Novel insights into transcriptional dysregulation in colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. Although CRC has been comprehensively characterized at the molecular level, the tumor heterogeneity hinders the identification of reliable diagnostic, prognostic and predictive biomarkers. Molecular stratification of CRC is based on prevalent gene mutations and transcription profiles but its significance for clinical practice remains obscure. Indeed, activating mutations in the genes KRAS, NRAS and BRAF are the only predictive biomarkers for anti-EGFR antibody therapy routinely tested the clinic for advanced stages of CRC. Gene expression signatures are important for clarifying the molecular mechanisms of CRC development and progression, but only two such tests for predicting recurrence risk are commercially available. The aim of our study was to propose a diagnostic approach based on mutation and gene expression analysis that can be routinely applied in the clinic for defining the most appropriate treatment strategy for each patient. We used qPCR to determine the presence of KRAS mutations and measure the transcription levels of a panel of 26 genes in 24 CRC patients. Statistical analyses were applied to check for associations between clinico-pathological and molecular parameters. Our results reveal novel data concerning CRC carcinogenesis: almost universal downregulation of EGFR; differential role of the pro-inflammatory cytokines TNF-α and IL-6; overexpression of the vitamin B12 transporter transcobalamin 1; tumor-suppressor function of SETD2, CA7 and GUCA2B. The practical application of these findings has yet to be clarified.

Protein and mRNA levels of YKL-40 in high-grade glioma.

Malignant gliomas are the most common type of primary malignant brain tumours, characterized by extreme proliferation and aggressive invasion. There is evidence for over-expression of the YKL40 gene in high-grade gliomas. The high serum levels of the glycoprotein are associated with poor prognosis of various inflammatory and tumour processes. We investigated the YKL40 mRNA level and protein expression in the tumour site and in the serum of high-grade glioma patients. The YKL-40 expression in 36 patients with glial tumours (astrocytoma grade III, glioblastoma) and 33 age-matched healthy persons was measured by gene analysis, immunohistochemistry and ELISA. YKL-40 serum levels in high-grade glioma patients compared to healthy subjects were significantly increased (P ≤ 0.05). A wide range of variability in YKL40 mRNA expression was found. YKL-40 staining in situ was more abundant in glioblastoma tissue than in anaplastic astrocytoma, with the lowest level in normal brain tissue. Our gene analysis revealed that in general, YKL40 mRNA in glioma patients was over-expressed versus normal brain. A significant correlation between YKL40 transcript and protein levels was observed (P ≤ 0.05). It could be speculated that the YKL-40 protein might contribute to glioblastomas' specific biological characteristics that distinguish them from grade III gliomas. A complex investigation of YKL40 expression was performed at the molecular and cellular levels in human high-grade gliomas. Serum YKL-40 concentrations increased with tumour grade and correlated positively with transcript rate, being the highest in glioblastoma. We provide evidence for a relationship between YKL40 expression and the malignancy of glial tumours.

Hair stem cells for bladder regeneration in rats: preliminary results.

BACKGROUND: A variety of tissue engineering techniques are currently under development or investigation for bladder augmentation, but so far no approach is clearly superior. The aim of this study was to compare the suitability for cystoplasty augmentation in rats of in vivo implanted acellular bladder matrices (BAM) previously seeded with hair follicle stem cells and that of matrices implanted without the cells. MATERIALS AND METHODS: The rat hair follicle stem cell line was positive for CD34, p63, and Ki-67. 1 x 10(6) cells from 34 to 40 passages seeded onto nine BAM scaffolds were cultured for one week. Nine other scaffolds were left unseeded. Scaffolds were grafted into a surgically created defect within the anterior bladder wall: nine rats with acellular matrices and nine with cell-seeded BAM. Rats observed for six months were killed in monthly intervals. We performed gross examination, X-ray cystography, and hematoxylin-eosin, cytokine (CK)-7, CK-20, myoglobin, and desmin staining of the excised bladders. RESULTS: Minimal adhesions were observed and urinary leakage was noted in one case. Two animals died in the acellular group. Rats developed stone disease in bladders reconstructed with acellular BAM. Bladder capacity was similar, but the shape was regular and characteristically oval only in bladders grafted with cell-seeded BAM. Muscle layers in the apical parts of the reconstructed bladder walls were extremely thin in the cases of acellular grafts and thicker in bladders reconstructed with cell-seeded grafts. Muscle layer regeneration was better in the cell-seeded group. Urothelium regenerated in all animals. CONCLUSIONS: We have shown that hair follicle stem cells may be used for rat bladder wall regeneration.

Primary cultures from rat vibrissae as a potential cell source for in vitro construction of urinary bladder wall grafts.

BACKGROUND: In vitro-constructed grafts can be used for human bladder augmentation. There are many diseases in which autologous cells cannot be used for this purpose. The aim of the present study was to examine the potential of rat vibrissae hair follicle cells to form cultures, which could serve as a source for in vitro creation of urinary bladder wall grafts. METHODS: Two hundred vibrissae were excised from young Wistar male rats. Two different digestions were performed, in dispase and in collagenase. All follicles were additionally incubated in trypsin and ethylenediamine tetraacetic acid. Two different culture media based on DMEM (Dulbecco's Modified Eagle's Medium) were used: the first was supplemented with keratinocyte growth factor (KGF) and the second with epidermal growth factor. Immunocytochemical detection of cytokeratin, CD34, p63, Ki-67 (proliferation index), and HMB45 (Human Melanoma Black 45) was performed. RESULTS: Forty-eight primary cultures of rat follicle vibrissae cells were established from 200 hair follicles (24% successful rate). Twenty-four primary cultures were obtained after dispase digestion and 24 after collagenase treatment. Each group was cultured in 2 different media. A heterogeneity of primary cultures was observed. Cells formed a monolayer within a period of 2 to 4 weeks. The 24 primary cultures established after dispase treatment exhibited monolayers of small cuboid cells expressing cytokeratin and CD34. In the 40th passage 20%-40% of cells expressed p63; 85% of these cells from late passages were positive for Ki-67, indicating preserved mitotic potential. Epithelial-like phenotype was observed after dispase digestion and cultivation in KGF-supplemented medium. After 3 weeks, the morphology of these cells changed into fibroblast-like. These cultures were negative for CD34. Fibroblast-like cell growth was observed after collagenase treatment in both KGF- and EGF-supplemented media. These cells were positive for the melanocyte cell marker (HMB45). CONCLUSIONS: Culture media and isolation conditions influence hair follicle stem cell differentiation. The stem cell niche within the hair follicles is a reservoir of cells, which can be potentially used for in vitro creation of urinary bladder wall grafts.

Histo-blood group antigen expression and proliferative activity of fibroblasts treated with dental monomers.

The present work is focused on examining the effect of the structurally similar dental monomers bis-GA and bis-GMA on the expression of histo-blood group antigens (HBGAs) in comparison with fibroblast vitality and proliferation. The fibroblast cell line McCoy-Plovdiv was cultivated in a serum-free medium and was treated with both monomers. Cell vitality was measured by the crystal violet test. Mitotic index and cell morphology were assessed. An immunocytochemical technique was applied to follow the expression of proliferative antigens PCNA and Ki-67 and of HBGA. The expression level of HBGA was measured by an improved pixel selection algorithm with proprietary software. The lowest concentration of 2.5 micromol/L did not significantly affect morphology, vitality, or proliferation activity. Interestingly, the quantitative analysis revealed augmented expression of HBGA B at 2.5 micromol/L. The higher concentrations of the dental monomers reduced cell vitality and mitotic indices and altered proliferative antigen expression. Bis-GA proved to be more toxic than bis-GMA and caused more prominent alterations including greater enhancement of HBGA B expression. We present novel evidence for altered expression of proliferative antigens and enhanced expression of HBGA B in fibroblasts treated with dental monomers bis-GA and bis-GMA suggesting that these substances affect cell morphology, proliferative activity, and antigenic profile.

Differential expression of ABH histo-blood group antigens and LAMPs in infantile hemangioma.

Although infantile hemangioma (IH) are the most common tumors of infancy, the mechanism of their proliferation and involution remains vague. Proliferation, differentiation and death of endothelial cells are the basic processes involved in their pathobiology. Here we hypothesize that the glycoconjugates ABH histo-blood group antigens (HBGA) and lysosome-associated membrane proteins (LAMPs) might be implied in both the differentiation and death of endothelial cells during vascular remodeling in IH. Proliferating and involuting IH were examined immunohistochemically for HGBA and LAMP expression together with vWF and CD31. Proliferative and apoptotic indices were determined. LAMPs were found in immature endothelium of proliferating IH. In involution an increased number of immunopositive cells stained with higher intensity was detected. The enhanced expression might be associated with augmented autophagy required for tissue remodeling during tumor involution. HBGA presented an opposite pattern of expression--they stained intensely the endothelium of mature capillaries, while the immature ones were positive for vWF. The presence of HBGA in endothelial cells of IH may be related to the differentiation process only, as well as to endothelial adhesion and angiogenesis. Novel evidence for differential expression of HBGA and LAMPs in proliferative and involutive phases of IH is presented.

[Thyroid nodules: histological approach using microbiopsies obtained with a biopsy gun: preliminary study]. / Nodules thyroïdiens.

Surgery remains the safest treatment of thyroid nodules, even though the frequency of the cancer is low. Modern diagnostic techniques are aimed at reducing the number of useless surgical interventions. These techniques rely on diagnostic patterns; cytology plays a predominant role. Nevertheless, it is difficult do obtain a sample with enough cellular material for the cytology study. The pathologist must also have skilled experience to provide a reliable reading. Surgical biopsies are no longer performed due to the risk of severe complications. Microbiopsy techniques have been greatly improved and are now widely used for many organs. In this preliminary study, we performed 18 microbiopsies of thyroid nodules measuring less than 1 cm in 18 patients using a 20-gauge biopsy gun. We had one complication: a cervical hematoma after the third biopsy in the series that resolved rapidly. The pathology examination identified follicular adenoma in 14 cases, and Hashimoto thyroiditis in 1. Three small samples contained subnormal thyroid vesicles with suspicion of adenoma for one. All samples were diagnostic, more cellular and easier to read than cytology specimens. This study points to the usefulness of these microbiopsy techniques for histological analysis, particularly applicable to thyroid nodules, and the obvious benefit in biopsy and reading efficiency.

Expression of human blood group antigens A and B in kidney and lung of some vertebrates.

Human blood group antigens (BGA) are genetically determined glycoproteins found in many cells and tissues of different mammals. Their major biological functions are still undefined. There are few investigations analysing the evolutionary aspect of BGA tissue distribution. The present study is aimed at examining the expression of human A and B antigens in the kidney and lung of some free-living vertebrates. The biotin-streptavidin-peroxidase immunostaining system was applied on kidney and lung paraffin sections derived from free-living representatives of five different vertebrate classes. Excluding the possibility of any non-specific staining by the application of inhibition tests, A and B antigens were demonstrated most constantly in epithelial cells of renal and respiratory tubules. They were also detected in chondrocytes of fish gills, in some muscular and endothelial cells. Single erythrocytes showed a positive cytoplasmic staining only in some higher vertebrates. Human BGA seem to be conserved carbohydrate structures with biological functions probably related to cell integrity and differentiation.

Evidence for protein splicing in the endoplasmic reticulum-Golgi intermediate compartment.

We present evidence that the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is a site for protein splicing of cathepsin C: (i) maturation of the enzyme in COS 7 cells is a two-step process starting within the ERGIC, and (ii) the intermediately processed polypeptide retains both termini of the proenzyme and lacks an internal fragment.

Expression of human blood group antigens A and B in stomach cells of C. carpio, C. auratus, R. ridibunda and H. sapiens.

Human blood group ABH antigens are found not only on red blood cell membranes, but in many other cell types as well. Their biological functions still remain unclear. The aim of the present study was to examine the cellular expression of these antigens in the stomach of representatives of different Vertebrates--Pisces and Amphibia and to compare it with their expression found in Man. The immunohistochemical technique applied was based on the biotinstreptavidin-peroxidase complex. Monoclonal antibodies to human A and B antigens were used as primary antibodies in the system. Stomach paraffin sections from Cyprinus carpio, Carassius auratus, Rana ridibunda and Homo sapiens were examined. Blood group antigens were found mainly in tunica mucosa of C. carpio, C. auratus and R. ridibunda. Tunica muscularis and tunica serosa were always immunonegative. The antigens were localized in the apical part of the cytoplasm of epithelial cells in lamina epithelialis. Strong positive reaction was seen in secretory granules of the stomach of C. auratus, while in R. ridibunda the antigens were expressed by single epithelial cells in cardial stomach glands. A and B antigens were not found in human stomach sections most probably due to the negative secretor status of the individuals studied. Our results show that ABH human blood group antigens are evolutionary conserved structures similarly expressed by different Vertebrates. The phylogenetic stability in their cellular expression possibly results from the important biological role they have. Future large scale systematic investigations could elucidate the undefined and disputable physiological functions of human blood group antigens.

Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells.

Lysosomal-membrane-associated glycoproteins (Lamps) 1 and 2 are rarely found on the plasma membranes of normal cells. There is evidence suggesting an increase in their cell-surface expression in tumor cells, with some data indicating that the adhesion of some cancer cells to the extracellular matrix is partly mediated by interactions between Lamps and E-selectin and between Lamps and galectins (endogenous-galactoside-binding lectins). The present study examined the expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in different human tumor cell lines. Indirect immunofluorescence staining revealed accumulation of Lamp molecules at the edges of A2058 human metastasizing melanoma cells suggesting that these glycoproteins could participate in cell adhesion. Flow cytometry showed the presence of cell-surface Lamps in A2058, HT1080 (human fibrosarcoma) and CaCo-2 (human colon-adenocarcinoma) cells. Treatment with 2 mM sodium butyrate for 24 and 48 hr resulted in a significant increase in Lamps surface expression. A strong binding of A2058 to recombinant galectin-3 was detected by FACS. The application of 2 and 5 mM butyrate for the same incubation period enhanced galectin-3 binding to Lamps-expressing cells. Our results support the idea that Lamps may be considered a new family of adhesive glycoproteins participating in the complex process of tumor invasion and metastasis.

ABH blood group antigen significance as markers of endothelial differentiation of mesenchymal cells.

The expression pattern of A, B and H blood group antigens was evaluated by staining frozen sections with specific monoclonal antibodies developed by us and using the indirect immunoperoxidase method. The expression of blood group antigens was ubiquitously upregulated in the endothelial cells of fetal organs. In the process of their differentiation to endothelial naive embryonic mesenchymal cells expressed cytoplasmic ABH antigens. They were assumed as products of the activation of the respective genes. ABH antigen expression was considered as suggestive evidence for the assumption that blood group antigens could serve as early immunomorphologic markers of endothelial differentiation of mesenchymal cells, thus specifying the location of future blood vessels. Extending the conceptual framework of blood group antigens' significance we consider them as being possibly involved in the process of fetal morphogenesis.

Arrangement of a cluster of three mouse type I keratin genes expressed sequentially during esophageal-type epithelial cell differentiation.

Keratins are intermediate filament proteins expressed in epithelial cells. They are divided into two groups, type I and type II, that must associate to form filaments. The genes encoding these proteins are clustered in two type-specific loci. In stratified epithelia, differentiation of the basal cells is accompanied by a switch in the expression of keratin genes. However, how this switch is controlled is not yet understood. We report here the cloning and mapping of a 55-kb region surrounding the keratin 19 (K19) gene in the mouse genome. This gene encodes a type I subunit expressed in simple and complex epithelia, notably in nonkeratinizing stratified epithelia of internal organs. In these tissues, it is expressed in basal cells and not in suprabasal cells, where the main type I subunit is keratin 13. Using probes corresponding to highly conserved sequences in intermediate filament proteins, we mapped two other genes downstream from the K19 gene. Restriction mapping and sequencing data indicate that they encode the mouse K15 and K13. The three genes are separated by about 5-6 kb, and they are in the same transcriptional orientation. Because the three genes are expressed together in stratified epithelia and because their order of expression during differentiation is the same as their order on the chromosome, we suggest that there is a relationship between their genomic organization and the control of their expression.

Immunohistochemical study on pigmented nevi (preliminary communication).

The human ABH blood-group antigen expression of cytokeratin, S-100 protein, and the melanoma-associated antigen was studied in 12 cases of pigmented nevi. The biotin-streptavidin immunostaining system was applied. ABH antigens were found both in the endothelial cells and in the germinative epidermal layer. The flat overlaying epithelium, single melanocytes and melanoblasts were positive for cytokeratin. Some basilar cells and the giant multinucleate cells in the Spitz nevi stained for S-100 protein. The melanoma-associated antigen was visualized in the upper epidermal layers as well as in some melanocytes. The present article discusses the specificity of the antigens studied as well as some immunologic traits of potential malignant development of Spitz nevi.

Expression of A, B and H blood-group antigens and carcinoembryonic antigen in human tumours.

The parallel expression of ABH blood-group antigens and of the carcinoembryonic antigen was examined applying the biotin-streptavidin immunostaining system. Monoclonal antibodies to the ABH antigens newly produced by us and a polyclonal antibody to the carcinoembryonic antigen (Ortho) were used as primary antibodies. Human tumours derived from six different organs were studied. ABH antigens showed a heterogeneous expression. They were entirely missing in some neoplastic tissues and found in single or in clustered tumour cells in others. The staining for the carcinoembryonic antigen revealed stronger intensity and covered large malignant areas. The possible functions of ABH blood-group antigens as tumour-associated structures are discussed. A number of tumour-associated antigens have been identified which may serve to improve the early diagnostics of tumour processes in man. Recently, the blood-group antigens (BGA) from the ABO/H system have attracted the attention of many investigators who regard them as differentiation antigens and as tumour-associated structures. Oncogenesis is accompanied by a block in BGA biosynthesis as a result of an altered ontogenetical programme. This process leads to: a) deletion of BGA-structures in malignant cells; b) neoantigen expression (onco-developmental markers) and c) appearance of BGA-incompatible antigens. The aim of the present investigation is to examine the coexpression of A, B and H BGA and of the carcinoembryonic antigen (CEA) in malignant human tissues. Using the monoclonal antibodies (MAbs) to ABH-BGA newly produced in our laboratory, some insufficiently explored organs like the mammary gland and especially its metastases were also tested.

[The immunobiological functions of human ABO (H)-system blood-group antigens]. / Imunobiologichni funktsii na choveshkite kruvnogrupovi antigeni ot sistemata ABO (H).

The human ABO (H) blood group antigens are genetically determined carbohydrate structures found in different cells and tissues. The modulation in their expression in the course of human onto- and oncogenesis gives grounds for speculations concerning the role of ABO (H) antigens as molecules, participating in cell interactions, adhesion and inhibition events, as well as in tumor metastases. The deletion of antigenic determinants observed in a number of human carcinomas, and the epitope homology between the blood group antigens and the carcinoembryonic antigen, focuses on clarifying the diagnostic and prognostic value of the ABO (H) antigens as tumor-associated markers.

Common identity of substrate binding subunit of vacuolar h-translocating inorganic pyrophosphatase of higher plant cells.

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H(+)-translocating inorganic pyrophosphatase (tonoplast H(+)-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (M(r)) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H(+)-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H(+)-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (M(r(Beta)) = 64,500; M(r(Vigna)) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [(14)C]-N-ethylmaleimide. Similarly, the M(r) 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the M(r) 66,000 subunit of the H(+)-PPase isolated from Vigna and the direct sequence data from Beta align precisely with the deduced amino acid sequence of cDNAs encoding the H(+)-PPase of Arabidopsis, all three enzymes are inferred to be highly conserved structurally. Accordingly, immunoblots of membranes prepared from Arabidopsis, Beta, Vigna, and Zea, probed with antibody affinity purified against the magnesium inorganic pyrophosphate-binding, M(r) 66,000 polypeptide of Vigna, reveal a single immunoreactive band at M(r) 64,500 to 67,000 in all four preparations. The M(r) 66,000 polypeptide of Zea membranes is, however, prone to proteolysis during membrane fractionation and selective aggregation during sample denaturation for SDS-PAGE. The anomalous M(r) 37,000 to 45,000 subunit pattern previously ascribed to the H(+)-PPase from Zea (A. Chanson and P.E. Pilet [1989] Plant Physiol 90: 934-938) is attributed to loss of the M(r) 66,000 subunit and the appearance of polypeptide fragments of M(r) 44,700 and 39,000 through the combined effects of sample aggregation before SDS-PAGE and proteolysis, respectively. It is, therefore, concluded that the substrate-binding subunit of the tonoplast H(+)-PPase has a common identity in all four organisms.

Vacuolar H(+)-translocating pyrophosphatases: a new category of ion translocase.

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.

Radiation-inactivation analysis of vacuolar H(+)-ATPase and H(+)-pyrophosphatase from Beta vulgaris L. Functional sizes for substrate hydrolysis and for H+ transport.

The functional sizes of the vacuolar H(+)-ATPase (V-ATPase; EC 3.6.1.34) and H(+)-pyrophosphatase (PPase; EC 3.6.1.1) from vacuolar membranes of red beet (Beta vulgaris L.) were estimated by radiation inactivation, both for substrate hydrolysis and for H+ transport. For the V-ATPase, the radiation-inactivation size for H+ transport was 446 (403-497) kDa and that for ATP hydrolysis was 394 (359-435) kDa. The low values of both of these estimates suggest that not all subunits which may co-purify with V-ATPases are required for either hydrolysis or transport. For the PPase, the radiation-inactivation size for hydrolysis was 91 (82-103) kDa, suggesting that the minimum functional unit for hydrolysis is the 81 kDa monomer. In contrast to the V-ATPase, the PPase gave a radiation-inactivation size for transport which was 3-4-fold larger than that for hydrolysis (two estimates for transport gave 307 and 350 kDa), indicating that a single catalytic subunit is insufficient for transport activity.

Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana.

The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H+ pumps: a vacuolar ("V-type") H(+)-ATPase (EC 3.6.1.3) and a H(+)-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1). The H(+)-PPase, like the V-type H(+)-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast H(+)-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H(+)-PPase of Arabidopsis thaliana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H(+)-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the Mr 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H(+)-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H(+)-PPase and any other characterized H+ pump or PPi-dependent enzyme implies a different evolutionary origin for this translocase.