Biomedical subjects

D Ehrlich

Publications and source records attributed to D Ehrlich.

At least 19 recordsLinked to original sources

PubMed1995

Characterization of the lactococcal abiD1 gene coding for phage abortive infection.

Lactococcal phage abortive infection (AbiD1) determined by plasmid pIL105 is active on both prolate- and small-isometric-head phages of the C6A and 936 phage groups, respectively, which are considered two different species. The Abi phenotype was found to be encoded by a single gene, designated abiD1. The abiD1-encoded protein (351 amino acids) does not show homology with any known protein and has a deduced isoelectric point of 10. It also possesses two helix-turn-helix structures and an unusually high content of asparagine, isoleucine, and lysine. A consensual promoter with a TGy extension to the -10 box was mapped 76 bp upstream of the start codon. Transcription initiated at this strong promoter stops at a terminator located 48 bp downstream from the promoter. The termination process is very efficient, and transcripts corresponding to the abiD1 gene were not visible in our experimental conditions with or without phage infection. Expression of abiD1 under the control of a T7 promoter induced a lag phase in Lactococcus lactis cell growth, suggesting that overproduction of AbiD1 could be toxic for the cells. AbiD1 protein was visualized in Escherichia coli by using a tightly controlled expression system.

Amino Acid Sequence↗

PubMed1995

Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1.

Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1. Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction. A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1s phages in AbiD1 cells when present in trans. Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4. The operon is transcribed 10 min after infection from a promoter presenting an extended -10 consensus sequence but no -35 sequence. Analysis of four independent AbiD1r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1. However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.

Amino Acid Sequence↗

PubMed1995

A review of microfabricated devices for gene-based diagnostics.

The next technologic revolution may well be the miniaturization of solution phase experimentation through the marriage of microelectronics and molecular biology. Already researchers leading this rapidly emerging technology are developing prototype biochemical "microlaboratories" that offer a millionfold reduction of scale; that is, biochemical analyses are being conducted with picoliter sample volumes rather than conventional microliter formats. Such miniaturization is achieved by exploiting the well-honed tools of microelectronics that accommodate highly parallel automated assays, ultrasensitive detection, high throughput, integrated data acquisition, computation, and distributed data storage/retrieval. The subject of this paper is to survey this evolving bioelectronic miniaturization technology with applications to gene-based diagnostics. Several pioneering microchips are described and compared to traditional biochemical methods. Specifically, miniaturized examples of various diagnostic processes such as sample preparation (PCR), assays (electrophoresis and probe arrays), and detection (integrated CCDs) are presented. Although these microdevices are rather embryonic they represent the first steps toward fabricating a fully integrated diagnostic system on a microchip.

Diagnosis↗

PubMed1994

Neural tuning for sound duration: role of inhibitory mechanisms in the inferior colliculus.

Duration is a biologically important feature of sound. Some neurons in the inferior colliculus of the big brown bat, Eptesicus fuscus, are tuned to sound duration, but it is unclear at what level the tuning originates or what neural mechanisms are responsible for it. The application of antagonists of the inhibitory neurotransmitters gamma-aminobutyric acid or glycine to neurons in the inferior colliculus eliminated duration tuning. Whole-cell patch-clamp recordings of synaptic currents suggested that inhibition produces a temporal frame within which excitation can occur. A model is proposed in which duration tuning arises when an early, sustained inhibitory input interacts with a delayed, transient excitatory input.

Acoustic Stimulation↗

PubMed1994

A microchip for quantitative detection of molecules utilizing luminescent and radioisotope reporter groups.

Through the marriage of microelectronics and molecular biology, a miniaturized device is presented for ultrasensitive detection of labeled molecules. The novelty of the approach is the direct integration of a charge-coupled device (CCD) and a probe-based assay. Specifically, a CCD detector serves as an active solid support that quantitatively detects and images the distribution of labeled target molecules near the spatially addressable pixels. The device exploits the inherent characteristics of microelectronics that accommodate highly parallel assays, ultrasensitive detection, high throughput, integrated data acquisition and computation. Hence, the technology presented offers substantial practical utility to both research and clinical diagnostic applications that require quantitative analysis of bound molecules. Specifically for probe-based assays such as reverse dot blots, hybridization of radiolabeled or fluorescently labeled, target DNA can be quantitatively assessed within seconds due to the high sensitivity and direct coupling employed.

DNA Probes↗

PubMed1993

Visual dysfunction in the spontaneously hypertensive rat.

We report two forms of visual defect in the spontaneously hypertensive rat (SHR) compared to its normotensive control strain (WKY). Ten-month-old male SHR and WKY were tested for intensity discrimination ability using a Y-maze and food reward. The SHR took more trials in the acquisition phase of the task using white light. In the test phase, in which the white light intensity difference was reduced, there was no significant difference in performance between SHR and WKY. Also, there was no significant difference between the strains when red light was used. The defect in SHR occurred in the blue range of the spectrum. Using blue light, the performance of SHR declined significantly at an intensity difference of 1.15 lux, whereas the performance of WKY did not decline significantly until the difference reached 0.01 lux. The defect in SHR for intensity discrimination was not related to cataract formation, but we detected a strain difference in the nuclei of the photoreceptor cells. Thus, this visual dysfunction may involve the rod photoreceptors. Further testing revealed a deficit in visual acuity in SHR aged 40-66 days, prior to the development of hypertension. Thus, the visual dysfunction of SHR is not secondary to their development of hypertension, but rather a discrete characteristic of the strain.

Animals↗

PubMed1993

Preclinical safety studies of glucose oxidase.

The purpose of this study was to develop preclinical safety data regarding the toxicologic and pharmacokinetic properties of glucose oxidase. Groups of adult BALB/c mice were injected with four doses of the enzyme ranging from 0.125 U of glucose oxidase/g b.wt. to 1 U/g b.wt., and the following responses were measured: survival, methemoglobin, glucose, blood urea nitrogen, creatine phosphokinase, erythrocyte count and body weight. We also compared the biodistribution of the enzyme in mice to the biodistribution of glucose oxidase conjugated to a monoclonal antibody. Finally, we assessed the histopathologic changes produced in mice by glucose oxidase and the binding of the enzyme to snap-frozen, human autopsy tissues. As expected, the acute toxicity of glucose oxidase was primarily due to methemoglobinemia (mean concentration 36% at the highest dose) and transient hypoglycemia (as low as 35 mg/dl). Furthermore, conjugated and unconjugated glucose oxidase had a blood half-life of less than 2 hr and concentrated in the liver and spleen. On the basis of our studies, we conclude that glucose oxidase has reasonably predictable toxicities and is, therefore, safe for human trials. The rapid uptake of conjugated and unconjugated glucose oxidase by the liver and spleen, however, may significantly limit the therapeutic targeting of glucose oxidase.

Animals↗

PubMed1992

Influence of acupuncture on physical performance capacity and haemodynamic parameters.

In a single blind study the question as to whether the needling of specific acupuncture points is able to produce an increase in physical performance capacity and better regulation of heart rate and blood pressure was examined. Thirty-six healthy young men were assigned at random to three groups, receiving either actual acupuncture, placebo acupuncture or no stimulation. Performance was determined by means of a spiro-ergometer test which was carried out at the beginning and at the end of five weeks of treatment consisting of one session per week. The subjects from the group which actually received acupuncture were able to increase maximum performance capacity significantly and also physical performance at the anaerobic threshold. This may be interpreted as a sign of functional improvement in haemodynamic and metabolic mechanisms. There was, on the whole, no noticeable effect produced by the placebo acupuncture. The control group, which received no stimulation, showed unfavourable changes in the values obtained compared with the results of the performance test at the commencement of the study.

Acupuncture Points↗

PubMed1992

New thermosensitive plasmid for gram-positive bacteria.

We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01. The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.

Amino Acid Sequence↗

PubMed1990

Effects of selective neurotoxins on eye growth in the young chick.

We have determined the extent of retinal cell damage and eye growth after treatment with different neurotoxins. Day-old chicks received an intraocular injection containing 2, 10, 50, 100 or 200 nmol of kainic acid (KA), an excitotoxic analogue of glutamate. After 21 days, with 2 nmol KA damage was confined to a small proportion of bipolar cells, whereas with 10-200 nmol KA there was dose-dependent damage to amacrine cells, ganglion cells and photoreceptors. There was an increase in eye weight and size of the vitreal chamber with 10 nmol KA or more. In a similar protocol using 5, 50 or 200 nmol quisqualic acid (QUIS), there was massive loss of amacrine cells and slight loss of horizontal cells, and a large increase in the anterior chamber depth. A single dose of 200 nmol N-methyl-D-aspartate lesioned amacrine cells, but did not alter eye size. Thus, excitotoxins which have different retinotoxic effects also have different effects on eye size. In another study, we examined the effects of KA and QUIS on occlusion-induced eye enlargement. Treatment with 200 nmol QUIS alone resulted in increased depth of the anterior chamber. QUIS combined with occlusion for 3 weeks did not interfere with occlusion-induced growth of the vitreal chamber. Therefore, the loss of a large proportion of amacrine cells did not interfere with occlusion-induced growth. KA results in eye growth in its own right, but restricts occlusion-induced growth. These effects are associated with damage to photoreceptors, amacrine and bipolar cells and a small proportion of ganglion cells. Finally, we examined the effects of an intraocular injection of tunicamycin, a purported photoreceptor-specific neurotoxin in amphibia. Tunicamycin (0.1 or 1 mumg/eye) resulted in a flattened anterior chamber but did not affect growth of the vitreal chamber. Tunicamycin inhibited occlusion-induced growth, and treated retinae displayed massive disruption and loss of all cell types apart from a single row of pigmented retinal epithelium. We conclude that (a) normal and occlusion-induced eye growth may have separate mechanisms of action, (b) the vitreous and anterior chambers have different growth mechanisms, and (c) photoreceptors may play a critical role in occlusion-induced growth of the eye.

Animals↗

PubMed1990

Differential effects of axotomy on substance P-containing and nicotinic acetylcholine receptor-containing retinal ganglion cells: time course of degeneration and effects of nerve growth factor.

The time course of degeneration of chick retinal ganglion cells was examined with Nissl stains and immunohistochemical methods for detection of substance P-like immunoreactive and nicotinic acetylcholine receptor immunoreactive neurons. Small lesions were made in the retinae, adjacent to the optic nerve head, and were subsequently sectioned parallel to the vitreal surface, permitting direct comparison of normal and axotomized retinal ganglion cells distal to the site of axon damage. At four and six days after surgery, a large number of degenerating cells with clear cytoplasm and pyknotic nuclei were seen. After eight, 10 and 14 days, many retinal ganglion cells displayed a chromatolytic response with dispersed Nissl granules, eccentric nuclei and the cells appeared crenulated. The number of apparently normal neurons in the ganglion cell layer in the axotomized region was reduced by about 50% six days following surgery, by about 70% on the 10th day and by about 75% on the 17th day. The remaining neurons in the ganglion cell layer were identified as displaced amacrine cells. From day 2 onwards, increased numbers of glial cells were present in the optic fibre, ganglion cell and inner plexiform layers. Many glial cells were enlarged and displayed extensive cytoplasmic processes, while others showed mitotic activity. Somata and proximal dendrites of retinal ganglion cells were intensely stained for substance P-like immunoreactivity at two and four days following surgery. At six, eight and 10 days, staining intensity was markedly reduced though still evident and at 14 and 17 days, substance P-like immunoreactivity had virtually disappeared. The persistence of limited substance P-like immunoreactive ganglion cells 10 days after surgery indicates that these cells have a relatively protracted response to axotomy. Nicotinic acetylcholine receptor-like immunoreactivity in the ganglion cells at two and four days following axotomy was substantially reduced. The majority of faintly stained nicotinic acetylcholine receptor-like immunoreactive ganglion cells, as visualized in counterstained sections, did not exhibit pyknosis in the immediate period following axotomy. Double label studies demonstrated that substance P-like immunoreactive ganglion cells were distinct from the nicotinic acetylcholine receptor-like immunoreactive ganglion cells. In a second set of experiments, nerve growth factor was then placed into the vitreous humor following intra-retinal axotomy. The somata, dendrites and proximal axons of lesioned substance P-like immunoreactive ganglion cells in these retinae were more intensely stained for a longer period of time and appeared more robust than cells from untreated retinae.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

A quantitative analysis of the effects of excitatory neurotoxins on retinal ganglion cells in the chick.

The present study examines the differential effects of three excitotoxins, kainic acid (KA), N-methyl-D-aspartate (NMDA), and alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) on neurons within the ganglion cell layer (GCL) of the chick retina. Two-day-old chicks were given a single, 5 microliters, intravitreal injection of KA, NMDA, or AMPA at a range of doses. Following treatment with 40 nmol KA, there was a 21% loss of neurons in the GCL. At 200 nmol KA, the loss increased to 46%. Exposure to KA eliminated mainly small neurons of soma area 5-15 microns2, and medium-sized ganglion cells of soma area 15-25 microns2. Large ganglion cells (greater than 25 microns2) remained unaffected. The vast majority of small cells were probably displaced amacrine cells. Exposure to 400 nmol NMDA resulted in a 14% loss of neurons, predominantly involving the large ganglion cells. At a dose of 3000 nmol NMDA, no further loss of cells was evident. Exposure to 200 nmol AMPA resulted in a 30% loss of large and some medium-sized ganglion cells. In a further series of experiments, exposure to excitotoxin was followed by a retinal scratch, which eliminated retinal ganglion cells within the axotomized region. The results indicate that only a small proportion of displaced amacrine cells are destroyed by NMDA and AMPA, whereas virtually all displaced amacrine cells are sensitive to KA. The findings of this study indicate the existence of subclasses of ganglion cells with specificity towards different types of excitatory amino acids (EAA).

Animals↗

PubMed1989

Organisation of the hyperstriatal projection to the ventral lateral geniculate nucleus in the chick (Gallus gallus).

In the chick forebrain, the hyperstriatum accessorium (HA), receives an orderly ascending projection from the dorsolateral thalamus, a primary visual centre. The study examines the topography of the descending projection from HA to the ventral lateral geniculate nucleus (GLv), another retino recipient region of the diencephalon. Discrete injections of horseradish peroxidase-labelled wheat germ agglutinin were placed in the HA, and reaction product was assessed in terminals within the GLv. The rostrocaudal axis of HA undergoes a 180 degrees rotation and is represented along the caudorostral axis of GLv. A comparison with other inputs to GLv, retina and optic tectum, shows that all inputs are in visuotopic register.

Animals↗

PubMed1989

Morphology of congenital microphthalmia in chicks (Gallus gallus).

This study examines the morphology of sporadic congenital microphthalmia in 1-day-old chicks, with particular emphasis on the neural retina. On the basis of the size of the eyeball it is possible to classify microphthalmia into two groups, severe and mild. In severe microphthalmia (less than 5 mm in equatorial diameter), the eyeball is severely malformed, but in most cases it shows evidence of an organized neural retina. Although ganglion cells and an optic nerve head are present in a small proportion of these retinae, we could not trace an optic nerve projection to the brain. These results indicate that some ganglion cells are able to be sustained after the period of naturally occurring cell death, suggesting either that those ganglion cells have established some contact with the central nervous system or that the presence of their axons in a rudimentary optic nerve is adequate for survival. In mild microphthalmia (greater than 5 mm in equatorial diameter), the most consistent abnormality is a defect in the pecten, which together with other abnormalities such as orbital cysts and colobomas indicates that the major abnormality occurs in the region of the choroid fissure. Associated with these defects are abnormalities within the ganglion cell layer. In some cases the number of ganglion cells was reduced, and in others the numbers of both ganglion and displaced amacrine cells were reduced. Unexpectedly, there were localized regions completely devoid of cells in the ganglion cell layer. The timing of the congenital defect may provide some clue as to the presence of a critical period in which displaced amacrine cells are formed or are sensitive to events related to ganglion cell loss.

Animals↗

PubMed1989

Differential effects of excitatory amino acids on photoreceptors of the chick retina: an electron-microscopical study using the zinc-iodide-osmium technique.

Although excitotoxins derived from acidic amino acids are known to damage neurons in the inner nuclear and ganglion cell layers of the retina, little is known about their effects on photoreceptors. This study examines the acute and long-term effects of excitotoxins on photoreceptors of the chick retina. The zinc-iodide-osmium (ZIO) technique, which darkly labels a substantial subpopulation of synaptic vesicles in normal photoreceptor terminals, was used to supplement routine electron microscopy. Two-day-old chicks received a single intraocular injection of either 10, 50, or 200 nmoles kainic acid (KA), 200 nmoles N-methyl-D-aspartic acid (NMDA), or 200 nmoles quisqualic acid (QUIS), and were allowed to survive for either 6 h, 7 d, or 21 d. At 6 h, following exposure to 10, 50, and 200 nmoles KA, there was swelling and disruption of photoreceptor lamellae of the outer segments. At 7- and 21-d survival, 50 and 200 nmoles KA resulted in rounded, condensed synaptic terminals, which contained a high density of synaptic vesicles. However, there was complete loss of ZIO-positive vesicles within these photoreceptors. Outer segments were still disrupted, although small patches of lamellae were evident, suggestive of regeneration. Following exposure to QUIS, there was extensive swelling of outer segment lamellae at 6 h survival. Synaptic ribbons in terminals were also swollen. At longer survival periods, QUIS exposure resulted in a reduction of ZIO-positive vesicles, as well as swollen lamellae in outer segments. NMDA exposure, at either short or long-term survival, did not alter photoreceptor morphology, including the pattern of ZIO stain. The prolonged effects of KA, and to a lesser extent QUIS, on photoreceptors suggests that these drugs have a long-term effect on photoreceptor function. The ZIO technique provides a novel and potentially useful approach for identification of damaged photoreceptors.

Amino Acids↗

PubMed1989

Excitatory amino acids interfere with normal eye growth in posthatch chick.

This study examines the effects of excitotoxic amino acids on eye growth and retinal morphology. Day old chicks received a single intraocular injection of either 200 nmoles kainic acid (KA), 200 nmoles quisqualic acid (QUIS) or 400 nmoles N-methyl-D,L-aspartate (NMDA). Following survival periods of 7, 14 and 21 days, eyeballs were removed and weighed. Measurements of axial length, equatorial length, anterior chamber depth and corneal diameter were taken. Treatment with KA increased eye weight and equatorial length. Treatment with QUIS increased the anterior chamber depth but decreased the equatorial length. Treatment with NMDA increased anterior chamber depth, but to a lesser extent than QUIS. The effects of QUIS and NMDA could be distinguished from those of KA since the former excitotoxins resulted in a marked increase in anterior chamber depth with no enlargement of vitreal chamber. Changes in eye size were evident by day 7 and were sustained throughout the duration of the experiment. Examination of retinae revealed that KA lesions amacrine cells, bipolar cells, some ganglion cells and photoreceptors. Exposure to QUIS lesions amacrine cells, horizontal cells and causes mild disruption of photoreceptor outer segments. In contrast, NMDA predominantly lesions amacrine cells. The results demonstrate that these neurotoxins have different effects on eye growth, which may be associated with differences in retinal pathology. It is proposed that photoreceptors are ideally suited to play a role in the control of eye growth.

Animals↗

PubMed1988

Development of the supraoptic decussation in the chick (Gallus gallus).

The developing supraoptic decussation (SOD), a major interhemispheric tract in birds, has been implicated in both transfer of visual information and in the modulation of brain asymmetry. Moreover little is known of its morphology during development. We have examined the development of the chick SOD, which consists of three subregions; dorsal, ventral and subventral SOD. In the dorsal SOD the total number of fibres reach 968,000 on the 19th day of incubation (E-19), falling to 570,000 by the 8th day after hatching (P-8). In the ventral SOD, the number of fibres at E-19 reach 660,000, followed by a gradual reduction in their number to about 490,000 at P-22. In the subventral SOD the number of fibres estimated was 87,000 at E-15 falling to about 36,000 P-1. Compared with adult levels, there is, respectively, a drop in the number of fibres of 44%, 25% and 69% in the dorsal, ventral and subventral SOD during development. At E-19 in both the dorsal and ventral SOD there is qualitative evidence of axonal loss; disrupted axonal profiles, increased extracellular space and cells containing lysosomal cytoplasmic inclusions indicative of macrophages. Differences were also observed in the pattern of myelination, the dorsal, ventral and subventral SOD were shown to myelinate at different rates. Thus, in a single named tract, the SOD, there are major differences in the onset, rate and extent of fibre loss and myelogenesis within its three subregions. The functional implications of these differences are considered.

Animals↗

PubMed1988

Neurotoxic effects of kainic acid on developing chick retina.

The neurotoxic effects of kainic acid (KA) on developing neurons in the chick retina was investigated in an in vitro preparation. Eyecups from chick embroyos at 6 (E-6), 8, 10, 12, 14, 16, and 20 days of incubation and from chicks on day 1 posthatch (D-1) were exposed to different doses of KA for 30 min and then processed for light microscopy. Neurotoxic damage was evaluated by the presence of swollen cell bodies, containing pale cytoplasm and pyknotic nuclei. At E-8, amacrine cells first became sensitive to KA and displayed neurotoxic damage at a threshold concentration of 20 microM. Their sensitivity to KA increased over the following 4 days, so that by E-12 they attained a threshold sensitivity of 5.0 microM KA. At E-14, one third of the retinae showed amacrine cell damage at 0.5-2.0 microM KA, less than the threshold dose of 5.0 microM KA required at D-1. Bipolar cells first become sensitive to KA at E-12, at a threshold concentration of 5.0 microM. The threshold concentration decreased over the following 10 days: 2.0 microM at E-16, 1.0 microM at E-20, and 0.5 microM at D-1. At E-8 and E-10, horizontal cells were susceptible to a relatively high concentration of 80 microM KA. The sensitivity to KA is evident prior to the formation of photoreceptor input. These results indicate that amacrine and horizontal cells are susceptible to KA at an earlier age than bipolar cells. Both amacrine and bipolar cells exhibit an age-dependent relationship with the threshold concentration of KA required to cause neurotoxicity; in general, the older the embryo, the lower the dose of KA. However, the increased susceptibility of amacrine cells at E-14 suggests a transient hypersensitivity to KA during this period which may reflect an overproduction of the receptor-ionic channel complex necessary for KA to exert its effect.

Animals↗