Cataract and optic disk drusen in a patient with glycogenosis and di George syndrome: clinical and molecular report.

BACKGROUND: We report the ophthalmic findings of a patient with type Ia glycogen storage disease (GSD Ia), DiGeorge syndrome (DGS), cataract and optic nerve head drusen (ONHD). CASE PRESENTATION: A 26-year-old white woman, born at term by natural delivery presented with a post-natal diagnosis of GSD Ia. Genetic testing by array-comparative genomic hybridization (CGH) for DGS was required because of her low levels of serum calcium. The patient has been followed from birth, attending the day-hospital every six months at the San Paolo Hospital, Milan, outpatient clinic for metabolic diseases and previously at another eye center. During the last day-hospital visit, a complete eye examination showed ONHD and cataract in both eyes. Next Generation Sequencing (NGS) was subsequently done to check for any association between the eye problems and metabolic aspects. CONCLUSIONS: This is the first description of ocular changes in a patient with GSD Ia and DGS. Mutations explaining GSD Ia and DGS were found but no specific causative mutation for cataract and ONHD. The metabolic etiology of her lens changes is known, whereas the pathogenesis of ONHD is not clear. Although the presence of cataract and ONHD could be a coincidence; the case reported could suggest that hypocalcemia due to DGS could be the common biochemical pathway.

Somatic genomic imbalances in 'tumour-free' surgical margins of oral cancer.

Up to 30% of oral squamous cell carcinoma (OSCC) patients develop local recurrence and distant metastasis. The molecular status of histologically cancer-free tumour margins could be a critical factor in predicting tumour behaviour. The aim of this study was to detect somatic genomic imbalances in OSCC with emphasis on the surgical margins. DNA was isolated from tumour tissues, margin tissues, and blood samples (used as control) obtained from 11 OSCC patients, and genome-wide array comparative genomic hybridization was performed. Imbalances were present in both tumours and margins, although, as expected, they were more prevalent in tumours (duplications, P = 0.0002; deletions, P = 0.0001). Duplications were more frequent than deletions in both tumours and margins, but without statistical significance. Fifteen imbalances in tumour tissues were recurrent and all of them were duplications. Four of these were found both in tumours and margins and involved chromosomes 1q, 8p, Xp, Yp, and Yq. Four imbalances were recurrent in margin tissue and all of them were duplications (autosomes 8 and 17 and both sex chromosomes). Histologically 'cancer-free' margins hide genomic alterations consistent with unexplained OSCC recurrences. Establishing the molecular status of the margins could improve outcome prediction.

Thymidine phosphorylase expression and benefit from capecitabine in patients with advanced breast cancer.

BACKGROUND AND AIM: Capecitabine is an orally bioavailable prodrug that is converted to 5-fluorouracil through several enzymatic steps, the last of which is mediated by thymidine phosphorylase (TP). TP has been reported to be expressed at higher levels in cancer tissue compared with normal counterpart. The present study aimed at evaluating the potential relationship between TP expression and benefit from capecitabine in patients with metastatic breast cancer (BC). METHODS: Immunohistochemistry for TP and other biological markers was carried out on paraffin-embedded cancer tissues of 61 patients with BC treated with at least three cycles of capecitabine as single agent for metastatic disease. All patients had received capecitabine 1000 mg/m(2) b.i.d. days 1-14 every 21 days. The following variables were analyzed as potential determinants of benefit from capecitabine: TP expression, estrogen receptor (ER) and progesterone receptor status, human epidermal growth factor receptor-2 (HER-2) status, MIB-1 expression, performance status at the beginning of capecitabine treatment, stage at diagnosis, grade, presence of visceral metastases at the beginning of capecitabine treatment, and previous chemotherapy. RESULTS: Overall, median time to progression (TTP) was 6.5 months (range 1.4-33). On multivariate analysis, ER status [hazard ratio (HR) for progression = 0.31; 95% confidence interval (CI) = 0.15-0.64; P = 0.002], presence of visceral metastases at the beginning of capecitabine treatment (HR = 2.30; 95% CI = 1.21-4.39; P = 0.01), and capecitabine as first- or second-line treatment (HR = 2.28; 95% CI = 1.21-4.32; P = 0.01) independently predicted TTP. TP was highly expressed in 34 of 61 cases (55.7%). In the subgroup of patients with TP-expressing tumor, TTP was significantly longer in patients who received anthracyclines and taxanes before capecitabine (median TTP 7.5 versus 3.3 months, P = 0.01, log-rank test). Similarly, patients with a TP-positive tumor showed a longer TTP if they received taxanes before capecitabine than patients with TP-positive tumor who did not receive this treatment (7.3 versus 3.4 months, P = 0.03). CONCLUSIONS: These data provide further evidence that TP expression in BC could represent a biomarker of sensitivity to capecitabine treatment. Prospective studies with translational approach are desirable to confirm the predictive and prognostic role of TP.

Tacrolimus Therapeutic Drug Monitoring in Stable Kidney Transplantation and Individuation of CYP3A5 Genotype.

The posology of tacrolimus (TAC) is usually guided by its therapeutic drug monitoring. Some patients reach target concentrations (CTs) quickly, others more slowly. In a retrospective study, 20 kidney transplant recipients were included (mean age, 50.7 ± 14.1 years; weight 64.0 ± 14.2 kg; patients clinically stable for over a year). We studied cytochrome CYP3A5 genotype, in particular CYP3A5 6986A>G, the most important polymorphism related to the metabolism of TAC (wild genotype CYP3A5 *1 genotype, and CYP3A5 *3 variants). One year after transplantation, the CTs were 5.0 to 8.0 ng/mL. The patients were divided into group A (TAC doses < 6.0 mg/d) and group B (TAC doses > 6.0 mg/d). All were tested for the CYP3A5 gene sequence to characterize their polymorphism. Patients with CYP3A5 *1/*1 and *1/*3 were extensive metabolizers, and those with CYP3A5 *3/*3 were poor metabolizers. In group A and group B, the average TAC doses at the time of therapeutic drug monitoring were 3.0 ± 1.4 ng/mL (0.05 ± 0.03 mg/kg) and 12.8 ± 3.7 ng/mL (0.2 ± 0.1 mg/kg), respectively (P < .001). Group A was the poor metabolizers genotype, while in group B, the extensive metabolizers genotype was present. Patients with the CYP3A5 *1/*1 or *1/*3 genotype required 1.5 to 2 times higher doses than patients *3/*3 to reach CT. This genetic test allows clinicians to know, before the kidney transplant, the patient's TAC metabolism pattern and then to optimize the drug exposure.

Thymidine phosphorylase expression is associated with time to progression in patients receiving low-dose, docetaxel-modulated capecitabine for metastatic breast cancer.

BACKGROUND: Preclinical data have indicated a synergistic interaction between docetaxel and capecitabine by means of taxane-induced up-regulation of thymidine phosphorylase (TP). On the basis of such premises, we conducted a phase II trial to determine the activity and tolerability of weekly docetaxel plus capecitabine in patients with metastatic breast cancer (MBC). Furthermore, we explored the relationship between TP tumor expression and benefit from this regimen. PATIENTS AND METHODS: Patients received docetaxel 36 mg/m(2) i.v. on days 1, 8, and 15 and capecitabine orally 625 mg/m(2) b.i.d. from days 8 to 21. Cycles were repeated every 4 weeks. In the correlative study, we evaluated the TP expression by immunohistochemistry and the TP messenger RNA expression by real-time RT-PCR in the primary tumor. RESULTS: Forty-seven women were enrolled. In the intention-to-treat analysis, objective responses were achieved in 24 patients (51%). Fourteen additional patients (30%) had stable disease. The median time to progression (TTP) was 6 months (range 1-44 months). Median survival was 17 months (range 1-48 months). Overall, the treatment was well tolerated. The most common clinical adverse events (all grades) were alopecia (55%), nail changes (53%), fatigue/asthenia (51%), nausea/vomiting (51%), neutropenia (49%), and neuropathy (49%). A significantly higher TTP was observed in patients with TP-positive tumors (log-rank test, P = 0.009). Interestingly, a subgroup analysis confirmed this TTP benefit in patients with TP-positive tumors obtaining a tumor response (log-rank test, P = 0.03), whereas the statistical significance was lost in nonresponders (log-rank test, P = 0.3). CONCLUSIONS: This study indicates that a regimen with low doses of capecitabine plus weekly docetaxel is active against MBC. The correlative analysis provides preliminary evidence that TP expression may be a predictive marker for therapeutic benefit.

Expression and function of the homeodomain-containing protein Hex in thyroid cells.

The homeodomain-containing protein Hex (also named Prh) is expressed in primitive endoderm (during the early phases of development), in some endoderm-derived tissues and in endothelial and hematopoietic precursors. Hex expression is exting-uished during terminal differentiation of endothelial and hematopoietic cells as well as in adult lung. Previous investigations have demonstrated that Hex is expressed during early thyroid gland development. No information has been reported on Hex expression in adult thyroid gland or on the function of this protein in follicular thyroid cells. These issues represent the focus of the present study. We demonstrate that Hex mRNA is present in rat and human adult thyroid gland as well as in differentiated follicular thyroid cell lines. In FRTL-5 cells TSH reduces Hex expression. In thyroid cell lines transformed by several oncogenes Hex expression is completely abolished. By using co-transfection assays we demonstrate that Hex is a repressor of the thyroglobulin promoter and that it is able to abolish the activating effects of both TTF-1 and Pax8. These data would suggest that Hex may play an important role in thyroid cell differentiation. Protein-DNA interaction experiments indicate that Hex is able to bind sites of the thyroglobulin promoter containing either the core sequence 5'-TAAT-3' or 5'-CAAG-3'. The DNA binding specificity of the Hex homeodomain, therefore, is more 'relaxed' than that observed in the majority of other homeo-domains.

An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation.

The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H(2)O(2)induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse-chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.

[Management of gynecological tumors associated with BRCA1 and BRCA2 germline mutations. Case report and literature review]. / Management dei tumori ginecologici associati a mutazioni germinali BRCA1 e BRCA2. Caso clinico e review della letteratura.

Genetic investigation of BRCA1 and BRCA2 germline mutations is, nowadays, a diagnostic procedure with practical clinical applications. The role of this genes in DNA repair and stability and in cancer development is now well recognised. Most involved are breast and ovarian cancers, but, less frequently, other gynecological cancers like cervical, corpus uteri and Fallopian tubes cancers and also other non gynecological malignancies. We report the case of a 67-year-old patient with strong familiarity for breast cancer, with a BRCA2 germline mutation, who developed in 30 months 4 primary malignancies: in chronological order, breast cancer, chronic lymphatic leukemia, and synchronous ovarian and endocervical adenocarcinoma. A better knowledge of the biological and clinical behaviour of BRCA related cancers is of strategical importance in the management of patients with strong familiar neoplastic history or with genetic test positivity. An adequate counselling can help in the management of these cancers in the prevention and early diagnosis taking also into consideration the possibility of a prophylactic surgery.

A unique combination of transcription factors controls differentiation of thyroid cells.

The thyroid follicular cell type is devoted to the synthesis of thyroid hormones. Several genes, whose protein products are essential for efficient hormone biosynthesis, are uniquely expressed in this cell type. A set of transcriptional regulators, unique to the thyroid follicular cell type, has been identified as responsible for thyroid specific gene expression; it comprises three transcription factors, named TTF-1, TTF-2, and Pax8, each of which is expressed also in cell types different from the thyroid follicular cells. However, the combination of these factors is unique to the thyroid hormone producing cells, strongly suggesting that they play an important role in differentiation of these cells. An overview of the molecular and biological features of these transcription factors is presented here. Data demonstrating that all three play also an important role in early thyroid development, at stages preceding expression of the differentiated phenotype, are also reviewed. The wide temporal expression, from the beginning of thyroid organogenesis to the adult state, is suggestive of a recycling of the thyroid-specific transcription factors, that is, the control of different sets of target genes at diverse developmental stages. The identification of molecular mechanisms leading to specific gene expression in thyroid cells renders this cell type an interesting model in which to address several aspects of cell differentiation and organogenesis.

Expression of thyroid-specific transcription factors TTF-1 and PAX-8 in human thyroid neoplasms.

TTF-1 and PAX-8 are tissue-specific transcription factors expressed in the thyroid follicular cells, contributing to the maintenance of the differentiated phenotype. In fact, it has been demonstrated that TTF-1 and PAX-8 are able to activate transcription from thyroglobulin and thyroperoxidase (TPO) promoters, the transcriptional activity of which is in vivo restricted only to the thyroid follicular cell. In order to gain insight into how these transcription factors control in vivo the differentiation of the thyroid cell and to have a better molecular characterization of human thyroid tumors, TTF-1, PAX-8, thyroglobulin, and TPO mRNA levels were measured in nonmalignant and malignant human thyroid tissues. Results indicate that the expression of TTF-1 and PAX-8 is not sufficient per se for the expression of the thyroid-differentiated phenotype. Furthermore, in follicular adenomas, PAX-8 mRNA levels are strictly related to TPO mRNA levels, suggesting that the amount of PAX-8 could play a role in the modulation of TPO gene expression. TTF-1 mRNA is always well detectable in papillary carcinomas and, in contrast, always absent in anaplastic carcinomas. Identical results were obtained when the expression of TTF-1 protein was investigated using immunohistochemistry. Thus, TTF-1 gene expression could be a molecular marker in order to distinguish these two types of thyroid neoplasms.

Insulin receptor expression and function in human breast cancer cell lines.

We have previously reported that insulin receptor expression is increased in human breast cancer specimens (V. Papa et al., J. Clin. Invest., 85:1503-1510, 1990). In the present study, in order to further understand the role of the insulin receptor in breast cancer, insulin receptor expression and function were characterized in three human breast cancer cell lines, MCF-7, ZR-75-1, and T-47D, and compared to a nonmalignant human breast epithelial cell line, 184B5. Insulin receptor content, measured by radioimmunoassay, was elevated 5- and 3-fold in MCF-7 and ZR-75-1 breast cancer cell lines, respectively, when compared to the nonmalignant cell line 184B5. In contrast, the insulin receptor content of T-47D cells was not increased. The increase in insulin receptor content in MCF-7 and ZR-75-1 cells was not due to amplification of the insulin receptor gene. Also, total insulin receptor mRNA content was not increased in breast cancer cells in respect to nonmalignantly transformed 184B5 breast epithelial cells. However, significant differences in the content of receptor mRNA species were observed. The insulin receptors in the breast cancer cell lines were functional: (a) In all 4 cell lines, high-affinity insulin-binding sites were detected, and, in concert with the insulin receptor radioimmunoassay data, binding capacity was highest in MCF-7 and then in ZR-75-1 cells. (b) In all cell lines, insulin stimulated insulin receptor tyrosine kinase activity. However, the effect of insulin was greater in breast cancer cell lines than in nonmalignant breast cells. (c) In all cell lines, insulin at concentrations of 1 nM or less stimulated [3H]thymidine incorporation. This effect of insulin was inhibited by 50% in MCF-7 cells and by 60% in 184B5 cells when alpha-IR3, a monoclonal antibody to the insulin-like growth factor I receptor, was present. In these cells, therefore, insulin was active via both its own receptor and the IGF-I receptor. In contrast, alpha-IR3 antibody was without effect in T-47D and ZR-75-1 cells, suggesting that in these cell lines insulin acted only via its receptor. In the breast cancer cells, MA-5, an agonist monoclonal antibody to the insulin receptor, stimulated [3H]thymidine incorporation. This present study indicates therefore that in breast cancer cell lines there are functional insulin receptors that regulate breast cancer cell growth.

Defective intracellular antioxidant enzyme production in type 1 diabetic patients with nephropathy.

There is an individual susceptibility to diabetic nephropathy, and oxidative stress is believed to play an important role in the pathogenesis of diabetic complications. Active oxygen species induce antioxidant enzyme expression in tissues, an effect considered to be a defensive mechanism. To test whether altered intracellular antioxidant enzyme production might explain the predisposition to diabetic nephropathy, we studied the effect of long-term (12 weeks) exposure to normal (5 mmol/l) or high (22 mmol/l) glucose concentrations on fibroblast antioxidant enzyme gene expression and protein activity in type 1 diabetic patients with and without nephropathy, nondiabetic nephropathic patients, and nondiabetic control subjects. Under conditions of normal glucose concentration in the culture media, CuZnSuperoxide-dismutase, MnSuperoxide-dismutase, catalase, and glutathione-peroxidase activity and mRNA expression were not different among the four groups. Under high-glucose conditions, CuZnSuperoxide-dismutase mRNA and activity increased similarly in all groups (P < 0.001 vs. basal), whereas MnSuperoxide-dismutase did not change. In contrast, catalase mRNA and activity as well as glutathione-peroxidase mRNA and activity increased in fibroblasts from type 1 diabetic patients without nephropathy (P < 0.001), in fibroblasts from nondiabetic nephropathic patients (P < 0.001), and in fibroblasts from nondiabetic control subjects (P < 0.001), but not in fibroblasts from type 1 diabetic patients with nephropathy. Exposure to high glucose concentrations significantly increased lipid peroxidation in cells, higher levels being found in cells from diabetic patients with nephropathy (P < 0.001). These data, while confirming that exposure to high glucose concentrations induces an antioxidant defense in skin fibroblasts from normal subjects, demonstrate a failure of this defensive mechanism in cells from type 1 diabetic patients with nephropathy, whereas skin fibroblasts from diabetic patients without complications or from nondiabetic nephropathic patients have an intact antioxidant response to glucose-induced oxidative stress.

A differential proteomic approach to identify proteins associated with thyroid cell transformation.

Tumour suppressor p53 is a transcription factor essential for DNA damage checkpoints during cellular response to stress. Mutations in the p53 gene are the most common genetic alterations found in human tumours; most pathogenetic modifications are missense mutations that abolish the p53 DNA-binding function. In the same cell type, distinct p53 missense mutations may determine different phenotypes. The PC Cl3 cell line retains several markers of thyroid differentiation in vitro. Introduction of the V143A mutant p53 allele, which abolishes the p53 DNA-binding function, leads to loss of differentiation markers as well as TSH dependency for growth. Conversely, PC Cl3 cells transfected with the S392A mutant p53 allele, presenting the mutation located outside the DNA-binding domain, show only loss of TSH dependency for growth. To identify molecular differences existing between PC Cl3 cell lines transformed by the V143A and the S392A mutant alleles, a differential proteomic approach was used. Two-dimensional gel electrophoresis analyses indicated that expression of a significant portion of protein species was modified by both p53 mutants. In fact, compared with wild-type PC Cl3 cells, modification of expression in V143A mutant cells occurred in 23.6% of the entire protein species. Conversely, modification of S392A mutant cells affected 14.0% of total proteins. Among these components, 8.3% were common to both mutants. Several of these proteins were identified by mass spectrometry procedures; some proteins, such as HSP90 and T-complex proteins, are already known to be related to p53 function.

A suppressor of transcriptional activity is present upstream from the rat c-Ha-ras promoter.

We report the presence of an element in the rat c-Ha-ras 5'-flanking region that has a suppressive effect on the promoter of this gene. Promoter activity was determined using constructs of different regions of the c-Ha-ras 5'-flanking region linked to the reporter gene chloramphenicol acetyl transferase (CAT) transfected into NIH-3T3 cells. The presence of the tract 400 base-pairs upstream from the promoter region reduced promoter activity tenfold. This suppressor element was effective in either orientation and was not produced by other DNA fragments of similar length. Transfection with different amounts of plasmid suggested that a trans-acting factor in limiting amounts was acting on the suppressor region. Gel-retardation assays demonstrated that a nuclear protein was present in 3T3 cells that specifically interacted with the 400-base fragment containing the suppressor element.

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells.

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.

Genetic thrombophilias and uterine artery Doppler velocimetry and preeclampsia.

OBJECTIVE: The aim of this study was to evaluate the correlation between genetic thrombophilic mutations, uterine artery Doppler at 24 weeks of gestation and preeclampsia. METHODS: In a case control study we performed the genetic analysis for Leiden mutation of factor V gene (FV), G20210A mutation of the prothrombin gene (PT) and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene in 103 women that had already attended routine ultrasonography scanner at 20 weeks at our Department. RESULTS: The frequency of heterozygous carriers of the factor V Leiden was 17.4% in the women with preeclampsia and abnormal artery Doppler compared with 3.12% in the patients with normal pregnancies. This difference was statistically significant (P<0.05). The frequency of mutation G20210A of prothrombin gene was 1.5 vs. 4.3% between women with normal pregnancies and with preeclampsia. This difference is not statistically significant. The frequency of homozygous patients for the C677T mutation of MTHFR gene among the patients with preeclampsia was 21.7% and in the control group was 10.3%, but this difference is not statistically significant. No thrombophilic gene variants were found in women with preeclampsia and normal uterine artery Doppler. CONCLUSION: We demonstrated the important association between factor V Leiden mutation, abnormal uterine Doppler at 24 weeks and preeclampsia in our population.

Missense mutations of human homeoboxes: A review.

The homeodomain (encoded by the homeobox) is the DNA-binding domain of a large variety of transcriptional regulators involved in controlling cell fate decisions and development. Mutations of homeobox-containing genes cause several diseases in humans. A variety of missense mutations giving rise to human diseases have been described. These mutations are an excellent model to better understand homeodomain molecular functions. To this end, homeobox missense mutations giving rise to human diseases are reviewed. Seventy-four independent homeobox mutations have been observed in 17 different genes. In the same genes, 30 missense mutations outside the homeobox have been observed, indicating that the homeodomain is more easily affected by single amino acids changes than the rest of the protein. Most missense mutations have dominant effects. Several data indicate that dominance is mostly due to haploinsufficiency. Among proteins having the homeodomain as the only DNA-binding domain, three "hot spot" regions can be delineated: 1) at codon encoding for Arg5; 2) at codon encoding for Arg31; and 3) at codons encoding for amino acids of recognition helix. In the latter, mutations at codons encoding for Arg residues at positions 52 and 53 are prevalent. In the recognition helix, Arg residues at positions 52 and 53 establish contacts with phosphates in the DNA backbone. Missense mutations of amino acids that contribute to sequence discrimination (such as those at positions 50 and 54) are present only in a minority of cases. Similar data have been obtained when missense mutations of proteins possessing an additional DNA-binding domain have been analyzed. The only exception is observed in the POU1F1 (PIT1) homeodomain, in which Arg58 is a "hot spot" for mutations, but is not involved in DNA recognition.

Comparative stability analysis of the thyroid transcription factor 1 and Antennapedia homeodomains: evidence for residue 54 in controlling the structural stability of the recognition helix.

The thyroid transcription factor 1 homeodomain (TTF-1 HD) shows a peculiar DNA-binding specificity which is partially dictated by several amino acids of the recognition helix. TTF-1 preferentially recognizes sequences containing the 5'-CAAG-3' core motif while most other homeodomains, such as Antennapedia (Antp), recognizes sites containing the 5'-TAAT-3' core motif. Since phenomena of 'induced fit' may occur during protein/DNA interaction, a primary role for high affinity binding and target discrimination has to be searched in the effect played by subtle structural determinants in these proteins. By using spectroscopic analysis in aqueous solution, we compared the structural stability of TTF-1 and Antp homeodomains. Although the three-dimensional structural architecture of homeodomains is conserved, some differences are detectable in terms of their structural stability. At 24 degrees C the TTF-1 HD is less structured than the Antp HD with 24 and 34% of the residues in the alpha-helical conformation, respectively. This poor folded structure reflects into different thermal and isothermal stability between the two homeodomains. TTF-1 HD exhibits a Tm of 39 degrees C and is stabilized by a delta GDH2O of +1487 cal/mol, calculated by Urea unfolding, while Antp HD exhibits a Tm of 48 degrees C and is stabilized by a delta GDH2O of +2742 cal/mol. By using mutants of both TTF-1 and Antp HDs we demonstrate that one of the major determinants in controlling the structural stability of the recognition helix is the residue at position 54. Since previous studies have shown that also residue at position 56 is involved in stabilization of the recognition helix, we conclude that the structure of this critical element is controlled by an interplay between residues at position 54 and 56 of the homeodomain.

Thyrotropin stimulates glucose transport in cultured rat thyroid cells.

Glucose transport by FRTL-5 cells, a rat thyroid cell line, was found to be TSH dependent. The effect of TSH on the uptake of 2-deoxy-D-glucose, a nonmetabolizable glucose analogue, was prompt, being 200% over basal value after 10 min and maximal after 12 h (600-700% increase). The TSH effect was dose dependent, with half-maximum stimulation at 10 microU TSH/ml, and maximum stimulation at 1 mU TSH/ml. TSH enhanced also the uptake of 3-O-methyl-D-glucose by FRTL-5 cells. The TSH activation of glucose transport had the following characteristics: it was mimicked by (Bu)2-cAMP (1 mM) and by agents that increase cAMP levels in thyroid cells, such as forskolin (10 microM) and cholera toxin (50 micrograms/ml); it involved the facilitated glucose transport system in that it was inhibited in a dose-related manner by both cytochalasin B and phloretin; it showed a glucose stereochemical sensitivity, being affected by D-glucose and 3-O-methyl-glucose, and not by L-glucose; it was characterized by an increase in the maximum velocity (Vmax) of glucose uptake (from 15.3 to 66.0 fmol/min X micrograms DNA) without change in the Michaelis-Menten constant (Km) (5.3 mM); the effect on the Vmax was due to an increase in the number of surface glucose transporters as indicated by the enhancement of the D-glucose-sensitive fraction of [3H]cytochalasin B binding sites that in thyroid plasma membranes of cells exposed to TSH for 2 and 8 h, increased from 5.0 (basal value) to 10.4 and 23.1 pmol/mg protein, respectively. These data indicate that in FRTL-5 cells TSH stimulates the glucose transport system by an enhancement of the number of functional glucose transporters in the thyroid plasma membrane.

Thyroid autoregulation: effect of iodine on glucose transport in cultured thyroid cells.

The nonmetabolizable glucose analogs, [3H]2-deoxy-D-glucose and [3H]O-methyl-D-glucose, were used to determine whether iodide influences glucose transport in porcine cells in primary culture. Incubation with iodide (3 h) decreased basal glucose transport with a half-maximum at NaI 3 X 10(-5) M and maximum at 10(-4) M. Iodide (10(-6) M to 10(-4) M) also abolished the stimulatory effect of TSH (1 mU/ml) on glucose transport. The iodide effect on [3H]2-deoxy-D-glucose transport had the following characteristics: 1) it was abolished 24 h after incubation in iodide-free medium; 2) it was prevented by methimazole (3 mM), and correlated with newly formed organic iodine, 3) and it affected the maximum velocity (Vmax) of glucose transport, reducing it from 25.1 to 14.4 and 12.0 nmol/(min mg protein) at 10(-5) M and 10(-4) M NaI, without affecting the Michaelis-Menten constant (Km) (6mM). Iodide-treated cells had a reduced specific binding of [3H]cytochalasin B (38% and 47% with respect to control cells at 10(-5) M and 10(-4) M NaI). These data suggest that iodide treatment reduces the functional carriers mediating glucose transport in the thyroid.