Sequence of the gene encoding an alkaline serine protease of thermophilic Bacillus smithii.

The complete nucleotide sequence of the gene encoding an alkaline serine protease from Bacillus smithii has been determined. Degenerate oligodeoxyribonucleotide primers were used to prime the amplification of a 507-bp sequence of the gene. This sequence was successively used for constructing new primers applied in inverse polymerase chain reaction, using as template self-ligated DNA fragments. The deduced amino-acid sequence is compared to serine proteases from B. amyloliquefaciens, B. licheniformis, B. subtilis and Thermus aquaticus.

BliI, a restriction endonuclease from Bacillus licheniformis.

From Bacillus licheniformis a site-specific restriction endonuclease, named BliI, has been purified and characterized. BliI was able to digest lambda DNA at pH 9.1 over a wide temperature range (25-65 degrees C). Digestion of lambda and psi X174 DNAs with BliI produced banding patterns identical to those seen with HaeIII. Therefore, BliI and HaeIII endonculeases are isoschizomers.

5-(5,6-Dichloro-2-indolyl)-2-methoxy-2,4-pentadienamides: novel and selective inhibitors of the vacuolar H+-ATPase of osteoclasts with bone antiresorptive activity.

The vacuolar H+-ATPase (V-ATPase), located on the ruffled border of the osteoclast, is a proton pump which is responsible for secreting the massive amounts of protons that are required for the bone resorption process. With the aim to identify new agents which are able to prevent the excessive bone resorption associated with osteoporosis, we have designed a novel class of potent and selective inhibitors of the osteoclast proton pump, starting from the structure of the specific V-ATPase inhibitor bafilomycin A1. Compounds 3a-d potently inhibited the V-ATPase in chicken osteoclast membranes (IC50 = 60-180 nM) and were able to prevent bone resorption by human osteoclasts in vitro at low-nanomolar concentrations. Notably, the EC50 of compound 3c in this assay was 45-fold lower than the concentration required for half-maximal inhibition of the V-ATPase from human kidney cortex. These results support the validity of the osteoclast proton pump as a useful molecular target to produce novel inhibitors of bone resorption, potentially useful as antiosteporotic agents.

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Genotypic characterization of thermophilic bacilli: a study on new soil isolates and several reference strains.

A genotypic study using amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA fingerprinting (RAPD) and ribosomal spacer analysis (RSA) in comparison with DNA-DNA reassociation experiments was carried out with 85 thermophilic Bacillus isolates from uncultivated soil of 14 different geographical areas and seventeen reference strains representing defined thermophilic Bacillus species. This approach permitted the attribution of 51% of the new isolates to the Bacillus thermoleovorans group and the identification of 40% of the new isolates as B. "thermodenitrificans". Moreover, 2 strains were assigned to B. pallidus species and 1 isolate to B. thermosphaericus species. The remaining 6% of our thermophilic isolates from soil, constituting 2 DNA-DNA homology groups, are still unidentified. A detailed genotypic characterization of the heterogeneous species of B. thermoleovorans and B. stearothermophilus was also presented.

Detection and characterization of naturally occurring plasmids in Bacillus licheniformis.

Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.

Specific identification of Lactobacillus helveticus by PCR with pepC, pepN and htrA targeted primers.

Specific regions in three genes coding for aminopeptidases C and N, and a trypsin-like serine protease were selected as species-specific primer sequences for rapid and reliable identification of Lactobacillus helveticus strains. The PCR procedures carried out gave specific 524-, 726- and 918-bp amplificates, with DNA isolated from L. helveticus. No PCR product was generated for closely related bacteria. The amplification products were also screened for their species specificity in dot blot hybridization with representatives of the most closely related genera and species and a number of other bacterial species.

Influence of functional groups on homologous antibody affinity of 11 alpha-hydroxyprogesterone derivatives. I. Synthesis and affinity evaluation.

Syntheses and cross-reactivities with progesterone toward the same specific antibody are reported for a series of amides of 11 alpha-hydroxyprogesterone 11-hemisuccinate. Some hypotheses are made regarding the effects of the chemical structure of the substituents on the immunological properties of derivatives.

Two new fluorescent derivatives of progesterone to use in fluoroimmunoassay.

Synthesis, fluorometric and immunological properties of two new fluorescent derivatives of progesterone are reported. Both compounds were obtained from 11 alpha-hydroxyprogesterone 11-hemisuccinate; the fluorescent molecules were joined to the steroid by bifunctional arms. The first of these is cysteamine whose thiol group was reacted with N-(3-fluoranthenyl) maleimide, and the second is tyramine whose phenolic group was reacted with 1-nitroso-2 naphthol.

Development of molecular RAPD marker for the identification of Pediococcus acidilactici strains.

A RAPD analysis performed using a single primer targeted to the pediocin AcH/PA-1 gene was carried out on several P. acidilactici strains and on some related species of lactic acid bacteria. The high degree of genetic variability detected in P. acidilactici strains did not allow the selection of a common RAPD fragment that could be chosen as a potential species-specific DNA marker. Nevertheless a 700 bp fragment, that was found to be peculiar of all potential pediocin producer strains analyzed, was cloned and sequenced with the aim to develop a species specific PCR marker. Sequence analysis of the cloned 700 bp fragment showed one putative small open reading frame (ORF1), with no significant homology with known genes, and a partial putative second coding region (ORF2) with a high degree of similarity with several methionyl tRNA synthesis (metS) genes. The two coding regions were separated by a short spacer region. Primers targeted to ORF2 plus part of the spacer region and primers designed for the amplification of the entire cloned RAPD fragment were found to be species-specific for the detection of P. acidilactici strains. Furthermore primers designed on the ORF1 sequence allowed the amplification of a 439 bp fragment only in some P. acidilactici strains, including pediocin producing strains.

Discrimination among Pediocin AcH/PA-1 producer strains by comparison of pedB and pedD amplified genes and by multiplex PCR assay.

Pediococcus acidilactici, Pediococcus parvulus and Lactobacillus plantarum pediocin AcH/PA-1 produces strains were studied with the aim to investigate their common genetic pediocin determinant using pedA, pedB, pedC and pedD gene-targeted PCR assay. Single Strand Conformation Polymorphism and restriction analysis of pedA and pedC amplified fragments from the three different species did not show any differences in sequence while these analysis carried out on pedB and pedD amplified fragments highlighted differences related to the three species analyzed harboring these plasmid encoded genes. Furthermore different multiplex PCR assay using IdhD, pedA and pedD as target genes were developed to clearly identify the pediocin AcH/PA-1 producer strains and to obtain the simultaneous identification of the P. acidilactici strains.

Contribution to phenotypic and genotypic characterization of Bacillus licheniformis and description of new genomovars.

A study of phenotypic and genotypic characteristics was carried out on 182 strains isolated from soil of different geographical areas; the type strains were B. licheniformis, B. subtilis, B. pumilus, B. cereus and B. coagulans. The results showed, primarily on the basis of phenotypic features, that all the isolates belonged to the B. licheniformis species, however DNA relatedness studies revealed only 161 to be genetically related to B. licheniformis, the DNA relatedness levels ranging from 66 to 100%. The other 21 isolates appeared to be genetically distinct not only from B. licheniformis but also from B. subtilis and B. pumilus, where there were low levels of DNA relatedness (from 4 to 37%). Nevertheless the ARDRA results indicate that the 21 atypical isolates were phylogenetically related to B. licheniformis. Our data and the phenotypic homogeneity found suggest the presence of three different genomovars.

Novel bone antiresorptive agents that selectively inhibit the osteoclast V-H+-ATPase.

The vacuolar proton pump (V-ATPase) located on the plasma membrane of the osteoclast is a potential molecular target for the discovery of novel bone antiresorptive agents useful for the treatment of osteoporosis. In order to design novel compounds able to selectively inhibit the osteoclast V-ATPase we firstly identified the minimal structural requirements of bafilomycin A1, a macrolide antibiotic which potently inhibits all V-ATPases. This information allowed the design of 2-(indole)pentadienamide derivatives whose optimization led to a novel class of potent inhibitors that demonstrated a high degree of selectivity for the osteoclast V-ATPase. The most interesting derivative, SB-242784, was able to inhibit bone resorption by human osteoclasts in vitro and to completely prevent ovariectomy-induced bone loss in rats when administered orally at 10 mg kg(-1) day(-1). Structure activity relationships of this class of compounds were investigated further by replacing the 2,4-pentadienoyl chain with suitable spacers able to maintain the correct orientation and distance between the indole ring and the amide moiety.

Selective delta opioid receptor agonists for inflammatory and neuropathic pain.

In the last decade a number of selective and potent non-peptidic agents became available to explore the usefulness of the delta-opioid receptor in modulation of pain of different origins. As a continuing effort in this field, potent and selective delta-opioid agonists based on the pyrrolomorphinan framework have been designed, synthesised and characterised biologically in our laboratories. In animal models, a selected compound of interest, SB 235863, has proved the concept that selective delta-opioid agonists may have great potential as pain relief agents in inflammatory and neuropathic pain conditions. Importantly, such a compound was free of the unwanted side effects usually associated with narcotic analgesics such as morphine.

Small rolling circle plasmids in Bacillus subtilis and related species: organization, distribution, and their possible role in host physiology.

Bacillus subtilis and related species (Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus mojavensis) represent a group of bacteria largely studied and widely employed by industry. Small rolling circle replicating plasmids of this group of bacteria have been intensively studied as they represent a convenient model for genetic research and for the construction of molecular tools for the genetic modification of their hosts. Through the computational analysis of the available plasmid sequences to date, the first part of this review focuses on the main stages that the present model for rolling circle replication involves, citing the research data which helped to elucidate the mechanism by which these molecules replicate. Analysis of the distribution and phylogeny of the small RC plasmids inside the Bacillus genus is then considered, emphasizing the low level of diversity observed among these plasmids through the in silico analysis of their organization and the sequence divergence of their replication module. Finally, the parasitic vs. mutualistic nature of small rolling circle plasmids is briefly discussed.

Occult metastatic lung carcinoma presenting as locally advanced uterine carcinosarcoma on positron emission tomography/computed tomography imaging.

Increasingly, positron emission tomography/computed tomography (PET/CT) is being used as a tumor surveillance modality for multiple tumor types. A 73-year-old postmenopausal female with stage IV nonsmall cell lung cancer presented after a PET/CT demonstrated focal uptake in the superior and lateral aspects of the uterus. The patient reported a history of intermittent postmenopausal bleeding and an endometrial biopsy documented uterine carcinosarcoma. Postoperative pathologic review and immunohistochemical staining with thyroid transcription factor-1 revealed metastatic adenocarcinoma consistent with her lung primary in her uterus and adnexa. Our case represents a rare occurrence in which lung cancer has metastasized to multiple female pelvic organs. Increasing use of PET/CT may lead to the discovery of occult metastases masquerading as a second primary malignancy.

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon.

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.

DNA restriction endonuclease cleavage patterns, DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti.

Physiological characteristics, deoxyribonucleic acid (DNA) base composition (% guanine + cytosine; + GC), DNA sequence similarity (% DNA-DNA hybridization) and DNA restriction endonuclease cleavage patterns of two strains of Lactobacillus helveticus and four strains of Lactobacillus jugurti were examined. All the strains investigated were closely related genetically, having DNA-DNA hybridization values ranging from 89-100%. Nevertheless, these strains can be differentiated from one another on the basis of the digestion of their DNA by specific restriction endonucleases, such as Bam HI, Eco RI and Hind III. The DNA of these strains shows clear, reproducible and distinct cleavage patterns. Cleavage patterns of DNA from strains L. jugurti S.35.19 and S.36.2 were found to be similar. These findings suggest that fingerprinting of DNA by restriction endonuclease cleavage might provide, in addition to the conventional methods, a useful tool for the characterization of closely related microorganisms at the strain level.

Complete sequence and structural organization of pFL5 and pFL7, two cryptic plasmids from Bacillus licheniformis.

The complete nucleotide sequences of two plasmids, pFL5 and pFL7, isolated from soil bacteria, Bacillus licheniformis FL5 and FL7, have been determined. The plasmids pFL5 and pFL7 were analyzed and found to be 9150 and 7853 bp in size with a G+C content of 41.0 and 43.6 mol%, respectively. Computer assisted analysis of sequence data revealed 11 possible ORFs in pFL5, four of which could be assigned no function from homology searches. Instead, eight putative ORFs were identified in pFL7, two of which appeared to have no biological function. All the ORFs were preceded by a ribosome binding site. The ORFs 9.5 and 6.7, each of 340 amino acids, were postulated to encode a replication protein similar to known replication proteins of rolling circle replicons, particularly those of the pC194 family. The structural organization of the two pFL plasmids is similar to the pTA plasmids family, with only a few putative coding regions that cannot be attributed to these plasmid backbone genes. In contrast to pTA plasmids, the majority of the genes have an orientation of transcription opposite to the direction of replication. The identified probable sso sequences seem to belong to a different group of those found in Bacillus plasmids; in fact, a significant level of homology was found with ssoA group sequences. These plasmids seem to be related to plasmids identified within the Bacillus subtilis group, confirming the low-level diversity among these replicons.