Biomedical subjects

J Kalinowski

Publications and source records attributed to J Kalinowski.

At least 19 recordsLinked to original sources

PubMed2006

Analysis of subcellular surface structure, function and dynamics.

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).

Animals↗

PubMed2005

Photophysics of an electrophosphorescent platinum (II) porphyrin in solid films.

We examine electronic processes in platinum (II) octaethyl porphyrin (PtOEP) embedded in an organic solid state matrix and in the form of vacuum-evaporated neat films in conjunction with potential applications of this compound to organic photovoltaic and electrophosphorescent devices. Absorption, photoexcitation, and luminescence spectra indicate the excitonic dimers to be dominant excited states, and their dissociation underlies the charge photogeneration process. Different charge separation distance (1.5 nm and 2.6 nm) in opposite charge carrier pairs preceding dissociation can be distinguished based on the fit of the three-dimensional Onsager theory of geminate recombination to electromodulated luminescence and photoconduction measurements. The near-positive electrode concentrated triplet dimer excitons, produced by strongly 370 nm absorbed light in neat PtOEP films, are efficiently quenched by electron transfer to the metal (Al), generating the positive charge with an efficiency eta+ exceeding 0.15 at high electric fields and dominating the measured photocurrent. Their dissociation efficiency in the bulk, eta- (negatively biased illuminated electrode), is more than one order of magnitude lower than eta+. The dissociation of singlet dimer states dominates the bulk photogeneration process induced by the weakly-absorbed light at 450 nm, with comparable eta+ and eta-. The "hot excited state" underlying the temperature-increasing emission at 540 nm has been attributed to the upper excitonic component Q+ of the first absorption band Q consistent with the exciton concept applied successfully to the interpretation of all dimer-underlain spectroscopic features of PtOEP samples studied.

Journal Article↗

PubMed2005

Role of the ssu and seu genes of Corynebacterium glutamicum ATCC 13032 in utilization of sulfonates and sulfonate esters as sulfur sources.

Corynebacterium glutamicum ATCC 13032 was found to be able to utilize a broad range of sulfonates and sulfonate esters as sulfur sources. The two gene clusters potentially involved in sulfonate utilization, ssuD1CBA and ssuI-seuABC-ssuD2, were identified in the genome of C. glutamicum ATCC 13032 by similarity searches. While the ssu genes encode proteins resembling Ssu proteins from Escherichia coli or Bacillus subtilis, the seu gene products exhibited similarity to the dibenzothiophene-degrading Dsz monooxygenases of Rhodococcus strain IGTS8. Growth tests with the C. glutamicum wild-type and appropriate mutant strains showed that the clustered genes ssuC, ssuB, and ssuA, putatively encoding the components of an ABC-type transporter system, are required for the utilization of aliphatic sulfonates. In C. glutamicum sulfonates are apparently degraded by sulfonatases encoded by ssuD1 and ssuD2. It was also found that the seu genes seuA, seuB, and seuC can effectively replace ssuD1 and ssuD2 for the degradation of sulfonate esters. The utilization of all sulfonates and sulfonate esters tested is dependent on a novel putative reductase encoded by ssuI. Obviously, all monooxygenases encoded by the ssu and seu genes, including SsuD1, SsuD2, SeuA, SeuB, and SeuC, which are reduced flavin mononucleotide dependent according to sequence similarity, have SsuI as an essential component. Using real-time reverse transcription-PCR, the ssu and seu gene cluster was found to be expressed considerably more strongly during growth on sulfonates and sulfonate esters than during growth on sulfate.

Bacterial Proteins↗

PubMed2003

Genome-wide analysis of the L-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation.

The genome sequence of Corynebacterium glutamicum, a gram-positive soil bacterium widely used as an amino acid producer, was analyzed by a similarity-based approach to elucidate the pathway for the biosynthesis of L-methionine. The functions of candidate ORFs were derived by gene deletion and, if necessary, by homologous complementation of suitable mutants. Of nine candidate ORFs (four of which were known previously), seven ORFs (cg0754 (metX), cg0755 (metY), cg1290 (metE), cg1702 (metH), cg2383 (metF), cg2536 (aecD), and cg2687 (metB)) were demonstrated to be part of the pathway while two others (cg0961 and cg3086) could be excluded. C. glutamicum synthesizes methionine in three, respectively four steps, starting from homoserine. C. glutamicum possesses two genes with similarity to homoserine acetyltransferases but only MetX can act as such while Cg0961 catalyzes a different, unknown reaction. For the incorporation of the sulfur moiety, the known functions of MetY and MetB could be confirmed and AecD was proven to be the only functional cystathionine beta-lyase in C. glutamicum, while Cg3086 can act neither as cystathionine gamma-synthase nor as cystathionine beta-lyase. Finally, MetE and MetH, which catalyze the conversion of L-homocysteine to L-methionine, could be newly identified, together with MetF which provides the necessary N(5)-methyltetrahydrofolate.

Amino Acid Sequence↗

PubMed2003

Self-reported efficacy of an all in-the-ear-canal prosthetic device to inhibit stuttering during one hundred hours of university teaching: an autobiographical clinical commentary.

This manuscript outlines the lifelong battle with severe stuttering and describes a new modality of effective amelioration of the disorder from the standpoint of a university professor and researcher in the field of stuttering. Childhood reactions to stuttering are discussed, along with the educational and vocational impact of stuttering. Ongoing therapy was received throughout the formative years and into adulthood, emphasizing reduced rates of speech. The use of Delayed Auditory Feedback (DAF) was found to induce fluent speech, but was considered only as a tool for decreasing the speech rate to achieve fluency. When fluency under DAF was discovered to be possible at faster speech rates, the possibility that the use of DAF and other forms of altered auditory feedback could themselves have an inhibitory effect on stuttering, without concomitant rate reduction was investigated. An In-The-Canal (ITC) fluency-enhancing device was used that provided DAF and Frequency Altered Feedback (FAF) to produce more fluent speech. After 10 months of use, the author was relatively free from stuttering. Speech was natural sounding, relatively spontaneous and unlaboured with an absence of fear. However, further testing (that is currently underway at various centres) is necessary before generalizations can be made.

Autobiographies as Topic↗

PubMed2002

The end-product of behavioural stuttering therapy: three decades of denaturing the disorder.

ISSUE: The state of stuttering therapy continues to be of great concern in the field of speech-language pathology. Despite decades of research and clinical practical, stuttering is still treated by ineffective and inefficient means. PROPOSAL: Our research group suggests that therapy failures stem from the use behavioural approaches; procedures that have resulted in only temporary and unnatural alleviation from the symptomatology by masking the disorder. These procedures 'denature' the disorder and induce pseudofluency rather than true fluency. It is proposed that in order to truly provide relief to those who stutter, professionals need to view both the overt and covert components of the disorder from the standpoint of attaining true fluency. Only then can the true syndrome-like nature of stuttering be addressed. In order to achieve true fluency the use of stuttering inhibitors must be incorporated such as choral speech and its permutations to derive natural sounding, stable and effortless speech in those who stutter. CONCLUSION: By doing so, the goal is to not only derive stable and natural-sounding speech, but also to help remove such factors as the expectancy, anticipation and the fear of stuttering that generally continue to shackle most people who stutter following behavioural therapy.

Behavior Therapy↗

PubMed2002

A common element in the immediate inducement of effortless, natural-sounding, fluent speech in people who stutter: 'the second speech signal'.

Recent attempts to find a common element in the inducement of fluent speech have focused on Wingate's notion that fluency can be induced via an altered manner of speaking by placing an emphasis on phonation. The problem with this notion of fluency enhancement is that it appears to be too expansive a scheme of fluency. The schemata allows for any forward flowing speech or speech-like act to be considered fluent despite the increased cognitive mediation, the extra effort involved in implementing the procedure, the unnaturalness of the resultant end product, and a lack of stability over time. It is proposed that the only significant characteristics of 'true fluency' are that it is indistinguishable from the typical fluent speech of people who do not stutter, and that it is effortless in nature. This is achieved via the use of a second speech signal which is not cognitive in nature, is operationally delineated, and is easy to manipulate.

Humans↗

PubMed2002

Reduction of stuttering: the dual inhibition hypothesis.

Treatment for stuttering attempts to reduce or eliminate the observable core markers of the disorder, specifically repetitions and prolongation. In this hypothesis, it is proposed that stuttering may be inhibited by two distinct yet related procedures: active inhibition and passive inhibition. Active inhibition is brought about when the person who stutters makes volitional changes to his or her speaking pattern, such as when employing behavioral modification techniques. Passive inhibition automatically inhibits the involuntary stuttering block and can be induced from an external source, such as altered auditory feedback, or by the use of sufficient active inhibition. It is suggested that passively inhibiting stuttering results in speech that is more automatic, natural sounding, and truly fluent speech than the speech that is derived primarily from active inhibition. Evidence of passive inhibition resulting from active inhibition can be seen when people who stutter exhibit uncontrolled fluency following behavioral therapy.

Humans↗

PubMed2001

Relaxed rrn expression and amino acid requirement of a Corynebacterium glutamicum rel mutant defective in (p)ppGpp metabolism.

The stringent response in Corynebacterium glutamicum was investigated. Sets of rrn-cat fusions were constructed in their native chromosomal position to examine the effects of amino acid starvation in a rel(+) strain and a Deltarel mutant defective in (p)ppGpp metabolism. The expression of the six rrn operons in the rel(+) control was stringently regulated and reduced to 79% upon induction of amino acid starvation. The Deltarel mutant displayed a relaxed regulation and was unable to reduce the rrn expression under amino acid depletion conditions. In addition, the Deltarel mutant grew more slowly in minimal medium than a rel(+) control. This growth effect was restored by a plasmid-encoded copy of rel or, alternatively, by supplementation of the minimal medium with the amino acid mixture casamino acids. In particular, the Deltarel strain of C. glutamicum displayed a requirement for the amino acids histidine and serine.

Amino Acids↗

PubMed2001

Producing the vowel/a/ prior to speaking inhibits stuttering in adults in the English language.

This study investigated the effects of producing and listening to the vowel /a/ on the frequency of overt stuttering moments in eight people who stuttered. Stuttering frequency counts were made for the speech produced in the control condition, and after each of these four experimental conditions: (a) producing a vowel /a/ for 4 s; (b) producing a vowel /a/ for 4 s and waiting for 4 s; (c) listening to a recording of the vowel /a/ for 4 s; and (d) listening to a recording of the vowel /a/ for 4 s and waiting for 4 s. A significant reduction in the stuttering frequency was only observed following production of the vowel /a/ without a 4 s delay (P=0.02), suggesting that the vowel production prior to speech, serves as a temporary fluency enhancer. Its similarity to the occurrence of overt stuttering moments (e.g. discrete part-word repetitions and prolongation's) and its relationship to the fundamental nature of the pathology are discussed.

Adolescent↗

PubMed2001

Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.

Amino Acid Sequence↗

PubMed2000

Stutter-free and stutter-filled speech signals and their role in stuttering amelioration for English speaking adults.

This study examined the power of an exogenously generated stuttered speech signal on stuttering frequency when compared to an exogenously generated normal speech signal. In addition, we examined the specific components of the second speech signal, which might be responsible for the inducement of fluency in people who stutter. Eight males and two females who stuttered participated in this study. Experiment I involved meaningful speech: normal continuous speech, normal interrupted speech, stuttered continuous speech, and stuttered interrupted speech, whereas Experiment II involved vowels and consonants: /a/, /a-i-u/, /s/, /s-sh-f/. The results indicated that stuttered and normal speech signals were equally effective in reducing stuttering frequency. Further, the vowels were more powerful than consonants in inducing fluency for people who stutter. It is suggested that acoustic manifestations of stuttering, rather than a problem, may be a natural compensatory mechanism to bypass or inhibit the 'involuntary block' at the neural level.

Adult↗

PubMed2000

Inducement of fluent speech in persons who stutter via visual choral speech.

A novel phenomenon of fluency enhancement via visual gestures of speech in the absence of traditional auditory feedback is reported herein. The effect on visual choral speech on stuttering frequency was investigated. Ten participants who stuttered recited memorized text aloud under two conditions. In a visual choral speech (VCS) condition participants were instructed to focus their gaze on the face, lips and jaw of a research assistant who 'silently mouthed' the text in unison. In a control condition, participants recited memorized text to the research assistant who sat motionless. A statistically significant (P=0.0025) reduction of approximately 80% in stuttering frequency was observed in the VCS condition. As visual linguistic cues are sufficient to activate the auditory cortex, one may speculate that VCS induces fluency in a similar yet undetermined manner as altered auditory feedback does.

Adult↗

PubMed2000

TetZ, a new tetracycline resistance determinant discovered in gram-positive bacteria, shows high homology to gram-negative regulated efflux systems.

The complete nucleotide sequence of the tetracycline resistance plasmid pAG1 from the gram-positive soil bacterium Corynebacterium glutamicum 22243 (formerly Corynebacterium melassecola 22243) was determined. The R-plasmid has a size of 19,751 bp and contains at least 18 complete open reading frames. The resistance determinant of pAG1 revealed homology to gram-negative tetracycline efflux and repressor systems of Tet classes A through J. The highest levels of amino acid sequence similarity were observed to the transmembrane tetracycline efflux protein TetA(A) and to the tetracycline repressor TetR(A) of transposon Tn1721 with 64 and 56% similarity, respectively. This is the first time a repressor-regulated tet gene has been found in gram-positive bacteria. A new class of tetracycline resistance and repressor proteins, termed TetA(Z) and TetR(Z), is proposed.

Amino Acid Sequence↗

PubMed2000

The 51,409-bp R-plasmid pTP10 from the multiresistant clinical isolate Corynebacterium striatum M82B is composed of DNA segments initially identified in soil bacteria and in plant, animal, and human pathogens.

The 51,409-bp DNA sequence of the multiresistance plasmid pTP10 from the gram-positive opportunistic human pathogen Corynebacterium striatum M82B has been determined. Fully automated genome interpretation led to the identification of 47 ORFs. Analysis of the genetic organization of pTP10 suggests that the plasmid is composed of eight DNA segments, the boundaries of which are represented by transposons and insertion sequences. The DNA segments of pTP10 are highly similar to (1) a plasmid-encoded erythromycin resistance region from the human pathogen Corynebacterium diphtheriae; (2) a chromosomal DNA region from Mycobacterium tuberculosis; (3) a plasmid-encoded chloramphenicol resistance region from the soil bacterium Corynebacterium glutamicum; (4) transposable elements from phytopathogenic gram-negative Pseudomonas, Xanthomonas and Erwinia species; and (5) a plasmid-encoded aminoglycoside resistance region from the gram-negative fish pathogen Pasteurella piscicida. The complete DNA sequence of pTP10 provides genetic information regarding the mechanisms of resistance to 16 antimicrobial agents that belong to six structural classes. In addition, the mosaic structure of pTP10 represents the evolutionary consolidation into a single plasmid molecule of antimicrobial resistances from microorganisms found in different habitats by means of mobile elements, resulting in the generation of a multiresistant bacterium that can infect humans.

Animals↗

PubMed1999

Pantothenate production in Escherichia coli K12 by enhanced expression of the panE gene encoding ketopantoate reductase.

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. coli double mutant was employed to isolate the E. coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate.

Alcohol Oxidoreductases↗

PubMed1999

The tetAB genes of the Corynebacterium striatum R-plasmid pTP10 encode an ABC transporter and confer tetracycline, oxytetracycline and oxacillin resistance in Corynebacterium glutamicum.

The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI-Sa/I DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated beta-lactam antibiotic oxacillin.

ATP-Binding Cassette Transporters↗

PubMed1999

The Corynebacterium glutamicum insertion sequence ISCg2 prefers conserved target sequences located adjacent to genes involved in aspartate and glutamate metabolism.

An IS element, termed ISCg2, was identified in the chromosome of Corynebacterium glutamicum ATCC 13032. After screening a cosmid library of the C. glutamicum ATCC 13032 genome, six copies of ISCg2 including their flanking regions were sequenced and analyzed. ISCg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. By comparisons with other IS elements, ISCg2 was classified as a member of the IS30 family of insertion sequences. The six copies of ISCg2 were identical at the nucleotide level and were located in intergenic, AT-rich regions of the chromosome. The regions in which the six copies of ISCg2 were inserted displayed significant similarities. This similarity extends over a region of 65 bp, which was assumed to be the target region for ISCg2. Interestingly, five of the six copies of ISCg2 were located adjacent to genes that may be involved in aspartate and glutamate metabolism or its regulation. Investigation of the distribution of ISCg2 showed that the IS element is restricted to certain C. glutamicum strains. Analysis of various integration regions indicates active transposition of ISCg2 in C. glutamicum.

Amino Acid Sequence↗