.00 +/- 6.44) relative to untreated animals. In a more severe rejection model (Brown-Norway RT1(n) rat kidney into Lewis RT1(1) rat), Met-RANTES significantly augmented low-dose cyclosporin A treatment to reduce all aspects of renal injury including interstitial inflammation (score 71.00 +/- 6.10 vs. 157.30 +/- 21.30). The majority of infiltrating cells in these models (60-70%) consisted of monocytes. Potential mechanisms of action of Met-RANTES were tested using monocyte attachment assays on microvascular endothelium under physiological flow conditions. Preexposure of microvascular endothelium to RANTES resulted in RANTES immobilization and RANTES-induced firm adhesion of monocytes only after prestimulation of the endothelium with IL-1beta. Met-RANTES completely inhibited this RANTES-mediated arrest. Thus, Met-RANTES may counter acute rejection by blocking leukocyte firm adhesion to inflamed endothelium.     

The critical structural role of a highly conserved histidine residue in group II amino acid decarboxylases

Glutamate decarboxylase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, belonging to the subset of PLP-dependent decarboxylases classified as group II. Site-directed mutagenesis of Escherichia coli glutamate decarboxylase, combined with analysis of the crystal structure, shows that a histidine residue buried in the protein core is critical for correct folding. This histidine is strictly conserved in the PF00282 PFAM family, which includes the group II decarboxylases. A similar role is proposed for residue Ser269, also highly conserved in this group of enzymes, as it provides one of the interactions stabilising His241.     

氨基酸制造工业的发展和展望

<正> (一) 国外氨基酸制造工业发展情况(1) 世界氨基酸制造工业当前世界氨基酸生产以及市场规模如表(1)。根据表(1)分析可以看出,从一九六九年～一九七九年的十年间世界氨基酸的产量以及市场规模增长情况,产量从一九六九年的25万吨增加到一九七九年的42万吨,增产率约1.7倍,每年的增产率约5～10%。在这十年中谷氨酸的增产率为1.3倍,而饲料用的L-赖氨酸、D、L-蛋氨酸以及医药用的其它氨基酸的     

The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation

Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols (trans- and cis-resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans- and cis-resveratrol 3-O-glucuronidation. Although the K(m) value for trans-resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K(m) values for cis-resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K(m) in trans-resveratrol, and V(max) and K(si) in cis-form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.     

A structural role of histidine 15 in human glutathione transferase M1-1, an amino acid residue conserved in class Mu enzymes.

His15 is a conserved amino acid residue in all known class Mu glutathione transferases. This His residue in human glutathione transferase M1-1 has been mutated into 17 different amino acid residues by means of site-directed random mutagenesis to determine if any substitutions are compatible with catalytic activity. The majority of the mutant proteins appeared unstable and could not be isolated in reasonable quantities by heterologous expression in Escherichia coli. Five mutant enzymes, H15C, H15K, H15N, H15Q and H15S were purified and more extensively characterized. The mutant proteins shared the same size as that of the wild-type enzyme but could be separated from the parental enzyme by reverse phase HPLC. For all the mutant forms except H15N, the sp. act. with 1-chloro-2,4-dinitrobenzene was less than 3% of the wild-type value--the H15N mutant enzyme displayed 29% of the wild-type activity. None of the catalytically active mutant enzymes showed any major alteration of the binding affinity for the substrate analog S-hexylglutathione, suggesting that His15 is not part of the active site of the enzyme. The high activity of the mutant H15N, also reflected in the kcat/Km, V and S0.5 values, rules out the possibility that His15 in the native enzyme contributes to catalysis by serving as a base. The role of His15, largely replaceable by Asn in the same position, appears to be structural, probably involving hydrogen bonds that maintain the protein in a stable and catalytically active conformation. A critical structural role of His15 in a buried position may explain the evolutionary conservation of this residue in the class Mu glutathione transferases.     

Dissociation of absorptions of calcium and phosphate after successful cadaveric renal transplantation.

Calcium and phosphate absorptions were studied by radiotracer techniques in 30 patients after successful cadaveric renal transplantation, and results were compared with those in a group of normal subjects and in groups of patients with chronic renal failure (CRF). Both calcium and phosphate absorptions were impared in patients with CRF, including those receiving haemodialysis. Abnormalities of calcium absorption, however, seemed to occur earlier in the course of advanced renal failure than abnormalities in phosphate absorption. Calcium absorption improved dramatically after successful renal transplantation, while phosphate absorption remained the same. A dissociation between calcium and phosphate absorptions is not often seen clinically, and the mechanisms for it are unknown. Phosphate malabsorption may be a further contributing factor in the development of persistent hypophosphataemia after transplantation.     

The effect of diamide on amino acid transport by rat renal cortex slices.

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of alpha-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4 degrees C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

Human-murine chimeras of ICAM-1 identify amino acid residues critical for rhinovirus and antibody binding.

Human ICAM-1 is the cellular receptor for the major group of human rhinoviruses (HRVs). Previous studies have suggested that the N-terminal domain of ICAM-1 is critical for binding of the major group rhinoviruses. To further define the residues within domain 1 that are involved in virus binding, we constructed an extensive series of ICAM-1 cDNAs containing single and multiple amino acid residue substitutions. In each case, substitutions involved replacement of the human amino acids with those found in murine ICAM-1 to minimize conformational effects. To facilitate the mutagenesis process, a synthetic gene encompassing the first two domains of ICAM-1 was constructed which incorporated 27 additional restriction sites to allow mutagenesis by oligonucleotide replacement. Each of the new constructs was placed into a Rous sarcoma virus vector and expressed in primary chicken embryo fibroblast cells. Binding assays were performed with six major group HRVs, including one high-affinity binding mutant of HRV-14, and two monoclonal antibodies. Results indicated that different serotypes displayed a range of sensitivities to various amino acid substitutions. Amino acid residues of ICAM-1 showing the greatest effect on virus and antibody binding included Pro-28, Lys-29, Leu-30, Leu-37, Lys-40, Ser-67, and Pro-70.     

Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding

Staphylococcal nuclease (SNase) is a model protein that contains one domain and no disulfide bonds. Its stability in the native state may be maintained mainly by key amino acids. In this study, two point-mutated proteins each with a single base substitution [alanine for tryptophan (W140A) and alanine for lysine (K133A)] and two truncated fragment proteins (positions 1-139 [SNase(1-139) or W140O] and positions 1-141 [SNase(1-141) or E142O]) were generated. Differential scanning microcalorimetry in thermal denaturation experiments showed that K133A and E142O have nearly unchanged DeltaH(cal) relative to the wild-type, whereas W140A and W140O display zero enthalpy change (DeltaH(cal) approximately 0). Far-UV CD measurements indicate secondary structure in W140A but not W140O, and near-UV CD measurements indicate no tertiary structure in either W140 mutant. These observations indicate an unusually large contribution of W140 to the stability and structural integrity of SNase.     

Regulation of amino acid transport system L by amino acid availability in CHO-K1 cells. A special role for leucine

Starvation of CHO-K1 cells for leucine leads to a 3–4-fold increase in transport system L activity, without modification of transport through systems A and ASC. The concentration of leucine must be below 10 μM before the enhancement of transport can be clearly seen. To achieve low concentrations of leucine such as 10 μM, extensive dialysis of fetal calf serum was required. The enhancement of transport was completed after 12–24 h of starvation and was fully reversed within 1 h of re-feeding with leucine. Starvation for isoleucine, valine or phenylalanine also produced an increase in system L transport activity, but the effect was only one half of that seen following leucine starvation.