New skeletal tuberculosis cases in past populations from Western Hungary (Transdanubia).

The distribution, antiquity and epidemiology of tuberculosis (TB) have previously been studied in osteoarchaeological material in the eastern part of Hungary, mainly on the Great Plain. The purpose of this study is to map the occurrence of skeletal TB in different centuries in the western part of Hungary, Transdanubia, and to present new cases we have found. Palaeopathological analysis was carried out using macroscopic observation supported by radiographic and molecular methods. A large human osteoarchaeological sample (n=5684) from Transdanubian archaeological sites ranging from the 2nd to the 18th centuries served as a source of material. Spinal TB was observed in seven individuals (in three specimens with Pott's disease two of which also had cold abscess) and hip TB was assumed in one case. The results of DNA for Mycobacterium tuberculosis were positive in seven of the eight cases identified by paleopathology, and negative in the assumed case of hip TB. However, the molecular results are consistent with highly fragmented DNA, which limited further analysis. Based on the present study and previously published cases, osteotuberculosis was found in Transdanubia mainly during the 9th-13th centuries. However, there are no signs of TB in many other 9th-13th century sites, even in those that lie geographically close to those where osteotuberculous cases were found. This may be due to a true absence of TB caused by the different living conditions, way of life, or origin of these populations. An alternative explanation is that TB was present in some individuals with no typical paleopathology, but that death occurred before skeletal morphological features could develop.

Appearance of hyperostosis frontalis interna in some osteoarcheological series from Hungary.

Hyperostosis frontalis interna (HFI) is a generalised pathological condition with an unknown etiology and variable clinical association. It is characterized by excess bone growth and manifested on the inner table of the frontal bone, occasionally extending onto the temporals, parietals and the occipital. The etiology of HFI is uncertain: it may be an unknown genetic predisposition, a common environmental exposure, or special metabolic diseases. The purpose of the present study is to report cases of HFI in some osteoarcheological series from Hungary and to emphasize the importance of the investigation of HFI in ancient populations. Twenty out of 803 adults with observable frontal bones exhibited HFI, ranging from early to mid-type, including 15 females and 5 males. Some overgrowths with edges were blending into the endocranial surface, and some were prominently protruding from the surface. Advanced cases of HFI (type C) were observed after age 40-60 years.

Nephrin localizes to the slit pore of the glomerular epithelial cell.

BACKGROUND: Recognition that mutation of the protein nephrin, encoded by the NPHS1 gene, singly results in the cellular alterations that result in foot process effacement, and nephrotic range proteinuria emphasizes the pivotal role that this protein plays in regulating glomerular filter integrity. This article reports the development of reagents necessary to study the biology of nephrin in mouse, and describes the initial characterization of the nephrin protein. METHODS: A cDNA including the full-length mouse nephrin open reading frame was cloned and sequenced. Immuno-affinity purified polyclonal antiserum directed against the cytoplasmic domain of mouse nephrin was developed. RESULTS: Nephrin identified in mouse glomerular extract was found to be a glycoprotein with an apparent molecular mass of 185 kDa. As detected by indirect immunofluorescence microscopy and immunogold electron microscopy, nephrin was located only in visceral glomerular epithelial cells, where it was targeted to intercellular junctions of mature podocyte foot processes. In developing glomeruli of newborn mouse, antinephrin immunolocalized to the earliest slit pore regions between differentiating podocytes, sites where slit diaphragms first become visible. CONCLUSION: As a putative cell adhesion molecule of the immunoglobulin superfamily, nephrin likely participates in cell-cell interactions between podocyte foot processes and may represent a component of the slit diaphragm.

Analysis of the inducer-responsive CAR1 upstream activation sequence (UASI) and the factors required for its operation.

Induced production of arginase (CAR1) enzyme activity and steady-state CAR1 mRNA in Saccharomyces cerevisiae requires wild-type ARG80/ARGRI and ARG81/ARGRII gene products. We demonstrate here that these gene products, along with that of the MCM1 gene, are required for the inducer-dependent USAI-A, UASI-B and UASI-C elements to function but they are not required for operation of inducer-independent CAR1 UASC1 or UASC2. Through the use of single and multiple point mutations, the CAR1 UASI-B and UASI-C elements were demonstrated to be at least 23 bp in length. Moreover, simultaneous mutation of both ends of an elements gave stronger phenotypes than mutations at either end. The center of the element was more sensitive to mutation than were the ends.

Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element.

Saturation mutagenesis is one approach for determining the contributions of individual base pairs to the structure and function of defined DNA sequence elements. In this paper, we describe a novel method for saturation mutagenesis involving PCR amplification with degenerate synthetic oligonucleotides as primers. The degeneracy is confined to a specific target within the primer by mixing a low percentage of the three non-wild type (non-WT) nucleotide precursors with WT at specific positions during primer synthesis. PCR amplification of WT template DNA with the degenerate primer and an opposing WT primer, followed by subsequent cloning using restriction sites designed into the primers, results in recovery of a population of randomly mutated products. Since primers with multiple mutations hybridize less efficiently to WT template DNA during PCR amplification, the recovery of mutants with multiple base changes is greatly reduced. The efficient generation of random point mutations with this method allows the construction of separate mutant populations, each mutagenized over a different portion of the DNA sequence element. If a phenotypic assay is available, these populations can be screened directly to define those regions within the element that are important for activity. Only those populations containing mutations in the important regions require further characterization by DNA sequence analysis.

Participation of RAP1 protein in expression of the Saccharomyces cerevisiae arginase (CAR1) gene.

Regulated expression of the inducible arginase (CAR1) gene of Saccharomyces cerevisiae has been shown to require three upstream activation sequences (UASs) and an upstream repression sequence, URS1. Two of the UAS elements, UASC1 and UASC2, operate in an inducer-independent manner, while the third, UASI, is inducer dependent. UASC1 and UASC2 were previously shown to contain ABF-1 binding sites that were required for normal transcription. In this work, we demonstrate that UASC1 and UASC2 also contain two and three sites, respectively, that are able to bind RAP1 protein. RAP1 binding to these sites, however, is significantly weaker than that to sites in TEF2 and HMRE. The effects of mutating the sites individually or in combination suggest that at least three of them, two in UASC1 and one in UASC2, probably participate in CAR1 expression.

Tripartite structure of the Saccharomyces cerevisiae arginase (CAR1) gene inducer-responsive upstream activation sequence.

Arginase (CAR1) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR1 contains four separable regulatory elements--two inducer-independent upstream activation sequences (UASs) (UASC1 and UASC2), an inducer-dependent UAS (UASI), and an upstream repression sequence (URS1) which negatively regulates CAR1 and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI-A, UASI-B, and UASI-C). Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction. The elements operate in all orientations relative to one another and to the TATA sequence. All three UASI elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins. The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRII deletion mutants. Similar sequences situated upstream of ARG5,6 and ARG3 and reported to negatively regulate their expression are able to functionally substitute for the CAR1 UASI elements and mediate reporter gene expression.

Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.

Localization of protective epitopes within the pilin subunit of the Vibrio cholerae toxin-coregulated pilus.

From a collection of monoclonal antibodies (MAbs) that recognize the native structure of the toxin-coregulated pilus of Vibrio cholerae, two protective MAbs (16.1 and 169.1) were used to localize the corresponding epitopes on the pilus. These MAbs were shown to specifically recognize the carboxyl half of the TcpA pilin subunit, as determined by their recognition of proteolytic fragments and hybrid pilin proteins. The positions of the epitopes were precisely determined through the use of overlapping synthetic peptides corresponding to this region of the pilin. The MAbs were found to recognize adjacent peptides, delineating a region between residues 157 and 199. Since the protective nature is specific for these two antibodies, the findings suggest that this region defines a domain that participates in toxin-coregulated pilus-mediated colonization and therefore represents a target for studies of its potential as an immunogen for incorporation into a component cholera vaccine.

Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae.

Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.