Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent protein

Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs.     

Effects of superovulation on endogenous LH secretion in cattle, and consequences for embryo production

Stimulation of follicular growth during superovulation is achieved by the injection of FSH or compounds with high FSH-bioactivities. However, some LH-activity is required for follicle maturation. It is of relevance to evaluate, therefore, the effect of superovulatory treatments on endogenous LH secretion. Luteinizing hormone is secreted in discrete pulses, and the pattern of pulsatile LH secretion during superovulation is reviewed. Four of five published studies have shown that LH pulse frequency is significantly reduced by injection of eCG or FSH preparations. This suppression appears within 8 h of treatment Effects of superovulation on LH pulse amplitude are less consistent. The reasons for the decrease in pulse frequency have been investigated, and although the answer is not definitive, it would seem that increased follicular estradiol, acting perhaps in synergism with progesterone, may play a role. Changes in plasma progesterone concentrations are not related to changes in LH pulse frequency. What is the significance of decreased LH pulse frequency? We attempted to investigate this by inducing LH pulses during superovulation, but the result was a major reduction in ovulation rate. More research is required to determine if modification of endogenous LH secretion can improve superovulatory responses.     

Biased T-cell receptor usage is associated with allelic variation in the MHC class II peptide binding groove

 A comprehensive analysis was carried out of the tri-molecular complex of peptide, major histocompatibility class II molecule, and T-cell receptor (TcR) involved in the recognition of the promiscuous HA (306–318) peptide, restricted by one of two closely related HLA-DR alleles, HLA-DRB1*0101 and HLA-DRB1*0103. These two DR molecules differ by only three amino acids at positions 67, 70, and 71, in the third variable region of the DRB1 chain. None of the HA (306–318)-specific T-cell clones restricted by these two DR molecules tolerated amino acid substitution at the peptide-binding position 71, despite the fact that the substitution did not interfere with peptide binding. The majority of the DRB1*0103-restricted clones tolerated substitution of the amino acid at the TcR-contacting position 70, while the DRB1*0101-restricted T cells did not. Biased usage of TRVA and TRVB segments was observed for the DRB1*0103-restricted clones; in contrast, apparently random usage was seen in the DRB1*0101-restricted T cells. Finally, limiting dilution analysis revealed a lower frequency of T cells reactive with the HA peptide in a DRB1*0103 compared with a DRB1*0101 individual. Taken together these data suggest that biased TcR gene usage may reflect a relatively low precursor frequency of T cells, and the need for clonal expansion of a limited set of high avidity T cells. Received: 7 August 1998 / Revised: 19 November 1998     

High expression level of a gene coding for a chloroplastic amino acid selective channel protein is correlated to cold acclimation in cereals

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.     

Effect of short-term testosterone treatment on leptin concentrations in boys with pubertal delay

Testosterone administration increases growth hormone (GH) secretion and decreases the plasma leptin concentration in men. We evaluated the effect of increased GH secretion due to short-term testosterone treatment on leptin concentrations. Ten boys aged 14.8 +/- 0.2 (mean +/- SE) years with transient GH deficiency caused by pubertal delay were evaluated before and after (3 months) 4 intramuscular injections of 100 mg testosterone heptylate, given at 15-day intervals. The leptin concentration decreased from 5.4 +/- 1.3 to 3. 6 +/- 1.1 microgram/l (p < 0.001), despite a weight gain of 3.4 +/- 0.5 kg. There were significant increases in body mass index (BMI), from -0.2 +/- 0.5 to 0.2 +/- 0.5 SD, p < 0.005, in GH peak after stimulation test, from 6.3 +/- 0.5 to 21.7 +/- 2.9 microgram/l, p < 0. 0003, in plasma testosterone, from 0.6 +/- 0.1 to 6.5 +/- 1.3 microgram/l, p < 0.001, in insulin-like growth factor-I (IGF-I), from 152 +/- 21 to 330 +/- 30 microgram/l, p < 0.0001, and in IGF-binding protein-3 (IGFBP-3), from 4.2 +/- 0.5 to 5.4 +/- 0.4 mg/l, p < 0.01. But there were no changes in blood glucose (4.7 +/- 0.1 and 4.8 +/- 0.1 mmol/l), or plasma fasting insulin (9.0 +/- 1.2 and 8.1 +/- 1.3 mIU/l). The leptin concentrations were positively correlated with the BMI before (p < 0.03) and after (p < 0.04) testosterone, but not with the GH peak after stimulation, or with plasma testosterone, IGF-I or IGFBP-3. The leptin and insulin concentrations after testosterone treatment were positively correlated (p < 0.04). Thus, short-term testosterone treatment of boys with pubertal delay decreases their leptin concentrations. The lack of correlation with GH secretion or with its changes, despite the dramatic increase in GH secretion, and the lack of change in insulin are additional features suggesting that testosterone increases the leptin concentration mainly by an effect on adipose tissue.     

Prognosis of children with malignant pheochromocytoma. Report of 2 cases and review of the literature

Malignant pheochromocytomas are rare in childhood and the prognosis of children with this tumor is not well known. We present 2 pediatric observations of malignant pelvic pheochromocytoma. Symptoms in both cases were headache and hypertension. The tumor invaded the sacral bone. Angiogram helped to localize the tumor and metastases, and allowed preoperative embolization of the tumor in 1 case. The first child underwent incomplete surgical resection, (131)I-MIBG therapy and radiotherapy, and is still alive 2 years after diagnosis. The second child died from metastatic invasion a few weeks after discovery of the tumor. We reviewed previous reports of children with malignant pheochromocytomas (30 cases). Primary tumor was extraadrenal in 50% of cases. The 3-year survival rate was 73 +/- 9% (mean +/- SD). Apart from surgical resection, no particular treatment appeared to be more effective than others in reducing mortality.