Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

Data obtainedduring the last two decades show that spontaneously hypertensive rats,an acceptable experimental model of primary human hypertension, possessincreased activity of both ubiquitous and renal cell-specific isoformsof theNa⁺/H⁺exchanger (NHE) andNa⁺-K⁺-2Clcotransporter. Abnormalities of these ion transporters have been foundin patients suffering from essential hypertension. Recent geneticstudies demonstrate that genes encoding the - and -subunits ofENaC, a renal cell-specific isoform of theNa⁺-K⁺-2Clcotransporter, and 3-, 1-, and 2-subunits of theNa⁺-K⁺pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis ofthe homology of genome maps, several other genes encoding these transporters, as well as theNa⁺/H⁺exchanger andNa⁺-K⁺-2Clcotransporter, can be predicted in QTL related to the pathogenesis ofhypertension. However, despite their location within QTL, analysis ofcDNA structure did not reveal any mutation in the coding region of theabove-listed transporters in primary hypertension, with the exceptionof G276L substitution in the1-Na⁺-K⁺pump from Dahl salt-sensitive rats and a higher occurrence of T594Mmutation of -ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities ofsystems involved in the regulation of their expression and/or function.Further analysis of QTL in F₂hybrids of normotensive and hypertensive rats and in affected siblingpairs will allow mapping of genes causing abnormalities ofthese regulatory pathways.     

gamma-Tubulin in Leishmania: cell cycle-dependent changes in subcellular localization and heterogeneity of its isoforms

A panel of six anti-peptide antibodies recognizing epitopes in different regions of the gamma-tubulin molecule was used for the characterization and localization of gamma-tubulin during cell cycle in Leishmania promastigotes. Immunofluorescence microscopy revealed the presence of gamma-tubulin in the basal bodies, posterior pole of the cell, and in the flagellum. Furthermore, the antibodies showed punctuate staining in the subpellicular microtubule. This complex localization pattern was observed in both interphase and dividing cells, where staining of posterior poles and the subpellicular corset was more prominent. In posterior poles, gamma-tubulin co-distributed with the 210-kDa microtubule-interacting protein and the 57-kDa protein immunodetected with anti-vimentin antibody. Immunogold electron microscopy on thin sections of isolated flagella showed that gamma-tubulin was associated with the paraflagellar rod (PFR) that runs adjacent to the axonemal microtubules. Under different extraction conditions, gamma-tubulin in Leishmania was found only in insoluble cytoskeletal fractions, in contrast to tubulin dimers that were both in soluble and cytoskeletal pool. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin. Posttranslational modifications of Leishmania gamma-tubulin might therefore have an important role in the regulation of microtubule nucleation and interaction with other proteins. The complex pattern of gamma-tubulin localization and its properties indicate that gamma-tubulin in Leishmania might have other function(s) besides microtubule nucleation.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Modulation of porphyrin binding to serum albumin by pH

In this study, we show that the difference in acidity of functional groups in porphyrin photosensitizers provides a meaningful avenue to achieve differential localization and retention of porphyrins in tissues and cells, and in the end could be a positive factor in the photodynamic treatment of cancer (PDT). We have demonstrated that meso-tetraphenylporphyrin derivative with four phosphonate (bond P(double bond O)(bond OH)(2)) moieties exists in aqueous solutions mainly in four forms that differ by a degree of protonation of the porphyrin ring and ionization of the phosphonate group. It is shown that each porphyrin form has different affinities toward the model protein (bovine serum albumin, BSA). Thus pH of the medium significantly modulates the affinity of the phosphonate porphyrin toward BSA. At lower pH (pH 6.0), the phosphonate porphyrin and BSA form a complex with affinity constant of K(b)=6.9 x 10(5) M(-1), while at pH 7.0 the K(b)=6.1 x 10(5) M(-1). At pH 8.0 the association is significantly lower. Because cancerous cells have generally lower pH (pH approximately 6.9) compared to healthy cells (pH approximately 7.4), the pH of such cells could be a decisive factor for cellular retention of the porphyrin in the form of an associate with intracellular proteins. Moreover, we have also demonstrated that the protonation/deprotonation equilibria do not negatively affect the photophysical properties or ability of phosphonate porphyrin to generate singlet oxygen.     

Coordinated regulation of replication protein A activities by its subunits p14 and p32

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.     

The structures of BtuCD and MscS and their implications for transporter and channel function

The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters. Although these transport families encompass a wide range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane. This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch-activated mechanosensitive channel of small conductance (MscS) and the ATP-dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function.     

Long-term follow-up of childhood-onset hypopituitarism in patients with the PROP-1 gene mutation

OBJECTIVE: The PROP-1 gene mutation is a rare disorder leading to combined pituitary hormone deficiencies over time. The aim was to analyze the clinical picture of 40 years of an almost untreated PROP-1 gene mutation. METHODS: We describe the clinical and hormonal data of 2 brothers from childhood to adulthood as well as imaging procedures (MRI of the pituitary gland, bone mineral density by QCT and DPX). The PROP-1 gene mutation (301-302delAG) was confirmed by DNA sequencing. RESULTS: Although long-standing untreated hypopituitarism was present, there was normal physical and professional activity. Bone mineral density was low only in 1 patient. Adrenocortical deficiency occurred late at 45 and 39 years. CONCLUSIONS: The biological evolution of the PROP-1 gene mutation illustrates the importance of continuous care for these patients. Hormonal deficiencies do not necessarily lead to the same phenotype as is obvious in differences of bone age and bone mineral density.     

RECODE 2003

The RECODE database is a compilation of translational recoding events (programmed ribosomal frameshifting, codon redefinition and translational bypass). The database provides information about the genes utilizing these events for their expression, recoding sites, stimulatory sequences and other relevant information. The Database is freely available at http://recode.genetics.utah.edu/.