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Biomedical subjects

D Johnston

Publications and source records attributed to D Johnston.

At least 19 recordsLinked to original sources
PubMed1996

Axonal action-potential initiation and Na+ channel densities in the soma and axon initial segment of subicular pyramidal neurons.

A long-standing hypothesis is that action potentials initiate first in the axon hillock/initial segment (AH-IS) region because of a locally high density of Na+ channels. We tested this idea in subicular pyramidal neurons by using patch-clamp recordings in hippocampal slices. Simultaneous recordings from the soma and IS confirmed that orthodromic action potentials initiated in the axon and then invaded the soma. However, blocking Na+ channels in the AH-IS with locally applied tetrodotoxin (TTX) did not raise the somatic threshold membrane potential for orthodromic spikes. TTX applied to the axon beyond the AH-IS (30-60 microm from the soma) raised the apparent somatic threshold by approximately 8 mV. We estimated the Na+ current density in the AH-IS and somatic membranes by using cell-attached patch-clamp recordings and found similar magnitudes (3-4 pA/microm2). Thus, the present results suggest that orthodromic action potentials initiate in the axon beyond the AH-IS and that the minimum threshold for spike initiation of the neuron is not determined by a high density of Na+ channels in the AH-IS region.

Action Potentials↗
PubMed1996

Multiple channel types contribute to the low-voltage-activated calcium current in hippocampal CA3 pyramidal neurons.

Hippocampal neurons exhibit low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium currents. We characterized the LVA current by recording whole-cell Ca2+ currents from acutely isolated rat hippocampal CA3 pyramidal neurons in 2 mM Ca2+. Long depolarizing steps to -50 mV revealed two components to the LVA current: transient and sustained. The transient phase had a fast decay time constant of 59 msec. The sustained phase persisted throughout the depolarization, even for steps lasting several seconds. The transient current was inhibited by the classic T-type channel antagonists Ni2+ and amiloride. The anticonvulsant phenytoin preferentially blocked the sustained phase, but ethosuximide had no effect. Steady-state inactivation of the transient component was half-maximal at -80 mV. Nimodipine, an L-type channel antagonist, partly inhibited the sustained current. BayK-8644, an L-type channel agonist, potentiated the sustained current. Calciseptine, another L-type channel antagonist, inhibited the sustained component. omega-Conotoxin-MVIIC, a nonselective toxin for HVA channels, had no effect on either of the LVA current components. omega-Grammotoxin-SIA, another nonselective toxin, partially inhibited the sustained component. The voltage dependence of activation of the nimodipine-sensitive current could be fit with a single Boltzmann, consistent with a homogenous population of L-type channels in CA3 neurons. Half-maximal activation of the nimodipine-sensitive current occurred at -30 mV, considerably more negative than the remaining HVA current. These results suggest that in physiologic Ca2+ more than one type of Ca2+ channel contributes to the LVA current in CA3 neurons. The transient current is carried by T-type channels. The sustained current is carried, at least in part, by dihydropyridine-sensitive channels. Thus, the designation "low-voltage-activated" should not be limited to T-type channels. These findings challenge the traditional designation of L-type channels as exclusively HVA and reveal a possible role in subthreshold Ca2+ signaling.

Animals↗
PubMed1996

Actions of endogenous opioids on NMDA receptor-independent long-term potentiation in area CA3 of the hippocampus.

The opioid peptides represent a major class of neurotransmitter in the vertebrate nervous system and are prevalent in the hippocampus. There is considerable interest in the physiological function of the opioids contained in the mossy fiber pathway. The release of opioids from mossy fibers shows a strong frequency dependence. Long-term potentiation (LTP) at this synapse, an NMDA receptor-independent form of LTP, also depends on high-frequency synaptic activity, and this has led to speculation that endogenous opioids may be a critical factor in LTP induction. Previous reports using extracellular recordings have provided evidence for and against a role for opioids in mossy fiber LTP. Using single-cell recording techniques, we have tested the hypothesis that endogenous opioids are required for mossy fiber LTP induction. We recorded from a defined population of synapses that had EPSCs with fast rise times, short latencies, and monophasic decays, consistent with a proximally terminating synapse. The opioid antagonist naloxone prevented mossy fiber LTP in the rat, but had no effect on the commissural/associational system, a nonopioid-containing pathway. The action of naloxone was not mediated through disinhibition because GABAA receptors were pharmacologically blocked in these experiments. We also tested the hypothesis that variations in postsynaptic receptor subtype distribution between species might explain previous controversies regarding the role of endogenous opioids. In contrast to the rat, LTP of the mossy fiber field potential in guinea pig was not blocked by naloxone. Our data suggest that opioids may be the presynaptically released, frequency-dependent, associative factor for mossy fiber LTP induction.

Animals↗
PubMed1996

Dendritic calcium channels and hippocampal long-term depression.

The authors examine the potential role of the different sources of dendritic Ca2+ influx in long-term depression (LTD) of synaptic efficacy. Recent data on the location and functional distribution of voltage-gated Ca2+ channels obtained from fluorescence imaging and patch clamp experiments are presented. This is followed by a discussion of the existing evidence for different sources of Ca2+ playing a role in the induction of LTD. The authors conclude that a number of key issues need to be resolved before any conclusions are drawn as to the involvement of any specific route of Ca2+ entry in LTD.

Animals↗
PubMed1996

Does eversion of the anorectum during restorative proctocolectomy influence functional outcome?

PURPOSE: The aim of this study was to determine the effect of eversion of the anorectum during restorative proctocolectomy (RP) for ulcerative colitis on functional outcome. METHODS: One hundred seventeen patients underwent RP with stapled end-to-end ileal pouch-anal anastomosis (EEA), without resection of the anal mucosa. Sixty-four underwent EEA with eversion of the anorectum, and 53 underwent EEA without eversion. Each patient underwent paired studies of anorectal function before and a median of 12 months after RP. RESULTS: One year after RP, median (interquartile range) maximum resting pressure was 69 (range, 51-88) cmH2O in those patients who underwent eversion vs. 80 (range, 64-90) cmH2O in patients without eversion (P < 0.04). Threshold sensation in the upper, middle, and lower thirds of the anal canal were 9.1, 7.4, and 6.8 mA after eversion vs. 6.9, 4.9, and 3.8 mA without eversion (P = 0.003, P < 0.001, P < 0.001, respectively). Before operation, all patients had a rectoanal inhibitory reflex; however, after RP, 54 of 64 patients in the eversion group and 50 of 53 patients with a stapled EEA without eversion had an inhibitory reflex (P = not significant). Leakage of mucus was experienced by 11 patients who underwent eversion, compared with 9 patients without eversion. Fifty-six of 64 patients with eversion could defer defecation for more than 30 min compared with 43 of 53 patients without eversion. Twenty-two of 64 patients in the eversion group retained perfect discrimination between flatus and feces compared with 38 of 54 without eversion (P < 0.001). Level of the anastomosis was 1 (range, 0.5-3) cm above dentate line after eversion compared with 1.5 (range, 0-6) cm without eversion. CONCLUSION: Clinical outcome after RP with eversion was not as good as outcome after stapled EEA without eversion. Such a conclusion requires confirmation in a prospective control trial.

Adult↗
PubMed1996

The calcium code.

Explore the source record for details and available documents.

Action Potentials↗
PubMed1996

Recurrent septicaemia in a neutropenic patient with typhlitis.

We report a case where life threatening gram negative sepsis developed in a patient with CLL in association with post chemotherapy neutropenia on three occasions. Bacterial typhlitis or neutropenic enterocolitis, which is a well described entity of bowel necrosis seen in immunosuppressed patients, was demonstrated at colonoscopy and was the probable portal of entry of micro-organisms. After spontaneous resolution of the typhlitis, further chemotherapy has been given without recurrent sepsis. Typhlitis should be considered as a cause of recurrent septicaemia in neutropenic patients.

Enterocolitis↗
PubMed1996

Comparative analysis of prostate specific antigen and its indexes in the detection of prostate cancer.

PURPOSE: Prostate specific antigen (PSA) density and age referenced PSA have been proposed in an attempt to improve the power of PSA in the detection of early prostate cancer. Reported results have been controversial and disappointing. Because the association of gland volume with PSA is stronger than that of age we developed a new index, volume referenced PSA, and compared it to PSA density and other indexes. MATERIALS AND METHODS: Volume referenced PSA was developed from a control group of 408 men without clinical evidence of prostate cancer using a standardized Z score. A retrospective analysis was performed comparing PSA and all its indexes in 580 men who underwent prostate biopsy. In addition to overall analysis, PSA and its indexes were evaluated with receiver operating characteristic curves by age and volume subcategories. RESULTS: Cancer was identified in 35% of the 580 men. The number of missed cancers using established thresholds significantly favored volume referenced PSA clinically and statistically compared to all other indexes but it was equivalent to PSA alone clinically. Age referenced PSA prevented more biopsies from being done than did volume referenced PSA (39% versus 31%) but resulted in the diagnosis of 48% fewer cancers. Receiver operating characteristic curve analysis demonstrated a significantly better performance for volume referenced PSA and PSA density compared to PSA alone and age referenced PSA. CONCLUSIONS: Volume based PSA indexes are superior to PSA and age referenced PSA statistically. However, clinically volume referenced PSA is comparable to PSA, and both are superior to age referenced PSA and PSA density in the detection of prostate cancer.

Adult↗
PubMed1996

The pathology of cornea in Tangier disease (familial high density lipoprotein deficiency).

AIMS: To clarify the underlying causes of corneal opacification in Tangier disease. METHODS: Both corneas were removed at death from a 62 year old man with Tangier disease, and were examined by direct and transmission electron microscopy, histochemistry, biochemical analysis by thin-layer and gas-liquid chromatography after extraction, and by differential scanning calorimetry. RESULTS: Membranous inclusions in the stroma were seen on transmission electron microscopy. Direct analysis confirmed enrichment with phospholipids and cholesterol, with acyl patterns and proportions as ester broadly similar to those of normal cornea. Tangier cornea showed major thermotropic phase transitions in the range 28-37 degrees C, peak 30-33 degrees C, extending above profiles of normal clear cornea and without the complexity of those seen with cornea with heavy arcus involvement. CONCLUSIONS: Lipid accumulation underlies corneal opacification in Tangier disease. The excess material is mainly phospholipid and cholesterol esters. As at other sites which are below body core temperature, notably tonsil, accumulation may be enhanced by local impaired mobilisation of material as the phase transitions of the excess lipid present extend above ambient corneal temperatures.

Calorimetry, Differential Scanning↗
PubMed1996

Active properties of neuronal dendrites.

Dendrites of neurons in the central nervous system are the principal sites for excitatory synaptic input. Although little is known about their function, two disparate perspectives have arisen to describe the activity patterns inherent to these diverse tree-like structures. Dendrites are thus considered either passive or active in their role in integrating synaptic inputs. This review follows the history of dendritic research from before the turn of the century to the present, with a primary focus on the hippocampus. A number of recent techniques, including high-speed fluorescence imaging and dendritic patch clamping, have provided new information and perspectives about the active properties of dendrites. The results support previous notions about the dendritic propagation of action potentials and also indicate which types of voltage-gated sodium and calcium channels are expressed and functionally active in dendrites. Possible roles for the active properties of dendrites in synaptic plasticity and integration are also discussed.

Action Potentials↗
PubMed1996

Dihydropyridine-sensitive, voltage-gated Ca2+ channels contribute to the resting intracellular Ca2+ concentration of hippocampal CA1 pyramidal neurons.

1. Whole cell recordings and high-speed fluorescence imaging were used to investigate the contribution of voltage-gated Ca2+ channels to the resting Ca2+ concentration ([Ca2+]i) in hippocampal CA1 pyramidal neurons. 2. Prolonged membrane hyperpolarization produced, in a voltage-dependent manner, sustained decreases in [Ca2+]i in the somatic and apical dendritic regions of the neuron. This hyperpolarization-induced decrease in [Ca2+]i occurred with a time constant of approximately 1 s and was maintained for as long as the membrane potential was held at the new level. Ratiometric measures showed that [Ca2+]i is significantly elevated at holding potentials of -50 mV compared with -80 mV. 3. The hyperpolarization-induced decrease in [Ca2+]i was reduced significantly by 200 microM Cd2+ and 10 microM nimodipine, but was only slightly inhibited by 50 microM Ni2+. The largest amplitude decrease in [Ca2+]i was observed in the proximal apical dendrites with the amplitude of the Ca2+ change decreasing with further distance from the soma. 4. Whole cell recordings from acutely isolated hippocampal pyramidal neurons reveal a slowly inactivating Ca2+ current with similar voltage dependence and pharmacology to the hyperpolarization-induced decrease in [Ca2+]i. 5. The data suggest that a population of dihydropyridine-sensitive Ca2+ channels are active at resting membrane potentials and that this channel activation significantly contributes to the resting [Ca2+]i. These channels appear to be present throughout the neuron and may be located most densely in the proximal apical dendrites.

Animals↗
PubMed1996

DNA image analysis of urinary cytology: prediction of recurrent transitional cell carcinoma.

To evaluate the utility of image analysis in monitoring patients with transitional cell carcinoma, we studied, by cytologic means and by image analysis, 78 urinary tract specimens from 66 patients, of whom 49 (74%) had a previous history of transitional cell carcinoma. The specimens consisted of 51 (65%) voided urine specimens, 12 (15%) bladder washings, 8 (10%) ureteral washings, 3 (4%) ureteral brushings, 2 (3%) renal pelvic washings, and 2 (3%) catheterized urine specimens. DNA histograms were classified into five patterns on the basis of their DNA index and the percentage of their cells with DNA content greater than 5c: diploid (single peak in the 2c region with no cells greater than 5c), intermediate (diploid with less than 10% of cells greater than 5c), aneuploid (single peak or multiple peaks between the 2c and 4c region or more than 10% of cells greater than 5c), tetraploid (at least 10% of cells in the 4c region and a corresponding peak at 8c), and polyploid (multiple peaks in the 2c, 4c, 8c, and 10c regions). Of the 78 cases, 22 were diploid, 24 were intermediate, 29 were aneuploid, one was tetraploid, and two were polyploid. Histologic confirmation or clinical follow-up was found in 29 aneuploid cases, 13 intermediate cases, and one diploid case. Most cases of carcinoma in situ (five of six) and invasive tumors (12 of 17) were aneuploid. The sensitivity was 100%, and the specificity was 73% when cytologic and image analysis results were combined. We conclude that image analysis, when combined with cytologic examination, is a reliable noninvasive diagnostic test for monitoring patients with transitional cell carcinoma; aneuploidy is specific for malignancy; and the presence of cells greater than 5c, although frequently associated with tumor recurrence, can be seen in non-neoplastic conditions.

Adult↗
PubMed1995

Isolation and characterization of a trypsin from the slipper lobster, Thenus orientalis (Lund).

Trypsin has been isolated and purified from the digestive glands of the slipper lobster, Thenus orientalis. It is a glycoprotein with a molecular mass of approximately 35 kDa as judged by both SDS-PAGE and gel filtration. The N-terminal amino acid sequence has strong homology to crustacean trypsins. This is confirmed by the cross-reaction of crustacean trypsins with antibodies to the T. orientalis enzyme. Despite a 40% identity with the bovine trypsin N-terminal sequence, there was no cross-reaction with the mammalian serine proteases. The optimum kcat and kcat/Km values for N-alpha-benzoylarginine-p-nitroanalide were 0.91 s-1 and 9.7 x 10(3) M-1 s-1, respectively, with this specificity constant being lower than those reported for other crustacean trypsins. Inhibition studies indicated the presence of serine and histidine at the active site and pKa of the catalytic histidine residue was found to be 5.7 in the free enzyme and 4.7 in the Michaelis complex.

Amino Acid Sequence↗
PubMed1995

Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons.

1. We have used dendrite-attached patch-clamp techniques to record single Na+ and Ca2+ channel activity from the apical dendrites (up to 350 microns away from soma) of CA1 pyramidal neurons in rat hippocampal slices (ages: 2-8 weeks). 2. Na+ channels were found in every patch examined (range: 2 to > 20 channels per patch). Channel openings, which had a slope conductance of 15 +/- 0.3 pS (mean +/- S.E.M.), began with test commands to around -50 mV and consisted of both early transient channel activity and also later occurring prolonged openings of 5-15 ms. All Na+ channel activity was suppressed by inclusion of TTX (1 microM) in the recording pipette. 3. Ca2+ channel activity was recorded in about 80% of the patches examined (range: 1 to > 10 channels per patch). Several types of channel behaviour were observed in these patches. Single channel recordings in 110 mM BaCl2, revealed an approximately 10 pS channel of small unitary current amplitude (-0.5 pA at -20 mV). These channels began activating at relatively hyperpolarized potentials (-50 mV) and ensemble averages of this low voltage-activated (LVA) channel activity showed rapid inactivation. 4. A somewhat heterogeneous population of high voltage-activated, moderate conductance (HVAm; approximately 17 pS), Ca2+ channel activity was also encountered. These channels exhibited a relatively large unitary amplitude (-0.8 pA at 0 mV) and ensemble averages demonstrated moderate inactivation. The HVAm population of channels could be tentatively subdivided into two separate groups based upon mean channel open times. 5. Less frequently, HVA, large conductance (27 pS) Ca2+ channel activity (HVA1) was also observed. This large unitary amplitude (-1.5 pA at 0 mV) channel activity began with steps to approximately 0 mV and ensemble averages did not show any time-dependent inactivation. The dihydropyridine Ca2+ channel agonist Bay K 8644 (0.5 or 1 microM) was found to characteristically prolong these channel openings. 6. omega-Conotoxin MVIIC (10 microM), did not significantly reduce the amount of channel activity recorded from the LVA, HVAm or HVA1 channel types in dendritic patches. In patches from somata, omega-conotoxin MVIIC was effective in eliminating a significant amount of HVAm Ca2+ channel activity. Inclusion of 50 or 100 microM NiCl2 to the recording solution significantly reduced the amount of channel activity recorded from LVA and HVAm channel types in dendritic patches. A subpopulation of HVAm channels was, however, found to be Ni2+ insensitive. Dendritic HVA, channel activity was unaffected by these low concentrations of Ni2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗
PubMed1995

p53 expression in dedifferentiated chondrosarcoma.

BACKGROUND: p53 acts as a tumor suppressor gene because of to its negative control of the cell cycle and its central role in programmed cell death. It frequently is mutated, as observed in a variety of human neoplasms. The mutations inhibit tumor-suppressor activities of p53, which may gain a new function of tumor promotion. In this study, p53 was investigated in various components of dedifferentiated chondrosarcoma and correlated with their proliferative activities. METHODS: Immunohistochemical assays for p53, Ki-67, and proliferating cell nuclear antigen (PCNA) were used in a series of eight dedifferentiated chondrosarcomas of bone. The cartilaginous component was low grade (Grade I-II) in five cases. It was predominantly low grade with foci of a high grade (Grade III) chondrosarcoma in the remaining three cases. The noncartilaginous (dedifferentiated) high grade component consisted of malignant fibrous histiocytoma in five cases and osteosarcoma in three. RESULTS: Regardless of the histological type, diffuse strong nuclear staining for p53 occurred in the high grade noncartilaginous component of all eight of the tumors. The low grade cartilaginous component of six cases was negative for p53, with focal weak staining in the two remaining cases. The high grade cartilaginous component showed strong positive staining for this protein in all three cases. Ki-67 and PCNA expression were similar to that of p53. CONCLUSIONS: The percentage of p53 positive staining roughly was parallel to the proliferating fraction of cells in various components of dedifferentiated chondrosarcoma. Moreover, p53 overexpression was consistently present in the high grade noncartilaginous (dedifferentiated) component of the tumor and was accompanied by increased proliferative activity.

Cell Differentiation↗
PubMed1995

Synaptic activation of voltage-gated channels in the dendrites of hippocampal pyramidal neurons.

Activation of dendritic voltage-gated ion channels by local synaptic input was tested by simultaneous dendrite-attached patch-clamp recordings and whole-cell somatic voltage recordings made from CA1 pyramidal neurons in hippocampal slices. Schaffer collateral stimulation elicited subthreshold excitatory postsynaptic potentials (EPSPs) that opened voltage-gated sodium and calcium channels in the apical dendrites. The EPSP-activated sodium channels opened near the peak of the EPSP, whereas low voltage-activated calcium channels opened near the EPSP peak and during the decay phase. Dendritic high voltage-activated channels required somatic action potential generation or suprathreshold synaptic trains for activation. Dendritic voltage-gated channels are, therefore, likely to participate in dendritic integration of synaptic events.

Action Potentials↗
PubMed1995

Mechanism of synergy of levamisole and fluorouracil: induction of human leukocyte antigen class I in a colorectal cancer cell line.

BACKGROUND: The use of the combination of fluorouracil (5-FU) and levamisole has been shown to improve the survival of patients with resected Dukes' stage C colon carcinoma. 5-FU is incorporated into RNA, which results in aberrant processing and turnover of RNA. Neither the mechanism of synergy between the two drugs nor the precise molecular mechanism of action of levamisole is known. Each drug has previously been shown to alter the expression of class I human leukocyte antigens (HLA class I) in colorectal cancer cell lines. PURPOSE: The purpose of this study was to explore the mechanism of interaction between 5-FU and levamisole by investigating the effect of this combination on HLA class I gene expression in the colorectal cancer cell line WiDr. METHODS: WiDr cells were treated either with 5-FU alone or with 5-FU and levamisole. Expression of HLA class I antigens was analyzed by flow cytometry using the monoclonal antibody W6/32. Specific DNA probes for HLA class I, beta 2-microglobulin, beta-actin, HLA class II, and p53 (also known as TP53) were used in Northern blot analysis of the steady-state level of messenger RNAs (mRNAs) and for "run-on" transcription analysis. RESULTS: 5-FU alone produced more than 50% increases in the expression of the HLA class I antigens, and levamisole caused a further 8%-18% increase. 5-FU caused the steady-state level of HLA class I mRNAs to increase by about 80%, and levamisole enhanced this effect of 5-FU by a further 70%. 5-FU did not increase the other mRNAs. In vitro run-on transcription revealed that 5-FU caused a 20%-57% reduction in RNA synthesis, while levamisole caused a 30%-190% increase in RNA synthesis. Levamisole therefore reversed the inhibition of RNA synthesis caused by 5-FU. Both drugs had a general effect on RNA synthesis that was not restricted to HLA class I transcription. CONCLUSIONS: The apparent synergy between levamisole and 5-FU is a result of the incorporation of 5-FU, which may stabilize HLA class I mRNAs, leading to their accumulation, while levamisole augments the accumulation of these stable mRNAs by increasing the rate of transcription. IMPLICATIONS: Levamisole reduces the toxicity of 5-FU caused by generalized inhibition of RNA synthesis, and at the same time augments the effects of 5-FU, which may be due to selective stabilization of certain mRNAs.

Blotting, Northern↗