Biomedical subjects

R Klinke

Publications and source records attributed to R Klinke.

At least 73 records · Page 4Linked to original sources

PubMed1988

Human interleukin-3 and granulocyte-macrophage colony stimulating factor: site-specific mutagenesis and expression in yeast.

The two human colony stimulating factors, interleukin-3 and granulocyte-macrophage colony stimulating factor, have been molecularly cloned and expressed as secreted proteins in yeast. In both cases, non-glycosylated and glycosylated forms of the molecules were produced. Removal of N-linked glycosylation sites from the genes by site-directed mutagenesis prevented addition of most of the sugar residues, but revealed a low level of residual O-linked glycosylation on a portion of the molecules. No difference in specific biological activity was found between the different forms of the proteins. It was found that a significant proportion of human granulocyte-macrophage colony stimulating factor was degraded by the yeast KEX2 protease that was cleaving after the dibasic sequence Arg-Arg at positions 23-24 of the mature protein. Site-specific mutagenesis was employed to change this sequence to Leu-Arg, and this change resulted in greatly increased expression levels of full length protein and biological activity.

Bone Marrow↗

PubMed1988

Production of recombinant human colony stimulating factors in yeast.

Efficient yeast expression and purification systems for production of recombinant human GM-CSF, IL-3 and G-CSF have been established. Though yeast-derived production of recombinant CSFs (through the use of secretion based system) allows for generation of native molecules which can then be readily separated from fermentation broth, in many instances, natural cDNAs have had to be altered to allow for efficient expression, as well as production of a less heterogeneous product. In the case of CSFs described herein, beneficial mutations (made through site-directed mutagenesis) have included elimination of potential N-linked glycosylation sites, removal of KexII protease recognition sites (notably alterations in dibasic sequences) and elimination of extraneous cysteine residues which might complicate isolation of a homogeneous product due to intermolecular disulfide bonding.

Amino Acid Sequence↗

PubMed1988

Quantitative assessment of torasemide ototoxicity.

Torasemide (1-isopropyl-3- ([4-(3-methyl-phenylamino)pyridine]-3-sulfonyl)urea), a new loop diuretic, was quantitatively tested for ototoxicity in cats. The toxic dose that causes a defined hearing loss in 50% of the animals (TD50) was determined. TD50 was calculated as 20.8 mg/kg. This is slightly but not significantly above the value for furosemide (18.37 mg/kg). Hearing function tended to recover after the acute effect. The main metabolite in man (1-isopropyl-3-([(3-carboxy-anilino)-3-pyridyl]sulfonyl)urea, M5) showed no ototoxic action even in excessive doses. To ensure complete recovery, hearing function was tested in animals pretreated with torasemide in doses higher than TD50. There was no indication for permanent hearing impairment in these pretreated animals.

Acoustic Stimulation↗

PubMed1987

Processing of interaural time and intensity differences in the cat inferior colliculus.

1. Binaural neurones were recorded in the central nucleus of the cat inferior colliculus and were stimulated with tone and noise bursts. Closed field sound systems were used to produce independent interaural time (ITD) and intensity (IID) differences. Particular attention was paid to high frequency (above 2 kHz) cells. 2. Three main types of binaural neurone were found: High frequency excitatory-inhibitory neurones (EI cells), excited by input from the contralateral ear and inhibited by ipsilateral input, high frequency excitatory-excitatory cells (EE cells), excited by inputs from either ear and low frequency cells sensitive to interaural phase differences (IPD cells). 3. The EI cells had characteristics similar to those of IE cells in the contralateral lateral superior olive. They were sensitive to envelope ITDs (most cells) and IIDs (all cells) favouring the contralateral ear. The response of these cells increased with increasing contra lead ITDs or contra loud IIDs up to values well outside the physiological range. 4. Low frequency binaural cells were sensitive to interaural phase differences (IPDs). The peak response was often in the contralateral physiological range and the response was unaffected by IIDs. 5. Many high frequency EE cells were sensitive to envelope ITDs. These units were relatively unaffected by IID. Although the ITD sensitivity of these cells was generally less than that of the IPD cells, the peak response of the ITD curve was also often in the contralateral physiological range. 6. Some of the high frequency EI and EE cells were sensitive to ongoing time differences (OTDs) in white noise signals, i.e. they showed ITD response curves to carrier only shifted noise bursts. 7. The EI cells often showed recovery from inhibition at large ipsilateral lead. This tendency was increased as the sound pressure level on the inhibitory side was lowered and by the use of click stimuli. Similarly, cycles of suppression could be seen to follow excitation in some EE cells. The time course of these effects was in the order of hundreds of microseconds. 8. Binaural characteristics (degree of ITD, IID or OTD sensitivity) showed considerable interunit variation within each cell type. These variations were also affected by signal type (tone or noise bursts) and did not appear to be correlated with best frequency, nature of the tuning curve or PSTH type. We suggest that the time course of the inhibitory and excitatory effects at each unit (and its interaction with the signal type) determines the type of ITD response and that this time course varies from cell to cell.

Acoustic Stimulation↗

PubMed1987

The effect of inferior colliculus lesions on auditory evoked potentials.

The brain-stem auditory evoked potential (BAEP) was recorded in Nembutal anaesthetized cats before and after aspiration of the inferior colliculus on each side. The fast P1-P4 waves and the binaural interactions of P4 were unaffected by inferior colliculus removal. P5 and the following slow negative wave were reduced by inferior colliculus lesion. When only one inferior colliculus was ablated, this reduction was greater when the ear contralateral to the lesion side was stimulated.

Animals↗

PubMed1987

Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

Animals↗

PubMed1987

Basilar membrane motion in the pigeon measured with the Mössbauer technique.

Vibration measurements were made of the basilar membrane (BM), limbi and columella footplate (CFP) of pigeon using the Mössbauer technique. Recordings were located at 0.23-1.33 mm from the basal end of the BM. The existence of a travelling wave mode, propagating from base to apex, was established for papillae in apparently good physiological condition. For these papillae the characteristic frequency (CF) of the BM isovelocity (0.08 mm X s-1) response was an exponential function of distance with a frequency mapping constant of 0.91 +/- 0.10 mm (equivalent to 0.63 +/- 0.07 mm X oct-1); BM CF at the base was 5.95 +/- 0.65 kHz. Travelling wave motion was not demonstrated for papillae in poor physiological condition; tonotopy of BM CF was still evident, although the correlation with distance was less (1.08 +/- 0.30 mm X oct-1; 4.35 +/- 0.73 kHz at the base). BM motion was linear and the isovelocity responses were less sensitive and less sharp than single unit threshold tuning curves: for papillae in good physiological condition the SPL at BM CF at 0.08 mm X s-1 was 51 +/- 6 dB SPL; Q10 dB was 1.24 +/- 0.38; high- and low-frequency slopes were 20 +/- 6 dB X oct-1 and -14 +/- 4 dB X oct-1, respectively. The response of the BM relative to the CFP for papillae in good physiological condition was reminiscent of a second order resonant system with damping constant of 0.33 +/- 0.06 and group delay at BM CF of 0.89 +/- 0.36 periods.

Acoustic Stimulation↗

PubMed1987

[Processing of acoustic stimuli in the inner ear--a review of recent research results].

This review presents recent findings on the micromechanics of the basilar membrane. Active processes are essential for basilar membrane motion. It may be that contractile proteins within the outer hair cells play an important role for this amplification. The coding of acoustic information within the auditory nerve depends on spectral analysis (place information) as well as on the time structure of the stimulus. This latter time analysis seems to play a major role.

Auditory Threshold↗

PubMed1986

Synchronized responses of primary auditory fibre-populations in Caiman crocodilus (L.) to single tones and clicks.

Measurements of the responses to tones and clicks were made from single primary auditory fibres of the caiman. The distribution of the amplitude and phase of the fundamental component of the response rate modulation over the best frequencies of the fibres is comparable to that reported in the cat, despite the fact that the basilar membrane in caiman is only 4.5 mm long. However, much higher intensities are needed in the caiman (75-85 dB SPL) than reported in the cat (20 dB SPL) to obtain systematic distributions of the phase of the responses, probably due to the larger scatter of the phase responses in the caiman. The slopes of the phase distributions are very similar to those in cat. Single unit phase responses as a function of stimulus frequency at 85 dB SPL can be approximated by one, or in fibres with low best frequency, two straight lines. At lower intensities the deviation of the phase-frequency responses from a straight line increases as the group delay at the best frequency becomes larger. The shortest latencies of click responses are obtained with rarefaction clicks. Group delay estimates obtained from the responses to clicks and from the straight line approximations of the phase-frequency responses are related in a way expected for linear filter systems and accurately predict the measured distributions of the phase of the responses over the neural best frequency. The obtained group delays and click latencies in the caiman are very similar to those reported by other workers in the cat, the squirrel monkey and the treefrog, despite large morphological and probably functional differences of their inner ears. The click latencies are also very similar to those in the pigeon. The results are consistent with the existence of a mechanical travelling wave reported previously on the basilar membrane of the caiman, but at the same stimulus level the phase characteristic of the present single unit responses is steeper and the wave length estimates from the neural population phase distributions are shorter than those observed directly in the motion of the basilar membrane. Since the neural responses are an indirect estimate of the basilar membrane motion it cannot be decided whether the difference between neural and mechanical data is due to deterioration of the basilar membrane responses during the direct measurements or whether the basilar membrane response is sharpened by additional tuning mechanisms.

Animals↗

PubMed1986

Neurotransmission in the inner ear.

The present view on cochlear neurotransmission can be summarized as follows: There are two main types of synapses on cochlear hair cells, afferent and efferent ones. Afferent synaptic structures are abundant on inner hair cells whereas similar structures on the outer hair cells are less frequent and appear to be rudimentary. Presynaptic vesicles seem to be rare in outer hair cells. For the inner hair cell--afferent terminal--the presence of a chemical transmission mechanism is generally accepted. The transmitter substance has not yet been unequivocally demonstrated. Glycine, catecholamines, GABA and 5-HT can be eliminated as candidates as these compounds do not activate afferent fibres. There are good reasons, however, to consider amino acids. Most of the experimental results support glutamate as the transmitter (e.g. effectiveness of glutamate, kainic acid, glutamate diethylester). Aspartate is less likely. It is not yet well understood, however, why glutamate has to be applied in concentrations of up to 10(-3) M intracochlearly in order to activate afferent fibres and why elevated glutamate levels could not be demonstrated in perilymph collected during acoustical stimulation, whereas this same perilymph was able to activate afferent nerve terminals when applied intracochlearly. Efferent endings use acetylcholine as a transmitter. Enzymes for synthesis and breakdown of acetylcholine are present; acetylcholine is effective at the synaptic junction, as are cholinergic compounds and specific blockers. However, there may be different types of efferent endings in both the cochlear and vestibular organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1986

Travelling wave motion along the pigeon basilar membrane.

The basilar membrane (BM) motion in the pigeon was measured using the Mössbauer technique. Tonotopic frequency mapping and travelling wave motion were observed over the basal 35% of the BM. The sensitivity and sharpness of the BM tuning depended on the physiological condition of the cochlea. The observed amplitude responses did not match the frequency threshold tuning curves of single primary auditory fibers.

Acoustic Stimulation↗

PubMed1986

[Changes in the reaction of the cardiovascular system to angiotensin II in pregnancy modified by aspirin].

The experiments were done in pregnant and in nonpregnant rabbits, anesthesized with urethane. Arterial blood pressure was measured directly in carotid artery, estimating changes of blood pressure after infusions and injections of angiotensin II. It was found, that in pregnant rabbits systolic blood pressure and diastolic blood pressure was higher than in non-pregnant animals. Aspirin was given into jugular vein and caused a significant decrease of blood pressure in both groups of animals during whole time of experiment. Aspirin augmented the elevations of blood pressure, caused by angiotensin II.

Angiotensin II↗

PubMed1985

Quantitative evaluation of ototoxic side effects of furosemide, piretanide, bumetanide, azosemide and ozolinone in the cat--a new approach to the problem of ototoxicity.

A new method for the quantitative assessment of acute ototoxic side effects of drugs is described. It is suitable for screening purposes. The method is based on the determination of the toxic dose (TD50) which causes a defined hearing loss in 50% of the animals tested. The hearing loss is defined as a complete suppression of the compound action potential (CAP) of the auditory nerve, elicited by clicks 30 dB above threshold. This is approximately equivalent to a clinical hearing loss of 30 dB. The TD50 is used to estimate the therapeutic range. With this approach ototoxic side effects of furosemide, piretanide and bumetanide were compared quantitatively in cats. The TD50 values for CAP suppression were 18.37 mg/kg for furosemide; 4.29 mg/kg for piretanide and 2.21 mg/kg for bumetanide. As equipotent diuretic doses are 2.61 mg/kg for furosemide, 0.26 mg/kg for piretanide and 1.16 mg/kg for bumetanide, it appears that the relative ototoxicity is least for piretanide and highest for bumetanide. Plasma concentrations, determined initially and when recovery of CAP to 50% of control had occurred, indicate that bumetanide may be more slowly eliminated from the cochlear spaces than furosemide and piretanide. In addition azosemide and ozolinone were tested. The TD50 for azosemide was less than 10 mg/kg. With ozolinone where there are two isomers, only the diuretic (-)ozolinone was ototoxic; the TD50 was less than 100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1985

Intra- and extracranially recorded auditory evoked potentials in the cat. II. Effects of interaural time and intensity differences.

The effects of interaural time differences and interaural intensity differences on binaural interactions in the brain-stem evoked response (BSER) and auditory field potentials (AFPs) in superior olive and inferior colliculus were studied. Interaural time differences of up to +/- 2048 microseconds and interaural intensity differences of up to +/- 30 dB were used. Binaural interactions were studied for waves P4 and P5 of the BSER and the corresponding AFP components. When binaural interactions were present (wave P4 and subsequent waves), the dichotic potential was less than the sum of left and right evoked potentials. At zero interaural intensity difference the maximum binaural interaction was seen at zero interaural time difference. When an interaural intensity difference was present, maximum interaction was shifted away from zero interaural time difference such that left louder gave maximal interaction at right lead and vice versa. The time intensity trading values for this shift were between 9 and 20 microseconds/dB. The trading ratios for the superior olive wave P4 component and BSER P4/P5 were in the same range, i.e., no extra effects could be seen in the BSER postsynaptic to the superior olive. These time intensity trading ratios correspond to those of medial superior olive cells but not to those of lateral superior olive cells (Caird and Klinke 1983). We suggest that these binaural effects are produced by binaural mechanisms in the medial superior olive and that the lateral superior olive does not significantly contribute to the BSER. The inferior colliculus AFP slow wave binaural interactions do not correspond to those of the BSER.

Acoustic Stimulation↗

PubMed1985

Intra- and extracranially recorded auditory evoked potentials in the cat. I. Source location and binaural interaction.

Auditory field potentials (AFPs) were recorded stereotactically from the superior olivary complex and from the exposed inferior colliculus in the Nembutal anaesthetized cat. The brain-stem evoked response (BSER) was recorded simultaneously between an electrode on the dura mater at the vertex and an electrode on one bulla or in the neck musculature. A closed condenser microphone sound system was used to deliver monaural and binaural clicks. The binaural difference potential (BDP) was calculated by subtracting the sum of both monaurally evoked potentials from the binaurally evoked potential. The first binaural interaction was a reduction of BSER wave P4, arising in the superior olivary complex. The large extracellular AFPs generated in both inferior colliculus and superior olivary complex do not correspond to BSER waves, whereas the small volleys preceding the main waves do. These small waves show a much smaller change with recording distance than do the main AFP waves, i.e., the sources and sinks generating these waves appear to be more widely separated. We suggest that, in contrast to extracellular field potentials, the BSER is generated by action potentials in fibre tracts rather than postsynaptic potentials in nuclei. The implications for stimulation and recording laterality of late BSER waves are discussed.

Animals↗

PubMed1985

Mechanics of the basilar membrane in Caiman crocodilus.

Vibration measurements were made at a number of positions near the proximal (basal) end of the basilar membrane, and on the columella footplate, of Caiman crocodilus using a capacitive probe. The measurements established the existence of a mechanical travelling wave in this species. They showed no significant change of mechanical tuning with temperature, and were highly significantly different from previous reports of neural temperature sensitivity (Smolders, J. and Klinke, R. (1984): J. Comp. Physiol. 155, 19-30). Thus the neural sensitivity to temperature change appears not to depend upon basilar membrane mechanics. One interpretation of this is that the basilar membrane passively precedes an active temperature-sensitive filter. It was also found that the limbus supporting the basilar membrane had a measurable, but unturned, vibration and that the effect of draining scala tympani for the measurements was to increase the basilar membrane tuning frequency by a factor of about 1.5.

Animals↗

PubMed1984

Discharge patterns of cat primary auditory fibers with electrical stimulation of the cochlea.

Intact and destroyed cat cochleae were electrically stimulated through round window electrodes. Intact cochleae provided information about fiber properties with acoustic stimuli. With sinusoidal currents thresholds for synchronization were 4-68 microA rms. Thresholds were independent of the fiber's characteristic frequencies and thus of their places of origin in the intact cochleae. This shows large current spread. Phase-locking of the responses to electric stimulation was much stronger than it was to acoustic stimulation. Destroyed cochleae had no spontaneous activity and showed even stronger phase-locking. Thresholds obtained using 0.2 ms per phase biphasic pulse stimuli were 60-350 microApp. Action potentials were found to be released with as little as 0.3 ms latency. The neuronal responses to any electric stimulus differed considerably from the responses to corresponding acoustic stimuli. Vestibular fibers were easily activated by electric stimulation.

Acoustic Stimulation↗

PubMed1983

Processing of binaural stimuli by cat superior olivary complex neurons.

A method was developed to record sterotactically from the cat Superior Olivary Complex (SOC) using glass micropipettes. Sound stimulation was given through a closed system that permitted independent variation of interaural time (delta time) and intensity (delta int) differences. The most common binaural units found (n = 34) were ipsilateral excitatory, contralateral inhibitory (EI1), cells of the Lateral Superior Olive (LSO). Some Medial Superior Olive (MSO) cells and presumed MSO ascending afferents were found but, as noted by other authors, we found it difficult to obtain single unit recordings from this nucleus. The LSO EI cells were mostly sensitive to higher frequencies and showed Peristimulus Time Histograms (PSTHs) consisting of a sharp "On" response followed by a plateau when stimulated with Best Frequency (BF) tone bursts or noise bursts. This "On" response was sensitive to delta time and delta int such that ipsilateral time lead or intensity increase resulted in a stronger response. The response reached a minimum around zero delta time or delta int. No sharp peaks or dips were seen in the physiological range needed for localization, instead the response increased with increasing ipsilateral lead or intensity to the maximum values tested (2048 microseconds delta time, 30 dB delta int). In the physiological range the delta time and delta int response were complementary (both increasing response as ipsilaterality was increased). Provided enough sound energy in the unit's sensitive region was present, the same delta time curves were produced when BF tone bursts, masked tone bursts, "sharp onset" tone bursts or noise bursts were used. Changing the delta time of the carrier of the tone burst alone had no effect (except for one cell with a BF of 560 Hz), only the relative time of arrival of the stimulus envelope seemed to be important. In contrast to these LSO EI cells MSO-type units showed EI or EE predominantly low frequency phase-locked responses. When stimulated with interaurally phase shifted (delta pha) BF tones the unit response was a cyclic function of delta pha. Some cells (all that were tested, n = 6 including the 560 Hz LSO EI cell) showed these cyclic responses when stimulated with noise bursts or non-BF tones. However, these "characteristic delays" were not necessarily in the physiological range, i.e. we could find no evidence that these units were responding to delta time/delta pha values corresponding to a particular sound source direction.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗