Comparison of peripherally inserted central venous catheters (PICC) versus subcutaneously implanted port-chamber catheters by complication and cost for patients receiving chemotherapy for non-haematological malignancies.

PURPOSE: Indwelling central venous catheters (CVCs) have been increasingly used to enable delivery of intravenous chemotherapy. We aimed to compare the safety and cost of two commonly used CVCs, peripherally inserted central venous catheter (PICCs) and ports, in the delivery of chemotherapy in patients with non-haematological malignancies. METHODS: Seventy patients were randomly assigned to receive either a PICC or a port. The primary endpoint was occurrence of major complications, which required removal of the CVC and secondary endpoints included occurrence of any complications. RESULTS: Port devices were associated with fewer complications compared with PICC lines (hazard ratio of 0.25, CI, 0.09-0.86, P = 0.038). Major complication rate was lower in the port arm compared to the PICC arm (0.047 versus 0.193 major complications/100 catheter days, P = 0.034) with 6 versus 20 % of patients experiencing major complications, respectively. Thrombosis, the most common complication, was significantly higher in the PICC arm compared to the port arm (25 versus 0 %, P = 0.013). Quality of life and cost estimates did not differ significantly between the two arms. CONCLUSIONS: Port devices are associated with a lower risk of complications, with no difference in cost, compared to PICC lines in patients with non-haematological malignancies receiving intravenous chemotherapy.

Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution.

The structure of a protein triple helix has been determined at 1.9 angstrom resolution by x-ray crystallographic studies of a collagen-like peptide containing a single substitution of the consensus sequence. This peptide adopts a triple-helical structure that confirms the basic features determined from fiber diffraction studies on collagen: supercoiling of polyproline II helices and interchain hydrogen bonding that follows the model II of Rich and Crick. In addition, the structure provides new information concerning the nature of this protein fold. Each triple helix is surrounded by a cylinder of hydration, with an extensive hydrogen bonding network between water molecules and peptide acceptor groups. Hydroxyproline residues have a critical role in this water network. The interaxial spacing of triple helices in the crystal is similar to that in collagen fibrils, and the water networks linking adjacent triple helices in the crystal structure are likely to be present in connective tissues. The breaking of the repeating (X-Y-Gly)n pattern by a Gly-->Ala substitution results in a subtle alteration of the conformation, with a local untwisting of the triple helix. At the substitution site, direct interchain hydrogen bonds are replaced with interstitial water bridges between the peptide groups. Similar conformational changes may occur in Gly-->X mutated collagens responsible for the diseases osteogenesis imperfecta, chondrodysplasias, and Ehlers-Danlos syndrome IV.

Increased spinal c-Fos expression with noxious and non-noxious peripheral stimulation after severe spinal contusion.

The effects of severe contusive spinal cord injury (SCI), at thoracic level 8 (T8), on lumbar c-Fos expression in the spinal cord was investigated. As hypothesized, chronic SCI has a significant effect on expression of c-Fos in the dorsal spinal sensory areas with noxious and innocuous peripheral stimulation of the sciatic nerve. This alteration to stimulation effects was measured using counts of c-Fos immunoreactive cells in the dorsal horn of the L5 lumbar spinal cord in injured animals at 90 days post-injury and in uninjured controls. The number of c-Fos immunoreactive cells increased in SCI rats only after noxious peripheral stimulation (electrical and chemical) suggesting a general increase in excitability in spinal pathways (central sensitization) associated with chronic SCI. These altered responses may represent a functional anatomical reorganization of spinal cord circuitry leading to increased dorsal horn c-Fos expression as a response to severe chronic contusive damage to the spinal cord sensory pathways.

Metal-backed versus all-polyethylene unicompartmental knee arthroplasty: Proximal tibial strain in an experimentally validated finite element model.

OBJECTIVES: Up to 40% of unicompartmental knee arthroplasty (UKA) revisions are performed for unexplained pain which may be caused by elevated proximal tibial bone strain. This study investigates the effect of tibial component metal backing and polyethylene thickness on bone strain in a cemented fixed-bearing medial UKA using a finite element model (FEM) validated experimentally by digital image correlation (DIC) and acoustic emission (AE). MATERIALS AND METHODS: A total of ten composite tibias implanted with all-polyethylene (AP) and metal-backed (MB) tibial components were loaded to 2500 N. Cortical strain was measured using DIC and cancellous microdamage using AE. FEMs were created and validated and polyethylene thickness varied from 6 mm to 10 mm. The volume of cancellous bone exposed to < -3000 µÎµ (pathological loading) and < -7000 µÎµ (yield point) minimum principal (compressive) microstrain and > 3000 µÎµ and > 7000 µÎµ maximum principal (tensile) microstrain was computed. RESULTS: Experimental AE data and the FEM volume of cancellous bone with compressive strain < -3000 µÎµ correlated strongly: R = 0.947, R2 = 0.847, percentage error 12.5% (p < 0.001). DIC and FEM data correlated: R = 0.838, R2 = 0.702, percentage error 4.5% (p < 0.001). FEM strain patterns included MB lateral edge concentrations; AP concentrations at keel, peg and at the region of load application. Cancellous strains were higher in AP implants at all loads: 2.2- (10 mm) to 3.2-times (6 mm) the volume of cancellous bone compressively strained < -7000 µÎµ. CONCLUSION: AP tibial components display greater volumes of pathologically overstrained cancellous bone than MB implants of the same geometry. Increasing AP thickness does not overcome these pathological forces and comes at the cost of greater bone resection.Cite this article: C. E. H. Scott, M. J. Eaton, R. W. Nutton, F. A. Wade, S. L. Evans, P. Pankaj. Metal-backed versus all-polyethylene unicompartmental knee arthroplasty: Proximal tibial strain in an experimentally validated finite element model. Bone Joint Res 2017;6:22-30. DOI:10.1302/2046-3758.61.BJR-2016-0142.R1.

An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy.

Changes in membrane and macromolecular fluidity which may accompany the differentiation processes of sporulation and germination in Bacillus megaterium K.M. are examined by electron spin and nuclear magnetic resonance spectroscopy. No change in membrane lipid fluidity is observed in isolated forespores up to stage VI. Between stage VI and release of mature spores, the ESR spectrum of doxylstearic acid spin labels becomes polycrystalline. This change in spectral fluidity is completely reversed during germination and is paralleled by the rapid release of Ca2+ from the spore. NMR studies also show that the mature spore has reduced macromolecular mobility and an increased nonexchangeable water pool compared with vegetative cells.

The effect of oral and parenteral typhoid vaccination on the rate of infection with Salmonella typhi and Salmonella paratyphi A among foreigners in Nepal.

We studied the incidence of enteric fever among travelers and foreign residents who attended an expatriate clinic in Kathmandu, Nepal, from February 1987 to June 1988. There were 42 cases of enteric fever; 20 were caused by Salmonella typhi and 22 by Salmonella paratyphi A. Among 18 unvaccinated foreigners who had enteric fever, S typhi was isolated from 67%, and S paratyphi A from 33%, a ratio similar to the local Nepalese population. Among 22 vaccinated foreigners, S typhi was isolated from 35%, compared with 65% with S paratyphi A. Nine percent of tourists had received the oral Ty21A typhoid vaccine. However, among seven vaccinated tourists who became infected with S typhi, four (57%) had received the oral vaccine. Typhoid vaccine efficacy for tourists was calculated and showed an overall protective rate of 90% against enteric fever in general, 95% protection against S typhi, and 72% to 75% protection against S paratyphi A. We conclude that typhoid vaccine should be recommended to all travelers to the Indian subcontinent, and since S paratyphi A is the predominant cause of enteric fever among vaccinated travelers, consideration should be given to an effective vaccine against S paratyphi A when that becomes available.

Hyperglycemia reduces functional expression of astrocytic Kir4.1 channels and glial glutamate uptake.

Diabetics are at risk for a number of serious health complications including an increased incidence of epilepsy and poorer recovery after ischemic stroke. Astrocytes play a critical role in protecting neurons by maintaining extracellular homeostasis and preventing neurotoxicity through glutamate uptake and potassium buffering. These functions are aided by the presence of potassium channels, such as Kir4.1 inwardly rectifying potassium channels, in the membranes of astrocytic glial cells. The purpose of the present study was to determine if hyperglycemia alters Kir4.1 potassium channel expression and homeostatic functions of astrocytes. We used q-PCR, Western blot, patch-clamp electrophysiology studying voltage and potassium step responses and a colorimetric glutamate clearance assay to assess Kir4.1 channel levels and homeostatic functions of rat astrocytes grown in normal and high glucose conditions. We found that astrocytes grown in high glucose (25 mM) had an approximately 50% reduction in Kir4.1 mRNA and protein expression as compared with those grown in normal glucose (5mM). These reductions occurred within 4-7 days of exposure to hyperglycemia, whereas reversal occurred between 7 and 14 days after return to normal glucose. The decrease in functional Kir channels in the astrocytic membrane was confirmed using barium to block Kir channels. In the presence of 100-µM barium, the currents recorded from astrocytes in response to voltage steps were reduced by 45%. Furthermore, inward currents induced by stepping extracellular [K(+)]o from 3 to 10mM (reflecting potassium uptake) were 50% reduced in astrocytes grown in high glucose. In addition, glutamate clearance by astrocytes grown in high glucose was significantly impaired. Taken together, our results suggest that down-regulation of astrocytic Kir4.1 channels by elevated glucose may contribute to the underlying pathophysiology of diabetes-induced CNS disorders and contribute to the poor prognosis after stroke.

Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe.

In a previous study the alteration in the amino acid sequence of Neurospora crassa NADP-specific glutamate dehydrogenase (GDH) resulting from two mutually compensating frameshift mutations was used to deduce the first 17 nucleotides of the coding sequence of the am gene. In the work reported here, a synthetic 17-mer corresponding to the deduced sequence was shown to hybridize strongly to a 9-kb HindIII fragment from N. crassa wild-type DNA but not to any corresponding fragment from the DNA of a mutant strain known to be deleted for most or all of the gene. Wild-type HindIII fragments were fractionated for size and a fraction centering around 9 kb was cloned in vector lambda L47. Two clones carrying the strongly hybridizing fragment were identified. The hybridization to the 17-mer was localized within a 2.7-kb BamHI fragment and, within this, to a 700-bp BamHI-Bg/II subfragment. 5' end-labelled polyadenylated RNA isolated from wild-type mycelium hybridized to the 2.7-kb BamHI fragment and not appreciably to flanking fragments. The partial sequence analysis of the BamHI-Bg/II fragment has confirmed that the 17-mer probe matches the coding sequence at the 5' end of the gene and has also revealed an intervening sequence 67 bp in length, interrupting codon 15. Both the 9-kb HindIII fragment and the 2.7-kb BamHI fragment have been shown to be capable of transforming the deletion mutant to prototrophy and ability to produce GDH. Analysis of one transformant showed that the am gene was integrated, together with a part of the long arm of the lambda vector, at an unusual locus. This transformant, in which the am gene does not show its normal linkage to the linkage group 5 marker inl, was found to produce GDH to about 20% of the normal level.

Depression in Chinese medical inpatients.

The authors studied depressive symptoms among 251 Chinese medical inpatients through the use of the Beck Depression Inventory. Assessment of 100 healthy Chinese volunteers validated the use of American score norms for Chinese subjects. A total of 47.8% of the 251 medical inpatients (N = 120) met the Beck scale criterion for depression. Beck scale scores varied with the occupation of patients and the severity of medical illness but did not vary with sex, age, marital status, duration of hospitalization, or medical diagnosis.

The regiochemical distribution of positive charges along cholesterol polyamine carbamates plays significant roles in modulating DNA binding affinity and lipofection.

We have quantified the effects of the regiochemical distribution of positive charges along the polyamine moiety in lipopolyamines for DNA molecular recognition. High affinity binding leads to charge neutralisation, DNA condensation and ultimately to lipofection. Binding affinities for calf thymus DNA were determined using an ethidium bromide displacement assay and condensation was detected by changes in turbidity using light scattering. The in vitro transfection competence of cholesterol polyamine carbamates was measured in CHO cells. In the design of DNA condensing and transfecting agents for non-viral gene therapy, the interrelationship of ammonium ions, not just their number, must be considered.

Distribution of aromatic L-amino acid decarboxylase mRNA in mouse brain by in situ hybridization histology.

Aromatic L-amino acid decarboxylase (AAAD) is the second enzyme in the sequence leading to the synthesis of catecholamines or serotonin. Antisense riboprobes for aromatic L-amino acid decarboxylase mRNA were used to map the gene in mouse brain by in situ hybridization. The substantia nigra, the ventral tegmental nucleus, the dorsal raphe nucleus, the locus coeruleus, and the olfactory bulb contained the highest signal for AAAD mRNA. After treatment with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the signal disappeared in the substantia nigra, decreased somewhat in the ventral tegmental area, and remained unchanged in the dorsal raphe nucleus. Hypothalamic and cerebellar Purkinje neurons known to contain histidine decarboxylase or glutamic acid decarboxylase, respectively, were unlabeled by the probes. However, neurons in the deep layers of the frontal cortex, many thalamic nuclei, and the pyramidal neurons of the hippocampus were lightly to moderately labeled for mouse AAAD mRNA. The presence of AAAD message in these neurons suggests that the enzyme has functions other than that for the synthesis of the classical biogenic amine neurotransmitters.

Lumbar transplants of immortalized serotonergic neurons alleviate chronic neuropathic pain.

The RN46A cell line was derived from embryonic day 13 rat medullary raphe cells by infection with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen. This cell line is neuronally restricted and constitutively differentiates following a shift to non-permissive temperature. Brain-derived neurotrophic factor (BDNF) induced the serotonergic phenotype and increased the survival of RN46A cells in vitro. After transfection of the rat BDNF gene into RN46A cells, an autocrine BDNF-secreting cell line, 46A-B14, was isolated and transplanted into the rat CNS. Transplanted 46A-B14 cells had increased survival and enhanced serotonin (5HT) synthesis compared to 46A-V1 cells, RN46A cells transfected with vector-alone. When 46A-B14 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after a chronic constriction injury (CCI) of the sciatic nerve, they survived longer than 6 weeks on the pia mater. Furthermore, the tactile and cold allodynia and thermal hyperalgesia induced by CCI was significantly reduced during a 4-6- week period. The maximal effect occurred 1 week after transplantation. 46A-V1 cells, transplanted after CCI, did not survive beyond 2-3 weeks and had no effect on the allodynia and hyperalgesia induced by CCI. Acute intrathecal injection of the 5HT receptor antagonist methysergide decreased the antinociceptive effects of the 46A-B14 cells to pre-transplant levels. These data suggest that a chronically applied, low local dose of serotonin near the dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters such as serotonin, in a model of chronic pain offers a novel approach to pain management.

Lumbar transplant of neurons genetically modified to secrete brain-derived neurotrophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction.

Chronic delivery of anti-nociceptive molecules by means of cell grafts near the pain processing centers of the spinal cord is a newly developing technique for the treatment of neuropathic pain. The rat neuronal cell line, RN33B, derived from E13 rat brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat brain-derived neurotrophic factor cDNA (BDNF), and the BDNF-synthesizing cell line, 33BDNF.4, was isolated. The 33BDNF.4 cells synthesized mature BDNF protein at permissive temperature (33 degrees C), when the cells were proliferating, and during differentiation at non-permissive temperature (39 degrees C) in vitro. The bio-active BDNF protein was also secreted by the cells during both growth conditions, as measured by ELISA analysis of BDNF content and secretion. The bio-activity of the BDNF in 33BDNF.4 cell conditioned media was assessed by neurite outgrowth from E15 dorsal root ganglion (DRG) cultures. A control cell line, 33V1, transfected with the vector alone, did not synthesize or secrete any significant BDNF at either growth condition. Both cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hindpaw. When 33BDNF.4 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord and the 33BDNF.4 cells continued to synthesize BDNF in vivo. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7 week period after grafts of 33BDNF.4 cells. The maximal effect on chronic pain behaviors with the BDNF grafts occurred 2-3 weeks after transplant and the anti-nociceptive effects of the BDNF cell grafts was permanent. Transplants of the control 33V1 cells had no effect on the allodynia and hyperalgesia induced by CCI and these cells did not synthesize BDNF in vivo. These data suggest that a chronically applied, low local dose of BDNF supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver anti-nociceptive molecules, such as BDNF, in a model of chronic pain offers a novel approach to pain management and such 'biologic minipumps' can be developed for safe use in humans.

Comparison of the effects of remoxipride and raclopride on nigrostriatal and mesolimbic dopaminergic neuronal activity and on the secretion of prolactin and alpha-melanocyte-stimulating hormone.

Raclopride and remoxipride, which are reported to be selective dopamine-D2 antagonists, are currently under clinical investigation as antipsychotic drugs. The present study compared the relative abilities of these two drugs to alter the activity of nigrostriatal and mesolimbic dopaminergic neurons, and plasma levels of hormones originating from the anterior and intermediate lobes of the pituitary in male rats. Although raclopride was consistently more potent than remoxipride, both drugs produced dose- and time-related increases in concentrations of 3,4-dihydroxyphenylacetic acid in the striatum and nucleus accumbens, which contain terminals of nigrostriatal and mesolimbic dopaminergic neurons, respectively. Both drugs also caused significant dose- and time-related increases in plasma levels of prolactin, but only raclopride increased plasma levels of alpha-melanocyte-stimulating hormone (alpha-MSH). These results suggest that although raclopride and remoxipride are both classified as D2 receptor antagonists they can be distinguished from one another by their relative ability to block the inhibitory dopaminergic control of alpha-MSH from melanotrophs in the intermediate lobe of the rat pituitary.

Characteristics of an MM 22 medical microtron 21 MV photon beam.

Beam quality, surface doses, depths of maximum dose, peak dose rates, beam profiles, and central axis percentage depth doses of a medical microtron's 21 MV photon beam were measured. The half-value layer of lead was 1.35 cm, the half-value layer of water was 23 cm, and the nominal acceleration potential was 17.7 MV. For a 10 X 10 cm2 field at 100 cm SSD, the maximum dose occurred at a depth of 3 g X cm-2 and the surface dose was 18% of the maximum. The highest dose rate at isocenter was approximately 800 cGy X min-1. The worst horns on beam profiles occurred at a depth of 5 cm for the 35 X 35 cm2 field where the dose rate 4 cm from the edge of the beam was 3% higher than that on the central ray. No horns were apparent for fields 25 X 25 cm2 or smaller.

Remoxipride and raclopride differentially alter the activity of incertohypothalamic dopaminergic neurons.

The effects of two dopaminergic (DA) antagonists, raclopride (S-(-)-3,5-dichloro-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy- 6-methoxybenzamide(+)-tartrate) and remoxipride (S(-)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2, 6-dimethoxybenzamide hydrochloride monohydrate), were compared on the DA receptor-mediated regulation of incertohypothalamic and nigrostriatal DA neurons. Both drugs produced dose- and time-related increases in concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, which contains the terminals of the nigrostriatal DA neurons. On the other hand, raclopride but not remoxipride increased concentrations of DOPAC in the medial zona incerta and dorsomedial hypothalamic nucleus, regions that contains cell bodies and terminals, respectively, of incertohypothalamic DA neurons. These results suggest that raclopride blocks a population of DA receptors that regulates the activity of incertohypothalamic DA neurons, whereas remoxipride does not.

Serotonergic neural precursor cell grafts attenuate bilateral hyperexcitability of dorsal horn neurons after spinal hemisection in rat.

Hemisection of the rat spinal cord at thoracic level 13 provides a model of spinal cord injury that is characterized by chronic pain attributable to hyperexcitability of dorsal horn neurons. Presuming that this hyperexcitability can be explained in part by interruption of descending inhibitory modulation by serotonin, we hypothesized that intrathecal transplantation of RN46A-B14 serotonergic precursor cells, which secrete serotonin and brain-derived neurotrophic factor, would reduce this hyperexcitability by normalizing the responses of low-threshold mechanoreceptive, nociceptive-specific, and multireceptive dorsal horn neurons. Three groups (n=45 total) of 30-day-old male Sprague-Dawley rats underwent thoracic level 13 spinal hemisection, after which four weeks were allowed for development of allodynia and hyperalgesia. The three groups of animals received transplants of no cells, 10(6) RN46A-V1 (vector-only) or 10(6) RN46A-B14 cells at lumbar segments 2-3. Electrophysiological experiments were done two weeks later. Low-threshold mechanoreceptive, nociceptive-specific, and multireceptive cells (n=394 total) were isolated at depths of 1-300 and 301-1000 micro in the lumbar enlargement. Responses to innocuous and noxious peripheral stimuli were characterized, and analyses of population responses were performed. Compared with normal animals, dorsal horn neurons of all types in hemisected animals showed increased responsiveness to peripheral stimuli. This was true for neurons on both sides of the spinal cord. After hemisection, the proportion of neurons classified as multireceptive cells increased, and interspike intervals of spontaneous discharges became less uniform after hemisection. Transplantation of RN46A-B14 cells restored evoked responses to near-control levels, normalized background activity, and returned the proportion of multireceptive cells to the control level. Restoration of normal activity was reversed with methysergide.These electrophysiological results corroborate anatomical and behavioral studies showing the effectiveness of serotonergic neural precursors in correcting phenomena associated with chronic central pain following spinal cord injury, and provide mechanistic insights regarding mode of action.

Immunocytochemical and in situ hybridization studies of the expression and distribution of three subunits of a complex with N-methyl-D-aspartate receptor-like properties.

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

Characterization of acid-sensitive ion channels in freshly isolated rat brain neurons.

Transient proton-activated currents induced by rapid shifts of the extracellular pH from 7.4 to < or =6.8 were recorded in different neurons freshly isolated from rat brain (hypoglossal motoneurons, cerebellar Purkinje cells, striatal giant cholinergic interneurons, hippocampal interneurons, CA1 pyramidal neurons and cortical pyramidal neurons) using whole-cell patch clamp technique. Responses of hippocampal CA1 pyramidal neurons were weak (100-300 pA) in contrast to other types of neurons (1-3 nA). Sensitivity of neurons to rapid acidification varied from pH(50) 6.4 in hypoglossal motoneurons to 4.9 in hippocampal interneurons. Proton-activated currents were blocked by amiloride (IC(50) varied from 3.6 to 9.5 microM). Reversal potential of the currents was close to E(Na), indicating that the currents are carried by sodium ions. The data obtained suggest that the proton-activated currents in the neurons studied are mediated by acid-sensitive ion channels. Strong acidification (pH<4) induced biphasic responses in all neuron types: the transient current was followed by a pronounced sustained one. Sustained current was not blocked by amiloride and exhibited low selectivity for sodium and cesium ions. Slow acidification from pH 7.4 to 6.5 did not induce detectable whole-cell currents. At pH 6.5, most of the channels are desensitized and responses to fast pH shifts from this initial level are decreased at least 10 times. This suggests that slow acidification which is well known to accompany some pathological states should rather desensitize than activate acid-sensitive ion channels and depress their function. Our results provide evidence for a widespread and neuron-specific distribution of acid-sensitive ion channels in the brain. The large amplitudes and transient character of currents mediated by these channels suggest that they could contribute to fast neuronal signaling processes.

Immunobead RT-PCR: a sensitive method for detection of circulating tumor cells.

Detection of circulating tumor cells and micrometastases in patients with cancer should prove useful in determining prognosis and in planning and monitoring systemic therapies. We have developed immunomagnetic isolation of carcinoma cells followed by reverse transcription polymerase chain reaction (immunobead RT-PCR) as a method for identifying very small numbers of breast cancer cells in blood. The expression of cytokeratin 19 (K19) was used as the marker by which the isolated tumor cells were identified. The immunobead RT-PCR technique allowed detection of one tumor cell per 10(6) leukocytes in whole blood. Immunobead RT-PCR is a highly sensitive method of detecting cancer cells in a hematopoietic environment.