Biomedical subjects

K Hammer

Publications and source records attributed to K Hammer.

At least 37 records · Page 2Linked to original sources

PubMed1998

The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters.

We constructed a library of synthetic promoters for Lactococcus lactis in which the known consensus sequences were kept constant while the sequences of the separating spacers were randomized. The library consists of 38 promoters which differ in strength from 0.3 up to more than 2,000 relative units, the latter among the strongest promoters known for this organism. The ranking of the promoter activities was somewhat different when assayed in Escherichia coli, but the promoters are efficient for modulating gene expression in this bacterium as well. DNA sequencing revealed that the weaker promoters (which had activities below 5 relative units) all had changes either in the consensus sequences or in the length of the spacer between the -35 and -10 sequences. The promoters in which those features were conserved had activities from 5 to 2,050 U, which shows that by randomizing the spacers, at least a 400-fold change in activity can be obtained. Interestingly, the entire range of promoter activities is covered in small steps of activity increase, which makes these promoters very suitable for quantitative physiological studies and for fine-tuning of gene expression in industrial bioreactors and cell factories.

Base Sequence↗

PubMed1998

Induced levels of heat shock proteins in a dnaK mutant of Lactococcus lactis.

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in Lactococcus lactis, we have constructed two dnaK mutants with C-terminal deletions in dnaK. The minor deletion of 65 amino acids in the dnaKDelta2 mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (dnaKDelta1), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in L. lactis. For Bacillus subtilis, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579-4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a dnaK insertion mutant has no effect on the expression of the heat shock proteins. The present data from L. lactis suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.

Bacterial Proteins↗

PubMed1998

The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically.

The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis, L. lactis is shown to possess only one carB gene; the same gene product is thus required for both biosynthetic pathways. Furthermore, arginine may satisfy the requirement for carbamoylphosphate in pyrimidine biosynthesis through degradation by means of the arginine deiminase pathway. The expression of the carB gene is subject to regulation at the level of transcription by pyrimidines, most probably by an attenuator mechanism. Upstream of the carB gene, an open reading frame showing a high degree of similarity to those of glutathione peroxidases from other organisms was identified.

Amino Acid Sequence↗

PubMed1998

Lipids infused into the jejunum accelerate small intestinal transit but delay ileocolonic transit of solids and liquids.

BACKGROUND: Various nutrients are known to alter small intestinal motility patterns although their effect on transit of fluids and solids in man is not clear. AIMS: To determine small intestinal transit of solids and liquids during perfusion with lipids, protein, and non-energy solutions. METHODS: Twenty eight healthy volunteers received a jejunal infusion (1 ml/minute for 30 minutes) of one of four solutions: a lipid or a protein solution (4.18 J/ml), a non-absorbable electrolyte solution containing polyethylene glycol, or 0.9% sodium chloride. As solid phase marker 1 g of amberlite resin pellets labelled with 111InCl3 was added; 99mTc DTPA was used as a fluid phase marker. Images were obtained on a gamma camera at 10 minute intervals for four hours or until all radiolabel was detected in the colon. RESULTS: Intestinal transit of solids and liquids from the duodenojejunal junction to the caecum was simultaneous, and independent of the energy content of the solution infused. Lipid infusion accelerated transit through the small intestine but delayed transport of chyme along the ileocolonic junction. After protein small intestinal transit was slowest; ileocolonic transit on the other hand was fastest with protein. Transit of the non-energy solutions was in between that of the nutrient solutions. CONCLUSIONS: Transit times through the small intestine and the ileocolonic junction were influenced by the luminal contents. In the small intestine fat induced significantly faster transit compared with proteins, but delayed ileocolonic transit. Once in the small intestine, solids and liquids transit the small bowel together, independent of the luminal content.

Adolescent↗

PubMed1998

Gi- and protein kinase C-mediated heterologous potentiation of phospholipase C signaling by G protein-coupled receptors.

We recently reported that activation of the highly efficient phospholipase C (PLC) stimulatory m3 muscarinic acetylcholine receptor (mAChR) can induce a long-lasting Gi-mediated heterologous potentiation of PLC stimulation in human embryonic kidney (HEK) 293 cells, which was accompanied by an increased cellular level of the PLC substrate phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Here, we examined whether such a potentiated PLC response is also induced by the rather poorly PLC stimulatory m2 mAChR and the endogenously expressed purinergic and lysophosphatidic acid receptors. Pretreatment of m2 mAChR-expressing HEK 293 cells for 2 min with carbachol, followed by agonist washout and measurement of PLC activity >/=40 min later, caused a long-lasting (up to approximately 90 min) heterologous potentiation of receptor- and G protein-mediated PLC stimulation. A similar heterologous potentiation of receptor-mediated PLC stimulation was induced by short term activation of lysophosphatidic acid and purinergic receptors. Either of the three receptor agonists increased the cellular level of PtdIns(4,5)P2 by approximately 50%. The mAChR-induced PLC potentiation was fully prevented by either pertussis toxin or the protein kinase C (PKC) inhibitors staurosporine and Gö 6976, which did not affect acute PLC stimulation. On the other hand, the rise in PtdIns(4,5)P2 was prevented only by combined treatment of HEK 293 cells with pertussis toxin and PKC inhibitors. In conclusion, we demonstrated that activation of poorly PLC stimulatory receptors can also induce a long-lasting Gi-mediated heterologous potentiation of PLC signaling in HEK 293 cells and that this novel PLC regulatory process is under the control of PKC.

Adenosine Triphosphate↗

PubMed1997

Neurons in the chicken ureter are innervated by substance P- and calcitonin gene-related peptide-containing nerve fibres: immunohistochemical and electrophysiological evidence.

Numerous ganglia or single neurones immunoreactive to protein gene-product 9.5 (PGP) were demonstrated in the chicken ureter. Ganglia were observed in the main nerve trunks accompanying the ureter (400-2,000 cells), in the adventitia (1-45 cells; density; 79 +/- 12 ganglia/cm2; mean +/- S.E.M.), in the circular muscle (1-9 cells; 76 +/- 10 ganglia/cm2) and in the longitudinal muscle (1-8 cells; 232 +/- 41 ganglia/cm2). Most of the PGP-positive neurones in the nerve trunk ganglia (approximately 66%) and in the smooth muscle layers (85%) were encircled by a dense plexus of varicose nerve fibres containing both substance P (SP) and calcitonin gene-related peptide (CGRP). SP-positive somata were rarely observed. Immunogold electron microscopy revealed that SP- and CGRP-immunoreactivity were colocalised in the same dense core vesicles. A strong reduction of SP-positive nerve fibres was observed in organ cultures of the ureter, indicating their extrinsic origin. The fibres might originate from the dorsal root ganglia, where SP and CGRP were colocalised in 20-30% of the neurones. The sensitivity of ureteric neurones to SP and CGRP was investigated in recordings obtained from mechanosensitive nerve fibres with cell bodies located in or adjacent to the ureter (U-G units). The majority (71%) of the U-G units was excited by local application of SP in a dose-dependent manner. The SP-sensitive U-G neurones had higher mechanical thresholds (29 +/- 5 mmHg) as opposed to the SP-insensitive ones (10 +/- 3 mmHg). Repeated applications of high doses of SP to the U-G units resulted in desensitisation and reduced the response to mechanical stimuli. None of the U-G units responded to local application of CGRP, but all U-G units were excited by acetylcholine. The data support the hypothesis that SP-containing primary afferents are involved in the modulation of the activity of ureteric neurons in the chicken.

Animals↗

PubMed1997

Screening for carpal tunnel syndrome in the workplace. An analysis of portable nerve conduction devices.

Several devices have been developed for rapid motor or sensory median nerve conduction testing. We evaluated the validity and reliability of the Neurosentinel (NS) and NervePace (NP) electroneurometer for sensory and motor testing, respectively, compared with formal electrodiagnostic studies (EDS), and examined their potential role in workplace screening for carpal tunnel syndrome (CTS). Thirty-two working subjects without CTS were examined and tested with the NS, NP, and EDS, and retested one week later. Subjects were selected who did not have CTS, other hand or nerve problems, or jobs with significant ergonomic risks, in order to decrease the likelihood of changes over time in median nerve function. Mean correlations of NP and NS with EDS latencies ranged from r = 0.069 to r = 0.85, with somewhat better correlation for NS (sensory) than NP (motor). Test-retest reliability was greatest for motor EDS (r = 0.86 to 0.91) and similar for sensory EDS, NS, and NP (r = 0.72 to 0.79); mean results were very similar. Based on the observed relationship between NS or NP and EDS results, confidence intervals were calculated to represent the range of EDS results consistent with a single NS or NP measurement. These intervals ranged from +/- 0.3 milliseconds (ms) for NS to +/- 0.6 msec for NP, with similar ranges for change over time in an individual. The magnitude of these intervals for a single test or individual implies that the NS and NP are unlikely to identify individuals with CTS or to detect changes over time that are not accompanied by symptoms or signs. The screening devices are not likely to be useful in confirming early CTS, when single latency values may be normal, and detailed EDS may be necessary to detect nerve entrapment. Compared with EDS, these devices have moderate validity and similar reliability; they are probably most useful for cross-sectional or longitudinal studies of groups, but care must be taken in using them for pre-placement or surveillance tests of individual workers. False-positive results may lead to discrimination, inappropriate referrals and interventions; false-negative tests can result in inappropriate reassurance and missed opportunities for intervention.

Adult↗

PubMed1997

Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis.

The bacterium Lactococcus lactis has become a model organism in studies of growth physiology and membrane transport, as a result of its simple fermentative metabolism. It is also used as a model for studying the importance of specific genes and functions during life in excess nutrients, by comparison of prototrophic wild-type strains and auxotrophic domesticated (dairy) strains. In a study of the capacity of domesticated strains to perform directed responses toward various stress conditions, we have analyzed the heat and salt stress response in the established L. lactis subsp. cremoris laboratory strain MG1363, which was originally derived from a dairy strain. After two-dimensional separation of proteins, the DnaK, GroEL, and GroES heat shock proteins, the HrcA (Orf1) heat shock repressor, and the glycolytic enzymes pyruvate kinase, glyceral-dehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase were identified by a combination of Western blotting and direct N-terminal amino acid sequencing of proteins from the gels. Of 400 to 500 visible proteins, 17 were induced more than twofold during heat stress. Two classes of heat stress proteins were identified from their temporal induction pattern. The fast-induced proteins (including DnaK) showed an abruptly increased rate of synthesis during the first 10 min, declining to intermediate levels after 15 min. GroEL and GroES, which also belong to this group, maintained a high rate of synthesis after 15 min. The class of slowly induced proteins exhibited a gradual increase in the rate of synthesis after the onset of stress. Unlike other organisms, all salt stress-induced proteins in L. lactis were also subjected to heat stress induction. DnaK, GroEL, and GroES showed similar temporal patterns of induction during salt stress, resembling the timing during heat stress although at a lower induction level. These data indicate an overlap between the heat shock and salt stress responses in L. lactis.

Amino Acid Sequence↗

PubMed1997

Construction of specific erythromycin resistance mutations in the temperate lactococcal bacteriophage TP901-1 and their use in studies of phage biology.

A method for the construction and isolation of specifically designed mutations of the temperate lactococcal phage TP901-1 has been developed. Two different erm-labeled mutants were isolated. One was shown to be defective in lysogenization and excision. The other, showing normal lysogenization, was used for host range studies.

Bacteriophages↗

PubMed1996

A genomic region of lactococcal temperate bacteriophage TP901-1 encoding major virion proteins.

Two major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome. This genomic region was sequenced. The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure. Upstream of each ORF, except ORF b2 and ORF x, potential ribosome-binding sites were found, suggesting independent translation. However, coupled translation is suggested for ORF x and as a possibility for ORF b3 and ORF c2, which have ribosome-binding sites located more distant from their start codons. ORF b2 may be translationally fused with mhp at a low frequency. The mhp and mtp genes are transcribed as a 3.7-kb mRNA with at least six additional ORFs. The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs.

Amino Acid Sequence↗

PubMed1996

Pitcher's arm: an electrodiagnostic enigma.

Every major league baseball pitcher, most minor league pitchers, but only few amateur pitchers that we have studied have had reduced sensory nerve action potentials in the throwing arm. We present 6 clinical cases which demonstrate the spectrum of "pitcher's arm." These cases suggest that the phenomenon is a pathologic process, probably an example of a repetitive use syndrome affecting the brachial plexus. Although it does not appear to impact performance, it has clear implications for the interpretation of electrodiagnostic studies in symptomatic pitchers.

Action Potentials↗

PubMed1996

Analysis of heat shock gene expression in Lactococcus lactis MG1363.

The induction of the heat shock response in Lactococcus lactis subsp. cremoris strain MG1363 was analysed at the RNA level using a novel RNA isolation procedure to prevent degradation. Cloning of the dnaJ and groEL homologues was carried out. Northern blot analysis showed a similar induction pattern for dnaK, dnaJ and groELS after transfer from 30 degrees C to 43 degrees C when MG1363 was grown in defined medium. The dnaK gene showed a 100-fold induction level 15 min after temperature shifting. Induction of the first two genes in the dnaK operon, orf1 and grpE, resembled the pattern observed for the above genes, although maximum induction was observed earlier for orf1 and grpE. Novel transcript sizes were detected in heat-shocked cells. The induction kinetics observed for ftsH suggested a different regulation for this gene. Experimental evidence for a pronounced transcriptional regulation being involved in the heat shock response in L. lactis MG1363 is presented. A gene located downstream of the dnaK operon in strain MG1363, named orf4, was shown not to be regulated by heat shock.

Bacterial Proteins↗

PubMed1996

Sequence analysis and identification of the pyrKDbF operon from Lactococcus lactis including a novel gene, pyrK, involved in pyrimidine biosynthesis.

Three genes encoding enzymes involved in the biosynthesis of pyrimidines have been found to constitute an operon in Lactococcus lactis. Two of the genes are the well-known pyr genes pyrDb and pyrF, encoding dihydroorotate dehydrogenase and orotidine monophosphate decarboxylase, respectively. The third gene encodes a protein which was shown to be necessary for the activity of the pyrDb-encoded dihydroorotate dehydrogenase; we propose to name the gene pyrK. The pyrK-encoded protein is homologous to a number of proteins which are involved in electron transfer. The lactococcal pyrKDbF operon is highly homologous to the corresponding part of the much-larger pyr operon of Bacillus subtilis. orf2, the pyrK homolog in B. subtilis, has also been shown to be necessary for pyrimidine biosynthesis (A. E. Kahler and R. L. Switzer, J. Bacteriol. 178:5013-5016, 1996). Four genes adjacent to the operon, i.e., orfE, orfA, orfC, and gidB, were also sequenced. Three of these were excluded as members of the pyr operon by insertional analysis (orfA) or by their opposite direction of transcription (orfE and gidB). orfC, however, seems to be the distal gene in the pyrKDbF-orfC operon.

Bacterial Proteins↗

PubMed1996

A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1.

The integration system of the temperate lactococcal phage TP901-1 was characterized in Lactococcus lactis subsp. cremoris LM0230 and MG1363 with the use of deletion derivatives of the integration vector pBC143 (B. Christiansen, M. G. Johnsen, E. Stenby, F. K. Vogensen, and K. Hammer, J. Bacteriol. 176:1069-1076, 1994). The phage-encoded elements necessary for integration were localized on a 2.8-kb NsiI-EcoRI fragment including the phage attachment site, attP. This fragment was DNA sequenced, and sequence analysis revealed three putatively expressed open reading frames, Orf1, Orf2, and Orf3 By the introduction of mutations within the orf1, orf2, and orf3 genes, it was shown that only Orf1 was necessary for the integration process. Furthermore, it was found that Orf1, attP, and a 425-bp region upstream of the orf1 gene are sufficient for integration. Orf1 contains 485 amino acids and is located just upstream of attP. The N-terminal 150 to 180 amino acids of Orf1 showed 38 to 44% similarity to the resolvase group of site-specific integrases, while no similarity to known proteins was found in the C-terminal end. Bacteriophage TP901-1 therefore contains a unique integration system that does not resemble the Int class of site-specific integrases usually found in temperate bacteriophages. The constructed integration vector, pBC170, integrates into the chromosomal attachment site very efficiently and forms stable transformants with a frequency corresponding to 20% of the transformation efficiency.

Amino Acid Sequence↗

PubMed1996

Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.

The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).

Amino Acid Sequence↗

PubMed1996

Ether, carbonate and urethane deoxynucleoside derivatives as prodrugs.

3'-Deoxythymidine and its 3'-azido derivative, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine have been acylated to form carbonates and urethanes in chemoselective reactions. The nucleosides have been N- and/or O-alkylated by alpha-chloroethyl or chloromethyl alkyl carbonates to form alpha-alkyloxycarbonyloxyethyl or alkyloxycarbonyloxymethyl derivatives. The products are lipophilic in order to facilitate transport through biological membranes and are designed to be cleaved by esterases with liberation of the bioactive nucleoside. Initial esterase cleavage of the alkylated derivatives produces hemiacetals or -aminals which subsequently dissociate to the active nucleoside.

Antiviral Agents↗

PubMed1995

Virion positions and relationships of lactococcal temperate bacteriophage TP901-1 proteins.

The major proteins of phage TP901-1 virion were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and structural relations were determined using specific antibodies, obtained by affinity purification from a polyclonal serum. A 23-kDa protein was identified as the major tail protein, and a 31-kDa molecule as the major head protein, respectively. Labeling experiments with antibodies against two proteins, with molecular masses of 20 and 19 kDa, indicated that they were baseplate-related components. A 72-kDa protein was found to be part of a neck passage structure, which includes a collar. Evidence for the presence of attached whiskers was also obtained. T7 RNA polymerase-mediated expression of the two major proteins confirmed the gene location of the previously sequenced region of the phage genome. The relation to other lactococcal phages was determined by DNA hybridization and antibody probing, showing that despite low DNA similarity, TP901-1 NPS epitopes were detected in both related and unrelated small isometric-headed phages.

Antibodies, Viral↗

PubMed1994

A subgroup of patients with chronic pancreatitis overexpress the c-erb B-2 protooncogene.

OBJECTIVE: Chronic pancreatitis (CP) is a chronic condition associated with pancreatic fibrosis. A small subgroup of patients with CP develop enlargement of the head of the pancreas (EHP). This study examined some of the mechanisms that may lead to the development of EHP. SUMMARY BACKGROUND: The c-erb B-2 protooncogene encodes a 185-kDa transmembrane growth factor receptor (p185) that regulates cell growth and differentiation. METHODS: The authors analyzed c-erb B-2 expression in samples obtained from the head of the pancreas from 26 patients with CP (5 women, 21 men) using immunohistochemical and molecular technique. A diagnosis of CP with EHP was made when the vertical pancreatic head diameter was greater than 4 cm (14 patients), as determined by contrast-enhanced computed axial tomography scan. Pancreatic tissues from 15 healthy organ donors served as control subjects. RESULTS: In all patients without EHP and in the healthy control subjects, p185 immunoreactivity was present at low levels. In contrast, strong p185 immunoreactivity was observed in acinar and ductal cells in all patients with EHP. By in situ hybridization, c-erb B-2 messenger ribonucleic acid (mRNA) grains were expressed at high levels in patients with CP with EHP in both ductal and acinar cells. Northern blot analysis demonstrated a 4.5-fold increase (p < 0.001) in c-erb B-2 mRNA levels in patients with EHP compared with patients without EHP and healthy control subjects. Southern blot analysis did not reveal c-erb B-2 gene amplification or rearrangement. CONCLUSIONS: These findings indicate the c-erb B-2 is not overexpressed in most patients with CP. However, its overexpression in patients with CP with EHP suggest that c-erb B-2 may contribute to the pathophysiologic processes that lead to pancreatic head enlargement.

Adolescent↗