Development of a PCR-based technique for detection of Helicobacter pylori

Abstract A primer-set was designed for specific detection of genes that encode for 16S rRNA of Helicobacter pylori , using direct polymerase chain reaction (PCR). The primers were selected in the hypervariable regions, derived from a complete small subunit 16S rRNA sequence of the reference strain H. pylori CCUG 17874. The primer-set amplified a 537 base pair (bp) sequence specifically from chromosomal H. pylori DNA. Amplification of purified chromosomal H. pylori DNA was achieved at concentrations as low as 1 femto gram (fg), equivalent to 5 bacteria. Furthermore, as few as 1 lysed H. pylori cell was detected by this PCR technique. The specificity of the primers was 100%, since purified chromosomal DNA was detected from all 32 various H. pylori isolates, whereas no other bacteria species were detected, whether related to Helicobacter or not. The 16S rDNA primers successfully detected H. pylori in antral biopsy specimens collected from infected patients.     

An angiotensin II antagonist with strongly prolonged action

A highly hydrophobic analogue of angiotensin II (AT), [Sar1,(2',3',4',5',6'-Br5)Phe8]AT exhibited strong and persistent specific antagonism against AT, both in vitro and in vivo. This peptide exhibited 32% of the binding affinity of [Sar1]AT towards membranes of bovine adrenal cortex, it was a specific AT antagonist of irreversible character on smooth muscle assays, and it also suppressed for over 120 min at 7.10(-8) M/kg the blood pressure response towards AT in the rat blood pressure assay. This compound harbours therefore the potential of a new class of AT-specific antihypotensive drugs.     

ADAM12 is a four-leafed clover: the excised prodomain remains bound to the mature enzyme

The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.     

Imprint cytology of sentinel lymph nodes in breast cancer. Experience with rapid, intraoperative diagnosis and primary screening by cytotechnologists

OBJECTIVE: To evaluate the intraoperative imprint diagnoses of smears from sentinel lymph nodes that had been primary screened by cytotechnologists and to assess the most important causes of false negative (FN) imprint diagnoses. STUDY DESIGN: Material consisted of 429 imprints from sentinel lymph nodes in 211 breast cancer patients that were sent for frozen section examination over 13 months. RESULTS: The mean number of imprints/lymph nodes per patient was 2.02. The mean screening time per imprint was 3.6 minutes. Sixty-six sentinel nodes (16%) from 51 women (24%) were metastatic. Imprints and/or frozen sections were positive in 54 nodes (82%). Imprints were positive in 38 nodes, representing 70% of intraoperative positive nodes and 58% of the total number of positive nodes. Twenty-six of 28 (93%) FN imprints were due to suboptimal sampling. Four of 9 FN macrometastases did not contain diagnostic or suspicious cells/cell groups even on rescreening, whereas a few, and then only 1 diagnostic group were identified in 2/9. There were no false positives. CONCLUSION: Primary screening by experienced cytotechnologists is both rapid and reliable and enabled the diagnosing pathologist to concentrate on the frozen section. The major cause of false negative imprints is sampling, even in macrometastases.     

Purification and cloning of piscicolin 61, a bacteriocin fromCarnobacterium piscicola LV61

Piscicolin 61, a bacteriocin produced byCarnobacterium piscicola LV61, inhibits the growth of strains ofCarnobacterium, Lactobacillus, andEnterococcus. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential hydrophobic interaction and reversed-phase chromatography. The N-terminal amino acid sequence of piscicolin 61 was determined by Edman degradation. The plasmid-located structural gene encoding piscicolin (psc61) was cloned and sequenced. It encoded a primary translation product of 71 amino acid residues, which is cleaved between amino acid residues 18 and 19 to yield the active bacteriocin. The calculatedM _r from the deduced protein sequence, 5052.6, agreed with that obtained by mass spectrometry. Piscicolin 61 did not show any sequence similarities to other known bacteriocins. However, the leader sequence resembled those of the pediocin-like bacteriocins. Piscicolin 61 may be able to form amphiphilic helices and may thus act on the membrane of sensitive cells.     

Cloning and nucleotide sequence of a gene from Lactobacillus sake Lb706 necessary for sakacin A production and immunity.

Sakacin A is an antilisterial bacteriocin produced by Lactobacillus sake Lb706. In order to identify genes involved in sakacin A production and immunity, the plasmid fraction of L. sake Lb706 was shotgun cloned directly into a sakacin A-nonproducing and -sensitive variant, L. sake Lb706-B, by using the broad-host-range vector pVS2. Two clones that produced sakacin A and were immune to the bacteriocin were obtained. A DNA fragment of approximately 1.8 kb, derived from a 60-kb plasmid of strain Lb706 and present in the inserts of both clones, was necessary for restoration of sakacin A production and immunity in strain Lb706-B. The sequence of the 1.8-kb fragment from one of the clones was determined. It contained one large open reading frame, designated sakB, potentially encoding a protein of 430 amino acid residues. Hybridization and nucleotide sequence analyses revealed that the cloned sakB complemented a mutated copy of sakB present in strain Lb706-B. The sakB gene mapped 1.6 kb from the previously cloned structural gene for sakacin A (sakA) on the 60-kb plasmid. The putative SakB protein shared 22% amino acid sequence identity (51% similarity if conservative changes are considered) to AgrB, the deduced amino acid sequence of the Staphylococcus aureus gene agrB. The polycistronic agr (accessory gene regulator) locus is involved in the regulation of exoprotein synthesis in S. aureus. Similar to the AgrB protein, SakB had some features in common with a family of transmembrane histidine protein kinases, involved in various adaptive response systems of bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of the 5'-nuclease PCR assay in evaluation and development of methods for quantitative detection of Campylobacter jejuni

Campylobacter jejuni is recognized as a leading human food-borne pathogen. Traditional diagnostic testing for C. jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable state. Nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, we present a 5'-nuclease PCR assay for quantitative detection of C. jejuni and describe its evaluation. A probe including positions 381121 to 381206 of the published C. jejuni strain NCTC 11168 genome sequence was identified. When this probe was applied, the assay was positive for all of the isolates of C. jejuni tested (32 isolates, including the type strain) and negative for all other Campylobacter spp. (11 species tested) and several other bacteria (41 species tested). The total assay could be completed in 3 h with a detection limit of approximately 1 CFU. Quantification was linear over at least 6 log units. Quantitative detection methods are important for both research purposes and further development of C. jejuni detection methods. In this study, we used the assay to investigate to what extent the PCR signals generated by heat-killed bacteria interfere with the detection of viable C. jejuni after exposure at elevated temperatures for up to 5 days. An approach to the reduction of the PCR signal generated by dead bacteria was also investigated by employing externally added DNases to selectively inactivate free DNA and exposed DNA in heat-killed bacteria. The results indicated relatively good discrimination between exposed DNA from dead C. jejuni and protected DNA in living bacteria.     

A severe traffic accident--250 years ago. Medical history presentation

During a scientific examination in July 1999 both crypts below the St. Martin's Church in Grünstadt, Germany, were opened and 9 coffins from the county family of Leiningen examined. This paper is concentrating on one of these persons: Georg Hermann (1679-1751), count of Leiningen-Westerburg-Altleiningen, who gave during the 18. century the city its barock character. He was also responsible for the rebuilding of the church. His skeleton revealed interesting pathological changes. Few years before his death the count had the accident to get run over by a heavy wagon which crushed the distal part of his legs. The fractures healed, but gave him an ancylotic and shortened left leg, which must have caused him a lot of suffering in his last years.     

Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.