A semi-automatic planning technique for whole breast irradiation with tangential IMRT fields.

PURPOSE: To implement a semi-automatic planning technique for whole breast irradiation with two tangential IMRT fields and to test the produced dose distribution against clinical 3DCRT plans, for introducing the technique in clinical practice. METHODS: The Auto-Planning module of the Pinnacle3 (Philips) treatment planning system was used for generating a Treatment Technique on left-sided breast cancer patients treated in free breathing or in deep inspiration breath hold (DIBH) and to right-sided breast cancer patients. The technique was evaluated against 3DCRT clinical plans in terms of dosimetric plan parameters. Plan robustness toward patient displacements was assessed on a subset of patients by inducing shifts to the isocenter. RESULTS: A statistically significant improvement in target coverage and dose homogeneity was observed for autoIMRT. No statistically significant differences were observed for ipsilateral organs, except for the ipsilateral lung in left DIBH, where slightly lower Dmean and V18% are registered for autoIMRT. Slightly higher Dmean doses (although far below the constraints) to contralateral organs were observed for autoIMRT plans. AutoIMRT plans were shown to be as robust as 3DCRT plans toward isocenter shifts, with a maximum decrease in CTV coverage of -2.2% and -2.1% for autoIMRT and 3DCRT, respectively. Average planning times were 40 min for 3DCRT and 6 min for IMRT plans. CONCLUSIONS: The developed autoIMRT technique was proven to be advantageous for target coverage and homogeneity and sufficiently robust towards isocenter displacements. The use of automated planning consistently reduces the planning workload with improvements in plan quality.

Automatic VMAT technique to treat glioblastoma: A two years' experience.

PURPOSE: The present work aims to guide the physicist in order to start automated planning for the VMAT treatment of glioblastoma multiforme (GBM) by giving a recipe that was set up and tested during a long-term (two years) evaluation. METHODS: An automatic technique in AutoPlanning module of the Pinnacle3 (Philips Medical Systems, Fitchburg, WI) treatment planning system was created and validated by comparing dose distributions of automatic plans (APs) and manual plans (MPs) and by performing a blind AP-MP comparison on a cohort of 20 patients. Automatic technique was then applied to 145 patients and failures were recorded i.e. the number of times for which dose distributions produced by the automatic module were not suitable for treatment. RESULTS: Each of the 20 APs considered in the validation step was clinically acceptable and proved to be better (15 cases) or equal (5 cases) respect to MPs. A statistically significant improvement in brain stem, optic pathways, cochleae, pituitary gland and scalp sparing was observed for APs, while no statistically significant differences were recorded in target coverage or plan parameters. For only 5 cases out of the 145 plans the operator intervention was needed in order to obtain a clinical acceptable plan, while for the remaining 140 plans the automatic created solution was suitable. CONCLUSIONS: A straightforward automatic procedure has been created and tested in our clinic. The AutoPlanning technique proposed represents a reliable tool to improve treatment planning efficiency and the recipe, here presented, could be simply imported to every radiotherapy center.

In phantom evaluation of targeting accuracy in MRI-based brain radiosurgery.

PURPOSE: To determine the targeting accuracy of brain radiosurgery when planning procedures employing different MRI and MRI + CT combinations are adopted. MATERIALS AND METHOD: A new phantom, the BrainTool, has been designed and realized to test image co-registration and targeting accuracy in a realistic anatomical situation. The phantom was created with a 3D printer and materials that mimic realistic brain MRI and CT contrast using a model extracted from a synthetic MRI study of a human brain. Eight markers distributed within the BrainTool provide for assessment of the accuracy of image registrations while two cavities that host an ionization chamber are used to perform targeting accuracy measurements with an iterative cross-scan method. Two procedures employing 1.5 T MRI-only or a combination of MRI (taken with 1.5 T or 3 T scanners) and CT to carry out Gamma Knife treatments were investigated. As distortions can impact targeting accuracy, MR images were preliminary evaluated to assess image deformation extent using GammaTool phantom. RESULTS: MR images taken with both scanners showed average and maximum distortion of 0.3 mm and 1 mm respectively. The marker distances in co-registered images resulted below 0.5 mm for both MRI scans. The targeting mismatches obtained were 0.8 mm, 1.0 mm and 1.2 mm for MRI-only and MRI + CT (1,5T and 3 T), respectively. CONCLUSIONS: Procedures using a combination of MR and CT images provide targeting accuracies comparable to those of MRI-only procedures. The BrainTool proved to be a suitable tool for carrying out co-registration and targeting accuracy of Gamma Knife brain radiosurgery treatments.

TomoTherapy treatments of multiple brain lesions: an in-phantom accuracy evaluation.

Aim of the present study was to evaluate the accuracy which can be obtained with helical TomoTherapy® (HT, Accuray) systems in the case of multiple intracranial targets treatments. Set-up accuracy was measured, for different registration options and MegaVoltage CT (MVCT) slice thickness, by applying known misalignments to an ad-hoc developed phantom. End-to-end (E2E) tests were performed to assess the delivery accuracy in phantoms containing multiple targets by using radiochromic films: measured dose distribution centroids were compared with physical and calculated target positions on axial and coronal planes. A Gamma index analysis was carried out on planned and measured planar dose maps. The bone and tissue algorithm with the fine MVCT reconstruction grid gave the best results among the automatic options. The most accurate registration modality resulted to be the manual one with a sub-voxel accuracy shifts and a capability in the detection of rotations within 0.3°. For the E2E along the coronal plane (six targets), a mean deviation between measured dose distribution centroids and physical barycenters of 0.6 mm (range 0.1 mm-1.3 mm) was observed. Along the axial plane (five targets), a mean deviation of 1.2 mm (range 0.7 mm-2.1 mm) was found for the centroids shifts. Gamma index (5%, 1 mm, local) passing rates higher than 87.5% between planned and delivered dose distributions were measured. These results demonstrate that multiple brain lesion HT treatments are feasible with an accuracy at least comparable to frameless linac-based delivery, when a set-up capable to assure angular corrections and a reliable patient immobilization is employed.

Use of an Amplatz Canine Ductal Occluder (ACDO) device to close an acquired aortopulmonary fistula with a hybrid approach in a dog.

A 2-year-old recently spayed female Rottweiler was referred as an emergency with cardiac tamponade and the presence of an anomalous retrograde flow in the pulmonary artery. Echocardiography and angiography demonstrated a left-to-right aortopulmonary fistula. Clinical history and data indicated a possible infectious aetiology. Antibiotics and heart failure medications were administered for 30 days before intervention. Initial attempt at insertion of an Amplatz occluder by means of a percutaneous catheterization technique was tried but a safe release of the device was judged to be not possible due to the angle and the fragile and irregular margins of the window. A decision was made to proceed with a hybrid technique combining thoracotomy and direct pulmonary artery catheterization. This hybrid approach was successful with resolution of congestive heart failure with only residual mild paraprosthetic leakage.

Monitoring the kinetic evolution of self-assembled SiGe islands grown by Ge surface thermal diffusion from a local source.

In this paper we experimentally study the growth of self-assembled SiGe islands formed on Si(001) by exploiting the thermally activated surface diffusion of Ge atoms from a local Ge source stripe in the temperature range 600-700 °C. This new growth strategy allows us to vary continuously the Ge coverage from 8 to 0 monolayers as the distance from the source increases, and thus enables the investigation of the island growth over a wide range of dynamical regimes at the same time, providing a unique birds eye view of the factors governing the growth process and the dominant mechanism for the mass collection by a critical nucleus. Our results give experimental evidence that the nucleation process evolves within a diffusion limited regime. At a given annealing temperature, we find that the nucleation density depends only on the kinetics of the Ge surface diffusion resulting in a universal scaling distribution depending only on the Ge coverage. An analytical model is able to reproduce quantitatively the trend of the island density. Following the nucleation, the growth process appears to be driven mainly by short-range interactions between an island and the atoms diffusing within its vicinities. The islands volume distribution is, in fact, well described in the whole range of parameters by the Mulheran's capture zone model. The complex growth mechanism leads to a strong intermixing of Si and Ge within the island volume. Our growth strategy allows us to directly investigate the correlation between the Si incorporation and the Ge coverage in the same experimental conditions: higher intermixing is found for lower Ge coverage. This confirms that, besides the Ge gathering from the surface, also the Si incorporation from the substrate is driven by the diffusion kinetics, thus imposing a strict constraint on the initial Ge coverage, its diffusion properties and the final island volume.

Patterning-induced strain relief in single lithographic SiGe nanostructures studied by nanobeam x-ray diffraction.

The continued downscaling in SiGe heterostructures is approaching the point at which lateral confinement leads to a uniaxial strain state, giving high enhancements of the charge carrier mobility. Investigation of the strain relaxation as induced by the patterning of a continuous SiGe layer is thus of scientific and technological importance. In the present work, the strain in single lithographically defined low-dimensional SiGe structures has been directly mapped via nanobeam x-ray diffraction. We found that the nanopatterning is able to induce an anisotropic strain relaxation, leading to a conversion of the strain state from biaxial to uniaxial. Its origin is fully compatible with a pure elastic deformation of the crystal lattice without involving plastic relaxation by injection of misfit dislocations.

BRCA1 and BRCA2 genetic testing in Italian breast and/or ovarian cancer families: mutation spectrum and prevalence and analysis of mutation prediction models.

BACKGROUND: Breast cancer is an extremely complex disease, characterized by a progressive multistep process caused by interactions of both genetic and non-genetic factors. A combination of BRCA1 and BRCA2 gene mutations appears responsible for about 20%-30% of the cases with breast cancer familial history. The prevalence of BRCA1/2 pathogenic mutations largely varies within different populations; in particular, the rate of mutations in Italian breast and/or ovarian cancer families is rather controversial and ranges from 8% to 37%. PATIENTS AND METHODS: Of the 152 breast/ovarian cancer families counseled in our centre, 99 were selected for BRCA1/2 mutation screening according to our minimal criteria. The entire coding sequences and each intron/exon boundary of BRCA1/2 genes were screened by direct sequencing (PTT limited to BRCA1 exon 11). For each proband, the a priori probability of carrying a pathogenic BRCA1/2 germline mutation was calculated by means of different mutation prediction models (BRCApro, IC and Myriad Table) in order to evaluate their performances. RESULTS: Our analysis resulted in the identification of 25 and 52 variants in the BRCA1 and BRCA2 genes, respectively. Seventeen of them represent novel variants, including four deleterious truncating mutations in the BRCA2 gene (472insA, E33X, C1630X and IVS6+1G>C). Twenty-seven of the 99 probands harbored BRCA1 (n = 15) and BRCA2 (n = 12) pathogenic germline mutations, indicating an overall detection rate of 27.3% and increasing by more than 15% the spectrum of mutations in the Italian population. Furthermore, we found the lowest detection rate (19.4%) in pure hereditary breast cancer family subset. All of the prediction models showed praises and faults, with the IC software being extremely sensitive but poorly specific, compared to BRCApro. In particular all models accumulated most false-negative prediction in the HBC subset. Interestingly preliminary results of a study addressing the presence of genomic rearrangements in HBC probands with BRCApro or IC prediction scores >/=95%, provided evidence for additional mutations undetectable with our conventional screening for point mutations. CONCLUSIONS: Altogether our results suggest that HBC families, the largest pool in our series, represent an heterogeneous group where the apparently faulty performances of the prediction models might be at least partially explained by the presence of additional kinds of BRCA1/2 alteration (such as genomic rearrangements) or by mutations on different breast cancer related genes.

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

EGF regulates a complex pattern of gene expression and represses smooth muscle differentiation during the neurotypic conversion of the neural-crest-derived TC-1S cell line.

EGF, known to sustain CNS neuronal progenitors, also promotes a neurotypic response in the thymic neural-crest-derived TC-1S cell line. We report here the use of TC-1S cells as a model to identify the genetic programs regulated during the neurotypic response induced by EGF and to isolate 23 EGF-responsive genes. Among them 5 represent novel cDNAs, while 18 are known genes, whose regulation by EGF is associated with the mitogenic or differentiating effects of the growth factor. The repression of smooth muscle alpha-actin and SM22alpha genes by EGF and their increase by TGFbeta suggest that the TC-1S line includes neural crest multipotent cells whose smooth muscle differentiation is repressed upon EGF treatment and stimulated by TGFbeta. Therefore, we identified a complex pattern of EGF-target genes and propose EGF as a novel signal able to recruit postmigratory neural-crest-derived cells along proliferation and cell lineage choice pathways.

Expression of the HMGI(Y) gene products in human neuroblastic tumours correlates with differentiation status.

HMGI and HMGY are splicing variants of the HMGI(Y) gene and together with HMGI-C, belong to a family of DNA binding proteins involved in maintaining active chromatin conformation and in the regulation of gene transcription. The expression of the HMGI(Y) gene is maximal during embryonic development, declines in adult differentiated tissues and is reactivated in most transformed cells in vitro and in many human cancers in vivo. The HMGI(Y) genomic locus is frequently rearranged in mesenchymal tumours, suggesting a biological role for HMGI(Y) gene products in tumour biology. HMGIs are both target and modulators of retinoic acid activity. In fact, HMGI(Y) gene expression is differentially regulated by retinoic acid in retinoid-sensitive and -resistant neuroblastoma cells, while HMGI-C participates in conferring retinoic acid resistance in some neuroblastoma cells. In this paper we show that HMGI and HMGY isoforms are equally regulated by retinoic acid in neuroblastoma cell lines at both RNA and protein levels. More importantly our immunohistochemical analysis shows that, although HMGI(Y) is expressed in all neuroblastic tumours, consistently higher levels are observed in less differentiated neuroblastomas compared to more differentiated ganglioneuromas, indicating that HMGI(Y) expression should be evaluated as a potential diagnostic and prognostic marker in neuroblastic tumours.

Molecular cloning and expression of an alternative hKLK3 transcript coding for a variant protein of prostate-specific antigen.

We report the molecular cloning of a full-length cDNA corresponding to a 2.1-kb hKLK3 mRNA. This mRNA results from the alternative splicing of intron 4, and its accumulation in prostatic LNCaP cells is stimulated by androgen. The cDNA encodes a prepro-prostate-specific antigen (PSA) variant containing 238 amino acids. The new protein, PSA-related protein 1 (PSA-RP1), differs from PSA at the COOH-terminal end and lacks the serine residue that is essential for catalytic activity. Prepro-PSA-RP1 was transiently expressed in COS1 cells fused to the V5 epitope of the paramyxovirus SV5. The recombinant fusion protein was detected in the spent medium by Western blot analysis using anti-V5 and anti-PSA antibodies. This indicates that PSA-RP1 is secreted and has PSA-like antigenic epitopes. A pro-PSA and a pro-PSA-RP1 having a mutated propeptide were overproduced in Escherichia coli fused to glutathione S-transferase. The recombinant PSA and PSA-RP1 were matured in vitro and identified by Western blot with molecular masses of 29 (PSA) and 27 (PSA-RP1) kDa. The data indicate that PSA-RP1, not complexed to serine protease inhibitors, could be present in biological fluids, thus contributing to the free PSA-immunoreactive fraction in serum.

HMGI(Y) and HMGI-C genes are expressed in neuroblastoma cell lines and tumors and affect retinoic acid responsiveness.

HMGI-C and HMGI(Y) are architectural DNA-binding proteins that participate in the conformational regulation of active chromatin. Their pattern of expression in embryonal and adult tissues, the analysis of the "pygmy" phenotype induced by the inactivation of the HMGI-C gene, and their frequent qualitative or quantitative alteration in experimental and human tumors indicate their pivotal role in the control of cell growth, differentiation, and tumorigenesis in several tissues representative of the epithelial, mesenchymal, and hematopoietic lineages. In contrast, very little information is available on their expression and function in neural cells. Here, we investigated the expression of the HMGI(Y) and HMGI-C genes in neuroblastoma (NB), a tumor arising from an alteration of the normal differentiation of neural crest-derived cells and in embryonal and adult adrenal tissue. Although HMGI(Y) is constitutively expressed in the embryonal and adult adrenal gland and in all of the NB cell lines and ex vivo tumors examined, its regulation appears to be associated to growth inhibition and differentiation because we observed that HMGI(Y) expression is reduced by retinoic acid (RA) in several NB cell lines that are induced to differentiate into postmitotic neurons, whereas it is up-regulated by RA in cells that fail to differentiate. Furthermore, the decrease of HMGI(Y) expression observed in RA-induced growth arrest and differentiation is abrogated in cells that have been made insensitive to this drug by NMYC overexpression. In contrast, HMGI-C expression is down-regulated during the development of the adrenal gland, completely absent in the adult individual, and only detectable in a subset of ex vivo NB tumors and in RA-resistant NB cell lines. We provide evidence of a causal link between HMGI-C expression and resistance to the growth arrest induced by RA in NB cell lines because exogenous HMGI-C expression in HMGI-C-negative and RA-sensitive cells is sufficient to convert them into RA-resistant cells. Therefore, we suggest that HMGI-C and HMGI(Y) may participate in growth- and differentiation-related tumor progression events of neuroectodermal derivatives.

2-Aminopurine unravels a role for pRB in the regulation of gene expression by transforming growth factor beta.

Transforming growth factor type beta (TGFbeta) is a pleiotropic factor that regulates different cellular activities including cell growth, differentiation, and extracellular matrix deposition. All the known effects of TGFbeta appear to be mediated by its interaction with cell surface receptors that possess a serine/threonine kinase activity. However, the intracellular signals that follow receptor activation and lead to the different cellular responses to TGFbeta are still largely unknown. On the basis of the different sensitivity to the protein kinase inhibitor 2-aminopurine and the phosphatase inhibitor okadaic acid, we identified two distinct pathways through which TGFbeta activates a genomic response. Consistently, 2-aminopurine prevented and okadaic acid potentiated the induction of JE by TGFbeta. The induction of PAI-1 and junB was instead potentiated by 2-aminopurine, after a transient inhibition and was unaffected by okadaic acid. The superinducing effect of 2-aminopurine required the presence of a functional RB protein since it was abolished in SV40 large T antigen-transfected cells, absent in the BT549 and Saos-2 RB-defective cell lines, and restored in BT549 and Saos-2 cells after reintroduction of pRB. The effects of 2-aminopurine on the TGFbeta inducible junB expression occur in all the cell lines examined suggesting that junB, and possibly other genes, can be regulated by TGFbeta through a distinct pRB-dependent pathway.

[The Internet and clinical medicine. An introduction to the biomedical information services available in electronic form on the "net of all nets"]. / Internet e medicina clinica. Una introduzione ai servizi informativi biomedici disponibili in forma elettronica sulla "rete delle reti".

Clinical users expectations from medical informatics are evolving due to the wide availability of biomedical information services on the Internet. Thanks to hypertexts and advanced browsing tools users with no informatical expertise can travel on the Internet and easily gain access to textual databases. With a multimedia computer other kinds of information can be grabbed: images, sounds and audiovisual documents. Basic Internet services (electronic mail, discussion lists, file transfer protocol, terminal emulation) can be accessed from a wide range of hardware equipment. However, the real power of a world-scale computer network like the Internet will be unleashed only when its global connectivity will be linked to the powerful retrieval ability of existing clinical and related databases. While biomedical journals editors and other medical information providers are in the way to offer electronic versions of their paper-based products, at the leading edge of this world-scale process is the USA National Library of Medicine, with the Internet-compatible version of its Grateful Med software which is expected to be launched during 1996.

The mechanism of binding of exogenous DNA to sperm cells: factors controlling the DNA uptake.

Mature sperm cells have the spontaneous capability of taking up exogenous DNA. Potential substrates for the interaction of the DNA with the sperm heads are specific classes of DNA-binding proteins. In the present work three major classes of DNA-binding proteins were identified by Southwestern analysis of sperm head protein extracts: a first class of about 50 kDa in molecular weight, a second one of 30-35 kDa, and finally a third one below 20 kDa. The latter group most probably contains sperm protamines. Our attention was particularly focused on the 30- to 35-kDa proteins as a substrate for DNA binding, as they represented the only group whose electrophoretic mobility was conserved among mammalian species. In addition they were the only class of DNA-binding proteins accessible to exogenous DNA in intact sperm cells. The purified 30- to 35-kDa proteins interacted in vitro with exogenous DNA and generated discrete protein/DNA complexes as determined by band shift assay. A factor blocking the binding of exogenous DNA to sperm cells was also identified in the seminal fluid of mammals and in echinoid spermatoza. The factor also exerted a powerful inhibitory effect on DNA uptake in sperm cells of heterologous species. The 30- to 35-kDa DNA-binding proteins appeared to be the specific target through which the inhibition was mediated. In the presence of the inhibitory factor, the 30- to 35-kDa lost the ability to bind exogenous DNA. Thus, the interaction of exogenous DNA with sperm cells does not appear to be a casual event but, on the contrary, relies on a molecular mechanism based on the cooperation of specific protein factors.

Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

The suggestion that acidic residues in the hydrophobic NH2-terminal domains of Mel permease (Asp-31 in helix I, Asp-51 and Asp-55 in helix II, Asp-120 in helix IV) may be essential components of a coordination network involved in cation recognition (Pourcher, T., Zani, M.L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215) is further analyzed using site-directed mutagenesis. To study whether nearby polar residues also contribute to the cation recognition process, Tyr-24, Tyr-27 and Tyr-28 (aligned with Asp-31) and Tyr-109 and Tyr-116 (aligned with Asp-120) were individually converted into a phenylalanine. The effect of replacing Arg-48 (aligned with Asp-51 and Asp-55) or Asn-83 (in the middle of helix III) by an alanine was also studied. The importance of the position of the carboxylate of the residue at position 31, 51, 55, or 120 was next examined by replacing each Asp by a Glu residue. Sugar binding and/or transport activity measurements indicate that all polar-->apolar or Asp-->Glu mutants use Na+ or Li+ for active sugar transport. Moreover, two groups of mutants could be distinguished. One group, composed of Y27F, Y28F, D31E, and Y109F mutants, retains wild type permease properties. A second group (Y24F, N83A, and Y116F and also D51E, D55E, and D120E) exhibits concomitant reduction of affinity for sodium and sugars and altered sugar specificity but conserves wild type cation selectivity profile. The data reinforce the notion that Asp-51, Asp-55, and Asp-120 residues and the position of their carboxyl side chains are of primary importance for cation recognition. Finally, since Mel permease properties are predominantly modified by mutagenizing residues located in the cytoplasmic half of the permease, we propose that Mel permease has a well-like shape opened toward the periplasmic space and is closed at its cytoplasmic extremity by a gate.