Biomedical subjects

J Kalinowski

Publications and source records attributed to J Kalinowski.

At least 55 records · Page 3Linked to original sources

PubMed1996

A physical and genetic map of the Corynebacterium glutamicum ATCC 13032 chromosome.

A combined physical and genetic map of the Corynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleases SwaI (5'-ATTTAAAT-3'), PacI (5'-TTAATTAA-3'), and PmeI (5'-GTTTAAAC-3') yielding 26,27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacent SwaI fragments, a genomic cosmid library of C.glutamicum was screened for chromosomal inserts containing SwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect the SwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blotted SwaI digests using as probes PacI and PmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partial SwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes to SwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.

Chromosomes, Bacterial↗

PubMed1996

Regulation of NFIL-6 and IL-6 expression by basic fibroblast growth factor in osteoblasts.

We determined whether basic fibroblast growth factor (bFGF) regulated the expression of IL-6 and NFIL-6 in osteoblasts. In mouse osteoblastic MC3T3-E1 cells, bFGF (10(-8) M) increased NFIL-6 mRNA 2-fold at 30 minutes and 3-fold at 2 h. IL-6 mRNA was increased by bFGF 10(-8) M after 1 h. IL-6 protein was detectable in control cultures but was significantly increased by bFGF (10(-8) M) at 4 h. Immunofluorescence analysis of MC3T3-E1 cells showed primarily cytoplasmic and perinuclear NFIL-6 staining in control cultures while bFGF-treated cells showed increased NFIL-6 staining at 2 and 4 h. Western blotting revealed that bFGF increased NFIL-6 protein at 2 h. In calvarial mouse osteoblasts, bFGF 10(-8) M induced IL-6 mRNA as early as 1 h and significantly increased IL-6 protein levels as early as 2 h. In conclusion, bFGF stimulates IL-6 and NFIL-6 mRNA in osteoblasts. The increase in NFIL-6 mRNA was associated with increased NFIL-6 protein. The increase in IL-6 mRNA was also associated with increased IL-6 protein. We propose that activations of NFIL-6 and IL-6 may be important mediators of the effects of bFGF on bone cells.

Animals↗

PubMed1996

Interleukin-6 expression and histomorphometry of bones from mice deficient in receptors for interleukin-1 or tumor necrosis factor.

We examined the roles of interleukin-1 Type I receptor (IL-1R1) and tumor necrosis factor receptor 1 (TNFR1) in bone metabolism using mice rendered deficient in these receptors by gene targeting. Sections of decalcified paraffin-embedded calvariae and humeri from 11- to 12-week-old mice deficient in IL-1 Type I receptor (IL-1R1-/-) or TNF receptor 1 (TNFR1-/-) were examined by histomorphometry. Wild-type mice (C57BL/6J x 129/J, WILD) served as controls. Interleukin-6 (IL-6) production in primary osteoblastic and bone marrow stromal cell cultures in response to parathyroid hormone (PTH, 100 ng/ml), IL-1 alpha (10 ng/ml), and TNF-alpha (10 ng/ml) was also examined. IL-1R1-/- and TNFR1-/- mice were viable and appeared phenotypically normal. However, the body weights of the IL-1R1-/- mice were 30% less than WILD, while the TNFR1-/- mice weighed 30% more than WILD mice of equivalent age. Calvariae and humeri of IL-1R1-/- and TNFR1-/- mice were normal with respect to trabecular bone volume, osteoclast number, osteoclast surface, growth plate widths, and cortical thickness. Receptor deficiency was confirmed by determining the ability of PTH, IL-1 alpha, and TNF-alpha to stimulate IL-6 in the media of primary calvaria-derived osteoblastic cell cultures from CD-1 and cytokine receptor-deficient mice. After 24 h of treatment, IL-1 alpha and TNF-alpha did not stimulate IL-6 production in osteoblasts from IL-1R1-/- and TNFR1-/- mice, respectively. In contrast, PTH increased IL-6 levels in the cells from all mice. IL-6 protein levels in bone marrow supernatants and conditioned media from untreated bone marrow stromal cells were undetectable in WILD, IL-1R1-/-, and TNFR1-/- mice. PTH, IL-1 alpha and TNF-alpha increased IL-6 mRNA and protein production in the WILD bone marrow stromal cells. In contrast, PTH and TNF-alpha increased IL-6 mRNA and protein levels in IL-1R1-/- bone marrow stromal cells while IL-1 alpha had no effect. These findings demonstrate that normal bone development in mice can occur in the absence of IL-1R1 or TNFR1 expression.

Animals↗

PubMed1996

Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy.

Interleukin-6 (IL-6) has been implicated as a mediator of postmenopausal bone loss. In vitro studies of bone and bone marrow cells have suggested that estrogen regulates bone turnover by controlling the production of IL-6, a potent stimulator of osteoclastogenesis and bone resorption. To investigate this hypothesis in an in vivo model, we examined the effect of ovariectomy or estrogen replacement on IL-6 mRNA and protein expression in adult mouse bone and bone marrow in vivo and in marrow stromal cell cultures. Eight-week-old CD-1 mice were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized and subcutaneously implanted with 21-day slow-release pellets containing 10 micrograms of 17 beta-estradiol (O + E). Placebo pellets were implanted in the SHAM and OVX mice. Uterine weights at 1, 2, or 3 weeks after surgery were significantly decreased (68-76%) in OVX animals compared with SHAM or O + E. In mice sacrificed at 1 or 3 weeks after surgery, we found by nonquantitative reverse transcribed polymerase chain reaction (RT-PCR), that SHAM, OVX, and O + E calvariae (CALV) constitutively expressed IL-6 mRNA. In contrast, IL-6 mRNA was either barely detectable or absent in the tibia (TIB) and bone marrow (BM). In the mice sacrificed 3 weeks after surgery, we determined by quantitative RT-PCR that IL-6 mRNA in the CALV from the OVX and O + E groups were decreased by 81 and 92%, respectively, compared with SHAM. IL-6 protein levels in the flushed bone marrow (BMSups) were detectable and were not significantly different among the groups. In bone marrow cells that were cultured for 1 week, basal levels of IL-6 protein were low and did not differ significantly among the SHAM, OVX, or O + E groups sacrificed 1, 2, or 3 weeks after surgery. After the addition of hrIL-1 alpha, IL-6 protein levels increased 100- to 1300-fold over control. IL-6 levels in cells from animals sacrificed 2 weeks after surgery were significantly lower in the hrIL-1 alpha-stimulated OVX and O + E groups than in hrIL-1 alpha-stimulated SHAM cell cultures. In conclusion, in this model we could find no increase in IL-6 production with in vivo estrogen withdrawal in calvaria, long bones, bone marrow, or marrow stromal cell cultures. If increases in IL-6 expression are involved in the effects of estrogen withdrawal on bone, the magnitude of these changes are relatively small and below the limits of detection of the assays that we employed.

Animals↗

PubMed1996

A Corynebacterium glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins.

Following the analysis of transposon Tn5432-induced mutants of Corynebacterium glutamicum ATCC 13032, a gene encoding a protein with a biotin-binding motif was cloned. The DNA sequence of this gene revealed an open reading frame encoding 591 amino acids with a calculated mol. mass of 63.4 kDa. The protein is composed of two domains, an N-terminal biotin carboxylase and a C-terminal biotin-carboxyl-carrier protein, that are highly similar to corresponding subunits from prokaryotic and eukaryotic biotin enzymes. Over 70% identity was found to a protein from Mycobacterium leprae proposed to be part of an acyl-CoA carboxylase. Since it was not possible to inactivate the C. glutamicum gene, the gene most likely encodes a subunit of the essential acetyl-CoA carboxylase, which catalyzes the committed step in fatty acid synthesis.

Amino Acid Sequence↗

PubMed1996

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes.

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B. lactofermentum ATCC 13869. The hom- and thrB-disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine.

Alanine↗

PubMed1996

Fluency effect of frequency alterations of plus/minus one-half and one-quarter octave shifts in auditory feedback of people who stutter.

The effect of frequency alterations in auditory feedback of people who stutter on stuttering frequency was investigated. Twelve participants who stutter read aloud under nonaltered auditory feedback (NAF) and four conditions of frequency-altered feedback ([FAF], plus/minus one-half and one-quarter octaves) at normal and fast speech rates. Stuttering frequency was significantly higher while reading aloud with NAF relative to the four conditions of FAF (p < 0.05). There were no differences among participants' stuttering frequency between the four FAF conditions (p > 0.05). Reductions in stuttering frequency of approximately 50% to 60% were found with FAF relative to NAF. More disfluencies occurred with the fast versus the normal speech rate condition (p = .0007) irrespective of auditory feedback condition. These findings suggest that slight alterations in the frequency of auditory feedback of people who stutter are fluency-enhancing.

Adult↗

PubMed1996

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation.

Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.

Amino Acids↗

PubMed1996

Fluent speech, fast articulatory rate, and delayed auditory feedback: creating a crisis for a scientific revolution?

In 1970 Kuhn argued that science does not progress through a process of accretion. It is typified, rather, by the successive emergence of different paradigms which during their reign dictate the direction of normal science's puzzle-solving activity. Normal science inevitably exposes an anomaly which violates expectations predicted by the reigning paradigm. The "crisis" evoking anomaly may induce a destructive/constructive paradigm change. Transformations from one paradigm to another constitute a scientific revolution and dictate the growth and maturation of a field. This paper suggests the recent finding, that stutterers experience enhancement of fluency while speaking under delayed auditory feedback at a fast articulatory rate, be viewed as an anomaly. By challenging the notion that a slowed speech rate is necessary for amelioration of stuttering, the anomalous finding may be perceived as a crisis in the study of stuttering.

Attention↗

PubMed1996

Effect of instruction to sing on stuttering frequency at normal and fast rates.

Singing as a fluency-enhancing mechanism is well-established. The fluency derived by singing has been attributed to a reduced speech rate, memorized material, semantically reduced content, and an imposed rhythm. In this study, we attempted to address each of these explanations. 12 participants who stuttered were instructed to read or sing each of four different passages under the following conditions: reading at a normal rate, reading at a fast rate, singing at a normal rare, and singing at a fast rate. Participants exhibited a statistically significant increase in disfluencies while reading, i.e., participants displayed a 75% reduction in disfluency in the singing condition relative to the reading condition. There was no difference in stuttering frequency with rate conditions. Current findings suggest that stutterers are capable of internally generating fluent speech production by imposing idiosyncratic melodic structures or some derivation of melody when asked simply to sing. There is no claim that these participants were singing, as skills and capabilities varied tremendously, only that participants achieved dramatic enhancement of fluency after they were just asked to sing. Thus, the only intervening variable was the instruction to sing, which suggests the attempt to follow the instruction, no matter how futile, generated fluent speech. Since fluency was maintained in both the normal and fast rates of production, alternate central mechanisms must be held accountable for these findings.

Adolescent↗

PubMed1996

Stuttering amelioration at various auditory feedback delays and speech rates.

The primary purpose of this study was to determine if the finding of Kalinowski et al. (1993) of dramatic reductions in stuttering under delayed auditory feedback (DAF) at normal and fast speech rates could be replicated. The second purpose was to determine if stuttering frequency is differentially affected by various delays in an attempt to identify the optimal delay for fluency enhancement for both normal and fast speech rates. Fourteen adult stutterers read eight different passages at either a normal or fast speech rate under non-altered auditory feedback (NAF) and DAF with delays of 25, 50 and 75 ms. Results showed that significant fluency enhancement occurred under DAF at both normal and fast speech rates at all DAF settings (p < 0.05). This finding corroborates the notion that a slowed rate of speech is not a necessary antecedent for fluency improvement under conditions of altered auditory feedback. In addition, the results indicated that 50 ms appears to be the shortest delay producing the maximum reduction in stuttering frequency.

Adolescent↗

PubMed1995

Isolation of insertion elements from gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker.

The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS-Bl and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.

Actinomycetales↗

PubMed1995

The erythromycin resistance gene of the Corynebacterium xerosis R-plasmid pTP10 also carrying chloramphenicol, kanamycin, and tetracycline resistances is capable of transposition in Corynebacterium glutamicum.

The clinical isolate Corynebacterium xerosis M82B carries the 50-kb R-plasmid pTP10 that confers resistance to the antibiotics chloramphenicol, kanamycin, erythromycin, and tetracycline. A detailed restriction map of pTP10 was constructed by cloning and analyzing restriction fragments of pTP10 in Escherichia coli. The resistance determinants of pTP10 were located by studying the phenotype of the recombinant plasmids in E. coli and Corynebacterium glutamicum. Restriction patterns of fragments encoding the kanamycin and erythromycin resistances revealed striking similarity to the kanamycin resistance of transposon Tn903 and the erythromycin resistance on plasmid pNG2 from Corynebacterium diphtheriae, respectively. Expression of the resistance determinants in E. coli and C. glutamicum ATCC 13032 led to high resistance levels in both strains, with the exception of the tetracycline resistance gene, which could be expressed only in C. glutamicum. Furthermore, the erythromycin resistance gene was found to be located on a transposable element which is functional in C. glutamicum strains.

Blotting, Southern↗

PubMed1995

The Corynebacterium xerosis composite transposon Tn5432 consists of two identical insertion sequences, designated IS1249, flanking the erythromycin resistance gene ermCX.

Analysis of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium xerosis M82B revealed that the erythromycin resistance gene, ermCX, is located on a 4524-bp composite transposable element, Tn5432. The ends of Tn5432 are identical, direct repeats of an insertion sequence, designated IS1249, encoding a putative transposase of the IS256 family. IS1249 consists of 1385 bp with 45/42 imperfect terminal inverted repeats. The nucleotide sequence of the 1754-bp Tn5432 central region is 99% identical to the previously sequenced erythromycin resistance region of the Corynebacterium diphtheriae plasmid pNG2. It encodes the erythromycin resistance gene, ermCX, and an ORF homologous to the amino-terminal end of the transposase of IS31831 from Corynebacterium glutamicum. Transposons with regions flanking the insertion sites were recovered from the C. glutamicum chromosome by a plasmid rescue technique. Insertion of Tn5432 created 8-bp target site duplications. A Tn5432-induced isoleucine/valine-auxotrophic mutant was found to carry the transposon in the 5' region of the ilvBNC cluster; in pTP10 the transposon is inserted in a region similar to replication and partitioning functions of the Enterococcus faecalis plasmid pAD1 and the Agrobacterium tumefaciens plasmid pTAR.

Amino Acid Sequence↗

PubMed1995

Effect of single and combined altered auditory feedback on stuttering frequency at two speech rates.

The purpose of this study was to determine if combining delayed auditory feedback (DAF) and frequency altered feedback (FAF) would enhance fluency more than either DAF or FAF alone. Ten stutterers read at normal and fast speech rates under nonaltered auditory feedback (NAF), DAF (i.e., a 50 ms delay), FAF (i.e., a one half octave downward shift), and a combination of DAF and FAF [(COMBO), i.e., a 50 ms delay plus a one half octave downward shift]. Results indicated that stuttering frequency was significantly reduced under all altered auditory conditions at high speech rates relative to the NAF condition. There were, however, no significant differences between the altered auditory feedback conditions (i.e., DAF, FAF, and COMBO). It is suggested that further studies be undertaken to explore the combination of altered auditory feedback conditions, as it may be the case that a floor effect was demonstrated with the singular presentations of DAF and FAF and further improvements in fluency enhancement could not be exhibited in the combined condition. Finally, these findings support the notion that a slowed rate of speech is not necessary for fluency enhancement under conditions of altered auditory feedback.

Adult↗

PubMed1994

Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli.

Efficient electroporation of Escherichia coli with plasmid DNA isolated from Corynebacterium glutamicum depends on the use of Mcr-deficient E. coli strains. The transformation frequency increased nearly 800-fold when the Mcr-deficient E. coli DH5 alpha MCR was used instead of E. coli DH5 alpha. We used E. coli strains with different mutations in the methyl-specific restriction systems to show that McrBC-deficiency is sufficient to generate this effect. The results imply that C. glutamicum DNA contains methylcytosine in specific sequences recognized by the E. coli McrBC system.

Cloning, Molecular↗

PubMed1994

Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum.

Here we describe small mobilizable vectors based on the Escherichia coli plasmids pK18 and pK19. We combined the useful properties of the pK plasmids (e.g., multiple cloning site, lacZ alpha fragment, sequencing with M13 primers) with the broad-host-range transfer machinery of plasmid RP4 and a modified sacB gene from Bacillus subtilis. The new pK derivatives can be transferred by RP4-mediated conjugation into a wide range of Gram- and Gram+ bacteria, and should facilitate gene disruption and allelic exchange by homologous recombination. As an application example, the generation of a defined deletion of the hom-thrB genes in the chromosome of the Gram+ bacterium Corynebacterium glutamicum is presented.

Chromosomes, Bacterial↗

PubMed1994

Interpreting results of the fluent speech paradigm in stuttering research: difficulties in separating cause from effect.

This paper examines difficulties inherent in interpreting results of studies that compare the fluent speech characteristics of stutterers and nonstutterers. The majority of these studies have reported stutterer/nonstutterer differences in temporal parameters of fluent speech production. Such differences have been interpreted as indicating that stutterers possess temporal-motor deficits that are ever-present in speech and, therefore, causal to stuttering. However, a problem for researchers studying the fluent speech of stutterers is that samples may be contaminated by the influence of stuttering. In this paper, evidence is reviewed which suggests that characteristics of the perceptually fluent speech of stutterers change as a function of a number of variables: (a) context of experimental samples, (b) treatment history of subjects, (c) stuttering severity of subjects, and (d) developmental history of stuttering. In addition, evidence is presented which can be interpreted to show that each of these variables reflect stuttering, either directly or indirectly. It is argued that because these variables are difficult to fully control, or account for, comparison of the characteristics of the perceptually fluent speech of stutters and nonstutterers as a method of studying stuttering causation is problematic. Alternative directions for research activity are discussed.

Age Factors↗