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Biomedical subjects

M John

Publications and source records attributed to M John.

At least 109 records · Page 6Linked to original sources
PubMed1996

DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.

Amino Acid Sequence↗
PubMed1996

Convergent pathways for lipochitooligosaccharide and auxin signaling in tobacco cells.

Lipochitooligosaccharides (LCOs) are a novel class of plant growth regulators that activate in tobacco protoplasts the expression of AXI1, a gene implicated in auxin signaling. Transient assays with a chimeric P(AXI)-GUS expression plasmid revealed that the N-octadecenoylated monosaccharide GlcN has all structural requirements for a biological active glycolipid, whereas the inactive N-acylated GalN epimer inhibits LCO action. Specific inhibition of LCO and auxin action shows that both signals are transduced within the tobacco cell via separate pathways that converge at or before AXI1 transcription. Cytokinin is suggested to be a common effector of LCO and auxin signaling. We also show that activation of AXI1 correlates with growth factor-induced cell division.

Arabidopsis Proteins↗
PubMed1996

Ten-year results of chemoradiation for anal cancer: focus on late morbidity.

PURPOSE: To evaluate (a) long-term survival and (b) the incidence and nature of long-term morbidity/ mortality related to chemoradiation using the anal cancer experience. METHODS AND MATERIALS: From January 1979 to April 1987,34 consecutive patients with Stage I (5 patients), II (15 patients), and II (14 patients) cancers of the anal canal were treated definitively with a chemoradiation regimen combining 41.4 Gy pelvic radiotherapy with two concurrent cycles of 5-fluorouracil and mitomycin C. Cumulative actuarial survival was calculated at 10 years and long-term morbidity was categorized per RTOG/EORTC late toxicity criteria. Specific criteria to grade anal toxicity were devised. RESULTS: Cumulative survival for all 34 patients was 92% at 5 years and 85% at 10 years. The most frequent late toxicity was chronic diarrhea in 17 (50%) patients. Five patients (15%) had Grade 3 or 4 late toxicities. Sexual dysfunction was present in 2 of 26 evaluable patients (7%). CONCLUSIONS: Excellent long-term survival and colostomy-free survival is possible for anal cancer patients treated definitively by chemoradiation. Late effects do not appear to be frequent or intense enough to deter the use of chemoradiation in anal cancer. The biologically expected increase in long-term toxicity when combining radiotherapy and chemotherapy is not substantiated by the results of this study.

Adult↗
PubMed1996

Final results of a phase II chemoradiation protocol for locally advanced cervical cancer: RTOG 85-15.

BACKGROUND: The lack of improved cure rates for advanced cervical cancer after three decades of megavoltage radiotherapy (RT) has prompted continued efforts in improved treatment delivery. Concurrent chemoradiation (CR) is one of the several avenues being explored to improve these results. METHODS: Sixty women with advanced cervical cancer (30 patients with unfavorable Stage IIB and 30 patients with Stages III and IVA) were treated with CR comprising of a combination of external and intracavitary RT delivering between 7000 to 7500 cGy total to point A and 5890 to 6015 cGy to point B along with one cycle of 5-FU and mitomycin C and a second cycle of 5-FU and cis-platinum. RESULTS: Grade 3 and 4 RT-related toxicities were 15 and 3%, respectively. Chemotherapy-related Grade 3 and 4 toxicities were 9 and 2%, respectively. The 5-year survival for unfavorable Stage IIB patients was 48%; for Stages III and IVA it was 39%. CONCLUSIONS: The toxicity of this particular CR regimen was acceptable and suggests that further qualitative and quantitative intensification of chemoradiation may be attempted. Retrospective comparisons with PCS studies and previous RTOG studies 79-20 and 80-05 suggest that this particular chemoradiation regimen may offer a modest survival advantage over RT alone for Stages III and IVA disease. A CR regimen with higher doses of radiotherapy and a greater number of active chemotherapeutic agents may yet result in acceptable toxicity and further improve cure rates in advanced and poor prognostic featured cervical cancer.

Adolescent↗
PubMed1996

Expression of inducible nitric oxide synthase in placenta of women with gestational diabetes.

Gestational diabetes is one of the most prevalent medical complications of pregnancy and causes increased fetal wastage. Investigation of placentas from diabetic mothers indicate chronic disturbances in intervillous, circulation, dilatation of capillaries, and a relatively immature villous structure. Abnormal levels of nitric oxide (NO) may contribute to maternal disorders such as the pathogenesis of diabetic vascular complications. In the normal placenta NO is generated only by endothelial NOS, which apparently serves to regulate vascular tone in the fetoplacental circulation. In contrast, studies have reported the absence of inducible nitric oxide synthase (iNOS) in human placentas under normal conditions. The aim of our study was to investigate whether iNOS is expressed in placentas from patients with gestational diabetes. Reverse transcription-polymerase chain reaction and Western blot analysis demonstrated iNOS mRNA and protein expression in placental tissue only from patients with gestational diabetes. Immunohistochemistry localized iNOS staining to endothelial cells and trophoblasts. We conclude that iNOS can be expressed in human placenta. Its expression might play an important role in placental pathophysiology.

Base Sequence↗
PubMed1996

Positive somatostatin receptor scintigraphy correlates with the presence of somatostatin receptor subtype 2.

Somatostatin receptor scintigraphy (SRS) is positive in approximately 75% of all patients with neuroendocrine gastroenteropancreatic tumours. This study aimed to identify specific somatostatin receptor (sstr) subtypes, which are responsible for the in vivo binding of the widely used somatostatin analogue, octreotide in human neuroendocrine gastroenteropancreatic tumours. Twelve patients underwent SRS with radiolabelled octreotide. After surgical resection, tumour tissues were analysed in vitro for somatostatin and octreotide binding sites by autoradiography. In addition, for the first time, sstr subtype mRNA expression was examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Tumour tissues from all SRS positive patients were positive by autoradiography. Semiquantitative RT-PCR revealed most prominently sstr2 expression in scintigraphically positive tumours. Two SRS negative tumours contained in vitro octreotide binding sites as well as high levels of sstr1 and sstr2 mRNAs. Positive SRS is mainly due to sstr2. sstr1, 3, 4, and probably 5 are less important for in vivo octreotide binding. False negative scintigraphic results seem to be influenced by factors independent of the expression of specific sstr.

Adult↗
PubMed1996

Behet's syndrome with pulmonary involvement-combined therapy for endobronchial stenosis using neodym-YAG laser, balloon dilation and immunosuppression.

We present a case report of a patient suffering from Behet's syndrome with pulmonary involvement. The development of endobronchial granulomatosis in the bronchus intermedius and right upper lobe bronchus led to severe bronchial stenosis and dyspnoea. Also aphthous ulcerations in the bronchial mucosa occurred. Treatment of the granulomatous stenosis consisted of Neodym-YAG laser resection and immunosuppression. On follow-up there was a recurrence of the stenosis, characterised by fibrotic strictures. Therefore a balloon dilation was performed and immunosuppression instituted, which led to an increase in airway diameter and a reduction in dyspnoea. These clinical results suggest that recanalisation therapy with Neodym-YAG laser and/or balloon dilation with additional immunosuppression is suitable in benign stenosis (granulomatous or fibrotic), caused by Behet's disease.

Adult↗
PubMed1996

Interleukin 13 inhibits macrophage inflammatory protein-1 alpha production from human alveolar macrophages and monocytes.

Interleukin 13 (IL-13) is a recently described protein secreted by activated T cells and is a potent in vitro modulator of human monocyte and B-cell functions. IL-13 shares some biologic properties as well as structural similarities with IL-4. Macrophage-inflammatory protein 1 alpha (MIP-1 alpha) is a product of activated monocytes and macrophages and an important activator of T cells, monocytes, and macrophages. We determined the effect of human recombinant IL-13 on lipopolysaccharide (LPS)- and IL-1 beta-induced MIP-1 alpha mRNA and protein expression from peripheral blood monocytes (PBM) and alveolar macrophages (AM). In PBM, basal MIP-1 alpha protein was 20 +/- 7 pM and increased following LPS and IL-1 beta to 1,520 +/- 193 (P < 0.001) and 233 +/- 50 (P < 0.003) pM. IL-13 (25 ng/ml) reduced these values by 55 +/- 10% [not significant (NS)], 43 +/- 9% (P < 0.03), and 44 +/- 15% (NS), respectively. LPS- and IL-1 beta-induced MIP-1 alpha mRNA expression was reduced by 43 +/- 5% (P < 0.01) and 41 +/- 4% (NS). In AM, IL-13 reduced LPS-induced MIP-1 alpha protein release of 2,030 +/- 242 pM by 32 +/- 8% (P < 0.05) and MIP-1 alpha mRNA by 27 +/- 1% (NS). For both PBM and AM, the inhibitory effect of IL-13 on MIP-1 alpha protein was maximal at 24 h, was dose dependent with a maximal effect at 100 ng/ml, and was similar to, although slightly less potent than, that seen with IL-4. In PBM, the inhibitory effect of IL-13 required de novo protein synthesis and was not due to enhanced mRNA decay. Thus, IL-13 has inhibitory effects on the transcription of MIP-1 alpha from monocytes and macrophages, and as is the case with IL-4 and IL-10, may be an important mediator for suppressing inflammatory responses.

Blotting, Northern↗
PubMed1996

Role of mitomycin in combination with fluorouracil and radiotherapy, and of salvage chemoradiation in the definitive nonsurgical treatment of epidermoid carcinoma of the anal canal: results of a phase III randomized intergroup study.

PURPOSE: Definitive chemoradiation (CR) has replaced radical surgery as the preferred treatment of epidermoid carcinoma of the anal canal. To determine the importance of mitomycin (MMC) in the standard CR regimen and to assess the role of salvage CR in patients who have residual tumor following CR, a phase III randomized trial was undertaken by the Radiation Therapy Oncology Group (RTOG)/Eastern Cooperative Oncology Group (ECOG). PATIENTS AND METHODS: Between August 1988 and December 1991, 310 patients were randomized to receive either radiotherapy (RT) and fluorouracil (5-FU) or radiotherapy, 5-FU, and MMC. Of 291 assessable patients, 145 received 45 to 50.4 Gy of pelvic RT plus 5-FU at 1,000 mg/m2/d for 4 days, and 146 received RT, 5-FU, and MMC (10 mg/m2 per dose for two doses). Patients with residual tumor on posttreatment biopsy were treated with a salvage regimen that consisted of additional pelvic RT (9 Gy), 5-FU, and cisplatin (100 mg/m2). RESULTS: Posttreatment biopsies were positive in 15% of patients in the 5-FU arm versus 7.7% in the MMC arm (P = .135). At 4 years, colostomy rates were lower (9% v 22%; P = .002), colostomy-free survival higher (71% v 59%; P = .014), and disease-free survival higher (73% v 51%; P = .0003) in the MMC arm. A significant difference in overall survival has not been observed at 4 years. Toxicity was greater in the MMC arm (23% v 7% grade 4 and 5 toxicity; P < or = .001). Of 24 assessable patients who underwent salvage CR, 12 (50%) were rendered disease-free. CONCLUSION: Despite greater toxicity, the use of MMC in a definitive CR regimen for anal cancer is justified, particularly in patients with large primary tumors. Salvage CR should be attempted in patients with residual disease following definitive CR before resorting to radical surgery.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

[Interventional pneumology: procedures for pulmonary hemorrhage and tumor-induced superior vena cava syndrome].

Interventional pneumology includes both bronchological and vascular methods of diagnostic and therapy, especially in case of pneumological emergency, such as pulmonary hemorrhage and superior vena cava syndrome. In massive pulmonary hemorrhage bronchological diagnosis is needed to determine the location and activity of the bleeding, as well as angiography of bronchial arteries, and of pulmonary arteries, respectively. Bronchus occlusion by aid of balloon catheter or double lumen tube are intermediate methods to bridge over till defenitive surgery or embolisation of bronchial or pulmonal arteries as complementary methods in patients with pulmonary hemorrhage. In patients suffering from superior vena cava syndrome caused by neoplasms venous angioplasty and Wallstent implantation provide immediate clinical improvement.

Angioplasty, Balloon↗
PubMed1995

Inhibition of macrophage inflammatory protein-1 alpha expression by IL-10. Differential sensitivities in human blood monocytes and alveolar macrophages.

IL-10 is a pleiotropic cytokine produced by monocytes and T cells that has potent inhibitory effects on monocyte/macrophage function. Because monocytes and macrophages are capable of releasing the C-C chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), which is a potent chemoattractant for activated T cells, we studied the effects of IL-10 on the expression of MIP-1 alpha in these cells. Low levels of MIP-1 alpha were detectable in resting monocytes and macrophages. Both LPS (1 micrograms/ml) and IL-1 beta (10 ng/ml) induced the expression of MIP-1 alpha mRNA and the release of MIP-1 alpha protein from these cells. The addition of exogenous human rIL-10 inhibited induced MIP-1 alpha mRNA expression as well as the release of MIP-1 alpha protein measured after 24 h. This inhibition was significantly higher in monocytes compared with alveolar macrophages. In monocytes, IL-10-induced inhibition of MIP-1 alpha was only partially accounted for by alterations in mRNA stability and was dependent on de novo protein synthesis. In the presence of an anti-human IL-10-neutralizing Ab, the release of MIP-1 alpha induced by LPS and IL-1 beta was further enhanced in monocytes but unchanged in alveolar macrophages. MIP-1 alpha mRNA was also increased in monocytes. There was no detectable release of IL-10 from alveolar macrophages after LPS or IL-1 beta in contrast to modest amounts released from monocytes. Thus, IL-10 is an inhibitor of the induced transcription of MIP-1 alpha mRNA and of the release of MIP-1 alpha protein, with a greater effect on monocytes as compared with alveolar macrophages. IL-10 may indirectly regulate effects on cells such as activated T lymphocytes partly through the inhibition of MIP-1 alpha expression from monocytes and macrophages.

Antibodies↗
PubMed1995

Prophylactic extended-field irradiation of para-aortic lymph nodes in stages IIB and bulky IB and IIA cervical carcinomas. Ten-year treatment results of RTOG 79-20.

OBJECTIVES: To investigate whether irradiation to the standard pelvic field only improves the response rate and survival in comparison with pelvic plus para-aortic irradiation in patients with high-risk cervical carcinoma, and to investigate patterns of failure and treatment-related toxicity. DESIGN: Randomized controlled trial from November 1979 to October 1986, with stratification by histology, para-aortic nodal status, and International Federation of Gynecology and Obstetrics (FIGO) stage. SETTING: Radiation Therapy Oncology Group (RTOG) multicenter clinical trial. PATIENTS: A total of 367 patients with FIGO stage IB or IIA primary cervical cancers measuring 4 cm or greater in lateral diameter or with FIGO stage IIB cervical cancers were randomized to RTOG protocol 79-20 to receive either standard pelvic only irradiation or pelvic plus para-aortic irradiation. INTERVENTION: Pelvic only irradiation consisted of a midplane pelvic dose of 40 to 50 Gy in 4.5 to 6.5 weeks with daily fractions of 1.6 to 1.8 Gy for 5 d/wk. Pelvic plus para-aortic irradiation delivered 44 to 45 Gy in 4.5 to 6.5 weeks with daily fractions of 1.6 to 1.8 Gy for 5 d/wk. A total dose of 4000 to 5000 mg/h of radium equivalent or 30 to 40 Gy was provided by intracavitary brachytherapy to point A. MAIN OUTCOME MEASURES: Response rate, overall and disease-free survival, patterns of failure, and treatment-related toxicities. RESULTS: Ten-year overall survival was 44% for the pelvic only irradiation arm and 55% for the pelvic plus para-aortic irradiation am (P = .02). Cumulative incidence of death due to cervical cancer was estimated as significantly higher in the pelvic only arm at 10 years (P = .01). Disease-free survival was similar in both arms; 40% for the pelvic only arm and 42% for the pelvic plus para-aortic arm. Locoregional failures were similar at 10 years for both arms (pelvic only, 35%; pelvic plus para-aortic, 31%; P = .44). In complete responders, the patterns of locoregional failures were the same for both arms, but there was a lower cumulative incidence for first distant failure in the pelvic plus para-aortic irradiation arm (P = .053). Survival following first failure was significantly higher in the pelvic plus para-aortic arm (P = .007). A higher percentage of local failures were salvaged long-term on the pelvic plus para-aortic arm compared with the pelvic only arm (25% vs 8%). The cumulative incidence of grade 4 and 5 toxicities at 10 years in the pelvic plus para-aortic arm was 8%, compared with 4% in the pelvic only arm (P = .06). The death rate due to radiotherapy complications was higher in the pelvic plus para-aortic arm (four [2%] of 170) compared with the pelvic only arm (one [1%] of 167) (P = .38). The proportion of deaths due to radiotherapy complications in the pelvic plus para-aortic arm was higher than in the pelvic only arm (four [6%] of 67 vs one [1%] of 85; P = .24). If the patient had abdominal surgery prior to para-aortic irradiation, the estimated cumulative incidence of grade 4 and 5 complications was 11%, compared with 2% in the pelvic only arm. CONCLUSIONS: The statistically significant difference in overall survival at 10 years for the pelvic plus para-aortic irradiation arm, without a difference in disease-free survival, can be explained by the following two factors: (1) a lower incidence of distant failure in complete responders and (2) a better salvage in the complete responders who later failed locally.

Abdomen↗
PubMed1995

In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure.

The Rhizobium common nod gene products NodABC are involved in the synthesis of the core lipochitooligosaccharide (Nod factor) structure, whereas the products of the host-specific nod genes are necessary for diverse structural modifications, which vary in different Rhizobium species. The sulfate group attached to the Rhizobium meliloti Nod signal is necessary for activity on the host plant alfalfa, while its absence renders the Nod factor active on the non-host plant vetch. This substituent is therefore a major determinant of host specificity. The exact biosynthetic pathway of Nod factors has not been fully elucidated. In particular, it is not known why some chemical modifications are introduced with high fidelity whereas others are inaccurate, giving rise to a family of different Nod factor structures produced by a single Rhizobium strain. Using protein extracts and partially purified recombinant NodH protein obtained from Escherichia coli expressing the R. meliloti nodH gene, we demonstrate here NodH-dependent in vitro sulfotransferase activity. Kinetic analyses with Nod factors, chitooligosaccharides, and their deacetylated derivatives revealed that Nod factors are the preferred substrate for the sulfate transfer. Moreover, the tetrameric Nod factor, NodRm-IV, was a better substrate than the trimer, NodRm-III, or the pentamer, NodRm-V. These data suggest that the core lipochitooligosaccharide structure must be synthesized prior to its host-specific modification with a sulfate group. Since in R. meliloti tetrameric Nod factors are the most abundant and the most active ones, high affinity of NodH for the appropriate tetrameric substrate guarantees its modification and thus contributes to the fidelity of host-specific behavior.

Bacterial Proteins↗
PubMed1995

Somatostatin receptor subtypes in neuroendocrine tumor cell lines and tumor tissues.

Somatostatin receptor scintigraphy (SRS) is positive in approximately 80% of all patients who have been found to have neuroendocrine (NE) gastroenteropancreatic (GEP) tumors. The reasons for negative results are unclear. The aim of the present study was identification of the specific somatostatin receptor (SSTR) subtypes that are responsible for the in vivo binding of the widely used somatostatin (SST) analogues octreotide and lanreotide in human neuroendocrine gastroenteropancreatic tumors. Ten patients were subjected to SRS with radiolabeled octreotide. Following surgical resection, tumor tissues were analyzed for SSTR subtype mRNA expression by the reverse transcription-polymerase chain reaction (RT-PCR). In addition, SSTR subtype transcripts were investigated by Northern blot analysis and RT-PCR in neuroendocrine tumor cell lines. Expression of SSTR at the protein level was studied by chemical cross-linking experiments. Three patients were negative by SRS. However, RT-PCR revealed most prominently SSTR 2 expression in all tumor specimens. In addition, all tumor tissues analyzed by chemical crosslinking exhibited SST-14 binding sites, indicating that at least some NE tumors were false-negative on SRS.

Animals↗
PubMed1995

Expression of functional GABAA receptors in neuroendocrine gastropancreatic cells.

Gastropancreatic neuroendocrine cells synthesize large amounts of gamma-aminobutyric acid (GABA). This amino acid neurotransmitter appears to be stored in and released from, vesicles similar to small synaptic vesicles. So far, the function of GABA in gastropancreatic, neuroendocrine cells has not been clarified. Previous work suggested that only pancreatic, glucagon-producing alpha 2 cells contain functional GABAA receptors. Using subunit-specific antibodies in sections of human antral mucosa, a human gastrinoma and rat pancreas, we show that expression of GABAA receptors is abundant in gastropancreatic, neuroendocrine cells. Using the patch-clamp technique in the whole-cell mode we demonstrate that both the rat insulinoma cell line RIN 38 and the amphicrine cell line AR42J express functional GABAA receptors, which are characterized by a relatively low benzodiazepine and Zn2+ sensitivity and by an insensitivity to the inverse benzodiazepine agonist 6,7-alpha-methoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). In contrast to neurons, activation of GABAA receptors leads to a membrane depolarization. This depolarization presumably activates voltage-gated Ca2+ channels, resulting in an increase in cytosolic Ca2+ concentration, [Ca2+]i, as shown with the fluorimetric dye fura-2. The combination of GABA release, GABAA receptor activation and the [Ca2+]i increase could constitute an autocrine mechanism, modulating the release of hormones such as gastrin, insulin and somatostatin.

Animals↗
PubMed1994

Electrical activity and calcium channels in neuroendocrine cells.

Similar to neuronal cells, neuroendocrine cells express voltage-dependent ion channels and fire action potentials. Ca2+ influx through voltage-dependent Ca2+ channels couples changes in membrane potential to Ca(2+)-dependent cellular processes, such as hormone release. Using the patch-clamp technique, we studied the spontaneous electrical activity as well as voltage-dependent Ca2+ channels in cholecystokinin-producing pancreatic cells (RIN 1056E cell line), in prolactin-secreting pituitary cells (GH3 cell line), and in calcitonin-secreting cells of the thyroid (rMTC 44-2 cell line). All three cell types displayed spontaneous electrical activity, that is, they spontaneously produced action potentials. RIN 1056E cells, GH3 cells, and rMTC cells exhibited (various types of) voltage-dependent Ca2+ channels that were regulated by various neurotransmitters and hormones, such as somatostatin.

Action Potentials↗
PubMed1994

Two pairs of oppositely charged amino acids from Jun and Fos confer heterodimerization to GCN4 leucine zipper.

The preferential assembly of Jun and Fos into heterodimers has been shown to be mainly driven by 16 amino acids (8 from each protein) situated in positions e and g of the leucine zipper coiled-coil structures of the two proteins (O'Shea, E. K., Rutkowski, R., and Kim, P. S. (1992) Cell 68, 699-708). Using a similar approach, we show that among these residues two pairs of oppositely charged amino acids account in fact for most of the additional free energy of heterodimerization in this system. These residues are 2 glutamic acid side chains in positions g1 and e2 of the Fos leucine zipper and 2 lysine residues in the equivalent positions of the Jun zipper. These amino acids were placed in the context of a GCN4 leucine zipper using peptide synthesis. These peptides contain unique cysteine residues enabling the formation of covalent dimers. The gain in heterodimer free energy has been determined both by cysteine-linked dimer formation under redox conditions and by thermal melting experiments of covalent dimers using circular dichroism experiments. The two pairs of oppositely charged residues (Glu,Glu and Lys,Lys) in positions g1 and e2 contribute at least -1.9 kcal/mol of additional free energy, accounting for a 50-fold excess of the heterodimer with respect to one of the homodimers. Thermal denaturation studies as a function of pH and ionic strength suggest that electrostatic effects should indeed be a major driving force for heterodimerization. On the contrary, peptides harboring the 12 amino acids from Jun and Fos in the other e and g positions (i.e. in e1, g2, e3, g3, e4, and g4) show only a moderate tendency to form heterodimers.

Amino Acid Sequence↗