Proton magnetic resonance spectroscopy of human brain: applications to normal white matter, chronic infarction, and MRI white matter signal hyperintensities. A modified ISIS method, for image-selected localized proton magnetic resonance spectroscopy (1H MRS), was used to determine the ratios and T2 relaxation times of proton metabolites in normal subjects and in patients with chronic infarction and MRI white matter signal hyperintensities (WMSH). First, in patients with cerebral infarctions, increased concentrations of lactate were found in the majority of patients, and N-acetyl aspartate (NAA) was reduced to a significantly greater extent than choline (Cho) or creatine (Cre). For TE = 270 ms, the raw ratios of Cho/NAA, Cre/NAA, and Lac/NAA were significantly (P less than 0.05) increased from 0.23 +/- 0.02 (mean +/- SE), 0.20 +/- 0.01, and 0.05 +/- 0.01, respectively in the normal group to 0.39 +/- 0.08, 0.37 +/- 0.05, and 0.48 +/- 0.15 in the stroke group. Also, the T2 relaxation time of creatine was significantly (P = 0.007) increased from 136 ms in normal white matter to 171 ms in cerebral infarcts. Second, in patients with WMSH, no significant change of the proton metabolite concentrations could be detected with the exception of the choline which was significantly (P = 0.003) altered. The Cho/NAA ratio, after T2 and excitation profile correction, increased from 0.47 +/- 0.02 in the normal group to 0.64 +/- 0.05 in the WMSH group. Third, in normal white matter, the concentration of N-acetyl aspartate, choline, and lactate was estimated to 11.5, 2.0, and 0.6 mM, respectively, by assuming a total creatine concentration of 10 mM.


Partial RF echo planar imaging with the FAISE method. I. Experimental and theoretical assessment of artifact. The fast acquisition interleaved spin-echo (FAISE) method is a partial RF echo-planar technique which utilizes a specific phase-encode reordering algorithm to manipulate image contrast (Melki et al., J. Magn. Reson. Imaging 1:319, 1991). The technique can generate "spin-echo" like images up to 16 times faster than conventional spin-echo methods. However, the presence of T2 decay throughout the variable k-space trajectories used to manipulate T2 contrast ensures the presence of image artifacts, especially along the phase-encode direction. In this work, we experimentally and theoretically examine the type and extent of artifacts associated with the FAISE technique. We demonstrate the existence of well-defined minima of phase-encode ghost noise for selected k-space trajectories, examine the extent of blurring and edge enhancement artifacts, demonstrate the influence of matrix size and number of echoes per train on phase-encode artifact, and show how proper choice of FAISE sequence parameters can lead to proton density brain images which are practically indistinguishable from conventional spin-echo proton density images. A comparison of contrast between FAISE and standard spin-echo methods is presented in a companion article referred to as II.


Partial RF echo-planar imaging with the FAISE method. II. Contrast equivalence with spin-echo sequences. The fast acquisition interleaved spin-echo (FAISE) sequence and its dual-echo version (DEFAISE) are partial RF echo-planar methods which utilize a specific phase-encode reordering algorithm to manipulate T2 contrast via an operator-controlled pseudo-echo time, pTE. The repetition time, TR, between successive applications of the Carr-Purcell-Meiboom-Gill (CPMG) echo trains used in FAISE may be reduced to introduce T1 weighting. To quantitatively determine the extent to which FAISE T1 and T2 contrast characteristics agree with spin-echo methods, signal intensities from FAISE acquisitions were compared with signal intensities from equivalent CPMG acquisitions. In phantoms and in human heads, the contrast characteristics of FAISE are found to be highly correlated with that obtained with equivalent CPMG sequences. However, conventional SE sequences generally utilize longer echo spacings than employed with FAISE/CPMG. Thus, echo spacing-dependent mechanisms such as spin-spin coupling and magnetic susceptibility lead to some differences in contrast between conventional SE and FAISE. Finally, FAISE appears to be more sensitive to magnetization transfer effects than conventional SE sequences since more off-resonance irradiation is applied to individual slices during multislice acquisitions.


Single-shot GRASE imaging without fast gradients. Based on the CPMG sequence, gradient- and spin-echo (GRASE) echo train length is limited by T2 decay rather than the T2* decay and phase error in echo-planar techniques, permitting a longer image acquisition period. An ultrafast GRASE sequence, utilizing a single excitation, generates a 128 x 56 true T2-weighted image in 200 ms on an unmodified commercial scanner without fast gradient switching, extreme field homogeneity, or fat signal suppression.


Recursive RF excitation.

Recursive RF excitation. We have investigated the properties of a recursive process in which the output signal from a given RF excitation pulse may be used as the input (excitation) pulse of a subsequent iteration. This recursive excitation technique increases contrast and improves feature segmentation for the purpose of motion tracking.


31P NMR spectroscopy of the human heart at 4 T: detection of substantially uncontaminated cardiac spectra and differentiation of subepicardium and subendocardium. 31P NMR spectroscopy of the human heart was undertaken at 4 T to investigate whether spectra localized exclusively to the myocardium can be obtained. Utilizing a Fourier series window approach to spectroscopic imaging, we find that at least two layers across the anterior left ventricle wall can be detected, with voxel sizes of about 8 cm3.


Rapid T1 estimation using tagged magnetization-prepared gradient-echo MR imaging. A technique for estimation of the longitudinal relaxation time of a large homogeneous object with an acquisition time of 4 s or less was developed by combining spatially selective rf tagging pulses with a T1-weighted magnetization-prepared gradient-echo sequence. Multiple 5-mm-wide tagged areas are laid orthogonal to the imaging section of interest. The contrast between each tag and the untagged regions differs because each tag is produced at a different time. The T1 value is determined from the nulling time at which tagged and untagged areas have no contrast.


Intracellular localization of Staphylococcus aureus within primary cultured mouse kidney cells. Staphylococcus aureus Cowan I was incubated with monolayers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among these of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.


Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction. Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.


Relationship between an 85 kDa protein and the protective effects of Mycoplasma pneumoniae. In the immunoblot analysis, sera from patients infected with Mycoplasma pneumoniae reacted with the 168 kDa (P1) and the 85 kDa proteins of virulent strain FH-P24 and P24-S1 mutant strain but not with the 85 kDa protein of P24-S11. Sera of hamsters and BALB/c mice, which had been immunized with live vaccines, were tested. In FH-P24 immunized animals, 100% or 80%, and in P24-S1, 40% of hamsters and 60% of BALB/c mice, developed antibodies against the 85 kDa protein, but antibodies were not detected in sera of P24-S11 immunized animals. The correlation between the development of antibodies to 85 kDa protein in the sera of vaccinated animals and the effects of protection by living vaccines were suggested.


Definition and application of a histopathological scoring scheme for an animal model of acute Mycoplasma pneumoniae pulmonary infection. A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model. A final score per animal (ranging 0-26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. The scoring scheme was then applied to test the ability of a heat-killed inoculum to induce pulmonary pathology and to the trial of a 43 kDa protein-associated antigen as a vaccine immunogen. A heat-killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P less than 0.01). Animals prevaccinated with the 43 kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P = 0.00007). Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture-negative media also were affected (mean score 5.4). Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r = 0.93; P = 0.000009). The scoring system has the ability to differentiate disease severity in small groups of animals.


Augmentation of host resistance to Candida albicans infection in ascites tumor-bearing mice. We found that the number of peripheral blood polymorphonuclear leukocytes (PMN) dramatically increased in both sarcoma 180 (S-180) and MM-46 mammary carcinoma (MM-46) ascites tumor-bearing mice, and mice required a remarkable resistance to Candida albicans infection via intravenous route. When the resistance was determined by number of cells of C. albicans in the kidney, a significant decrease in the number of fungal cells was observed in the kidneys of infected ascites tumor-bearing mice. An increase of active oxygen levels of PMN from ascites tumor-bearing mice was observed, suggesting that this factor is important in developing of resistance in ascites tumor-bearing mice. Additionally, a culture supernatant of tumor cells co-cultivated with bone-marrow cells in vitro increased the number of granulocytes and macrophages differentiated from the bone-marrow cells.


An activity which restores theta toxin activity in some theta toxin-deficient mutants of Clostridium perfringens. Group a mutants of Clostridium perfringens are deficient in theta toxin but release a dialyzable substance ("substance A"), which restores theta toxin activity to group b mutants, into a culture supernatant; group b mutants are defective in "substance A" release. "Substance A" activity appeared in the exponentially growing phase of group a mutants and disappeared in the stationary phase. "Substance A" activity was most stable at pH 5.0 and 0 C and even increased threefold in the first 5 hr, but gradually decreased during the following 15 hr. It was quickly inactivated at neutral and higher pHs at 0 C.


Differentiation between Mycobacterium avium and Mycobacterium intracellulare by thin-layer chromatography of lipid fraction after incubation with [35S]methionine. Mycobacterium avium and Mycobacterium intracellulare are differentiated from each other by thin-layer chromatography of lipid fraction extracted after incubation with [35S]methionine. The former contained a petroleum ether-soluble sulfolipid and the latter did not.


Existence of phosphoenolpyruvate: carbohydrate phosphotransferase systems in Lactobacillus fermentum, an obligate heterofermenter. The presence or absence of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) in obligately heterofermentative group III lactobacilli including Lactobacillus brevis (3 strains), L. buchneri (2 strains) and L. fermentum (3 strains) was surveyed systematically for a series of sugars utilizable by these organisms. Contrary to common expectation, PTSs were found in two strains of L. fermentum: sucrose-PTS in one strain; sucrose- and mannose-PTSs in the other. All these activities were found to be constitutive.


Genetic variation among Malaysian isolates of Salmonella typhi as detected by ribosomal RNA gene restriction patterns. Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.


Conspicuous ingestion of Staphylococcus aureus organisms by murine fibroblasts in vitro. A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts-adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophyticus. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non-professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.


The role of aromatic hydrocarbons in the genesis of breast cancer. The incidence of breast cancer in women has increased dramatically over the last decade. Epidemiological markers of this increased incidence include: endocrine related phenomena (early menarche, age of first parity and age of menopause); exposure of the breast to X-radiation; and a group of seemingly disparate factors--urban residence, dietary selection and alcohol consumption. Although experimental breast cancer may be induced by estrogenic hormones, X-radiation and aromatic hydrocarbons, only aromatic hydrocarbons have not been previously implicated in human mammary carcinogenesis. The seemingly unrelated human factors can best be understood by examining the role of breast tissue in aromatic hydrocarbon metabolism. Aromatic hydrocarbons are important environmental chemicals produced by the incomplete combustion of hydrocarbons for use in energy production. Benzene, benz(a)pyrene, dibenz(ah)anthracene and 1-nitropyrene, known experimental breast carcinogens, are produced in this way. Human exposure to aromatic hydrocarbon metabolites induces and promotes altered DNA by mechanisms described as increased intracellular pro-oxidant production as well as direct adduction to DNA. The breast is anatomically embedded in a major fat depot which stores and concentrates aromatic hydrocarbons and can metabolize these hydrocarbons to carcinogenic metabolites. Ductal cells concentrate these metabolites and themselves become target cells for carcinogenesis. Some lifestyle factors increase the amount of carcinogens produced or enhance their activity. A unitary model for mammary carcinogenesis in humans as well as in experimental carcinogenesis is hypothesized. If correct, the hypothesis would account for some of the increase in breast cancer incidence in industrial countries--and would suggest environmental and dietary modifications that would inhibit hydrocarbon induced mammary carcinogenesis.


Possible roles for nitric oxide in AIDS and associated pathology. The endogenous free radical, nitric oxide (NO), plays a neurotransmitter-like role in vascular endothelium, a second-messenger role in N-methyl-D-aspartate (NMDA)-responsive neurons in the central nervous system (CNS), a neurotoxic role after its release from these neurons, and a cytotoxic role after its release by macrophages. NO also derives from exogenous sources, such as the nitrite inhalants, amyl, butyl and isobutyl nitrite. There is evidence that abuse of nitrite inhalants can affect immunomodulation, and epidemiological studies suggest that such abuse may be a cofactor in the pathogenesis of acquired immunodeficiency syndrome (AIDS). Hitherto, however, the potential role of NO in such pathogenesis has not been examined. This paper presents some current evidence that implicates both endogenous and exogenous sources of NO in AIDS and associated pathology.


Mechanisms of adaptive evolution. Darwinism and Lamarckism restated. This article discusses the conceptual basis of the different mechanisms of adaptive evolution. It is argued that only two such mechanisms may conceivably exist, Lamarckism and Darwinism. Darwinism is the fundamental process generating the diversity of species. Some aspects of the gene-centered approach to Darwinism are questioned, since they do not account for the generation of biological diversity. Diversity in biological design must be explained in relation to the diversity of interactions of organisms (or other higher-level units) with their environment. This aspect is usually overlooked in gene-centered views of evolution. A variant of the gene-selectionist approach has been proposed to account for the spread of cultural traits in human societies. Alternatively, I argue that social evolution is rather driven by what I call pseudo-Lamarckian inheritance. Finally, I argue that Lamarckian and pseudo-Lamarckian inheritance are just special cases of faithful replication which are found in the development of some higher-order units, such as multicellular organisms and human societies.


A gene for alcohol toxicity, not drinking behavior. Apparently contradictory results concerning association of an allele of a human dopamine receptor gene with alcoholism are explained by the gene's being involved with ethanol toxicity, not drinking behavior.


Addiction to nicotine is due to high intrinsic levels of dopamine. Smokers are less likely to develop Parkinson's disease (PD) than is true of non-smokers, and PD is the only disease inversely related to smoking. PD is associated with a reduced level of dopamine. Although nicotine can affect the receptors, including dopamine receptors, it seems unlikely that use of nicotine is protective in PD. An alternative explanation is that just as PD is associated with a lower than normal level of dopamine, addiction to smoking is linked with a higher than normal level of dopamine. This high innate level of dopamine facilitates addiction to nicotine.


Cell proliferation and carcinogenesis may share a common basis of permeable plasma membrane clusters. Wound potentials increase the surface potential of exposed areas of nearby cells. In these cells, soluble cytoplasmic bases are assumed gradually to move nearer the exposed area. Acidic molecules on the cell surface migrate to points opposite the bases. The image-charged species are mutually attracted to form transmembrane clusters. At clusters, membrane permeability increases and the cell is stimulated to cycle. When the wound heals, its clusters disperse, leaving a small 'permanent' residuum. Permanent clusters initiate cells to malignancy. They have (or develop) lipophilic molecules on both surfaces that help fix them in the membrane. Exposed cells contaminated with polycyclic aromatic hydrocarbon carcinogens (PAH) readily form permanent clusters. At mitosis, clusters on parental plasma membrane pass with that membrane to a daughter cell. Promotion results from many short-term or a single long-term exposure of initiated membranes to abnormal surface charge. Permanent clusters increase on the membrane after repeated wounding, proximity of charged foreign bodies like plastic film or asbestos, or oxidation of surface molecules. Progression requires acceleration of cluster growth so the daughter cell membranes become as leaky at maturity as was the parent membrane. One mechanism suggested involves reversible phosphorylation by membrane-bound kinases; another involves attraction of a basic protein (p36) to the membrane.


Sensitivity of latent cancer to chemotherapy. The paper is a review of some cancer patients who were treated successfully by surgery followed by chemotherapy. The cases suggest the hypothesis that early cancer growth following surgery is more sensitive to chemotherapy than the active cancer growth of the original tumor.


Hyperfractionated, twice-a-day, radiotherapy may decrease IQ deterioration due to prophylactic cranial irradiation in childhood acute lymphoblastic leukemia: a radiobiological analysis. High cure rates in childhood acute lymphoblastic leukemia (ALL) are being achieved with aggressive systemic chemotherapy and treatment to sanctuary sites including prophylactic cranial irradiation. However, IQ deterioration is a dreaded complication of prophylactic cranial irradiation. IQ deterioration is a late sequela. Since there is evidence--both radiobiological and clinical--to suggest that acute tissue (including tumor) response and late tissue response can be separated by hyper-fractionation, we propose a twice-a-day radiotherapy in prophylactic cranial irradiation of childhood ALL to decrease delayed toxicity. Analysis based on current radiobiological models favors such a treatment scheme. However, only a prospective clinical trial can confirm whether IQ deterioration can be prevented or decreased with hyper-fractionated radiotherapy.


Colic, sleep inertia, melatonin and circannual rhythms. Colic is periodic behavior occurring at the end of the day during the first 3 months of life characterized by crying. It is hypothesized that the crying at the end of the day is due to sleep inertia or a state dissociation during which the infant is simultaneously partially awake and partially asleep because of the absence of a melatonin diurnal rhythm. The melatonin timing mechanisms, which codes for day length, is initiated prenatally by the maternal pineal gland, and after 3 months postnatally, the melatonin nocturnal secretion rhythm is maintained by the infant's pineal gland. To record the seasonal variation in day length away from the equator, 12 months are required to complete the melatonin chemical calendar. This circannual process is only 9 months completed at the time of birth, and 3 additional months are needed during which the infant may have difficulty reconciling cues for the timing of evening sleep due to discrepancies between the expected photoperiod derived from the prenatal maternal pineal melatonin circadian rhythm and the postnatally experienced photoperiod. Data is presented showing that the incidence of colic increases with increasing latitude in support of the hypothesis that infant crying at the end of the day during the first 3 months represents the last quarter of a circannual photoperiodic development process.


Proposed role of septohippocampal and pallido-habenulo-raphe systems in photoperiodic time measurement. The neuroanatomic substrate for mammalian photoperiodic time measurement (PTM), a primary requirement for appropriately timed seasonal reproductive function, is unknown. It is suggested that the 'comparator' of successive day lengths previously postulated as necessary for PTM may be located within the septohippocampal system, output from which is then directed to reproductive-system response elements by way of several possible routes including the pallido-habenulo-raphe conduction line. PTM thus requires the integration of an invariant, context-free circadian function subserved by the well-known melatonin-generating system with a flexible, context-dependent extracircadian function subserved by the septohippocampal system. The context-dependent neuronal network endows reproductive-system responses with the ability to be modulated by previous photoperiodic history, an ability not possessed by the context-free circadian network proper. Several suggestions for testing the validity of the proposed model are offered.


L-arginine: a therapeutic option for AIDS/HIV infection? Numerous studies implicate cellular immunological effector systems in the partial containment of virus replication during the early stages of HIV infection. Immunostimulatory therapeutic regimes designed to enhance virus clearance are therefore theoretically attractive, but are accompanied by the risk of concomitant activation of HIV replication. Supra-normal levels of L-arginine have been shown to induce broad immune stimulation in vitro and in vivo, but do not increase HIV gene expression in vitro. These observations, together with the lack of toxicity of this agent, suggest a novel therapeutic approach to HIV disease.


Transgenic domestic animals provide an animal model for rheumatoid arthritis. Production of transgenic domestic animals by microinjection of egg nuclei has permitted the introduction of exogenous genes into the transgenic animal. Pursell et al (1) report severe synovitis, cardiomegaly, dermatitis, renal disease and gastric ulcers in pigs with an expressed bovine growth hormone transgene. I have hypothesized that rheumatoid arthritis (RA) is a disease with initial symptoms to the gamete (2, 3). This hypothesis is supported by data showing a reduced rate of RA in women using oral contraceptives (4, 5) and remission of RA in pregnancy (6). RA in animals resulting from trauma to the gamete would be consistent with this hypothesis. This paper hypothesizes that some of the pathology associated with genetic engineering of livestock can be attributed to a rheumatoid arthritis-like spectrum of symptoms. This systemic reaction could be sufficiently similar to human RA to permit the transgenic pig to serve as an animal model of the disease.


Oxidized LDL ceroid, and prostaglandin metabolism in human atherosclerosis. A noted histological feature of human atherosclerotic lesions upon dissection is the presence of a 'pigment' referred to as ceroid; however the significance of ceroid in human fatty streaks and atherosclerotic plaques is not certain. The research focus to this point has presumed that ceroid synthesis and intracellular accumulation is harmful and may have adverse effects on lesion progression or reversibility. Alternatively, ceroid production may be a defense mechanism employed by cells in the artery wall to prevent uncontrolled synthesis and release of prostaglandins (PGs) or prostaglandin-precursors locally. Export of PGs or PG precursors may promote blood platelet aggregation at the site of export. A feedback inhibition mechanism will arrest the cellular export of prostaglandins, thus ending a potentially disastrous premature clotting event.


Calcific aortic stenosis and autonomic dysfunction. With an aging population, calcific aortic stenosis is becoming more important in medical practice. Its cause is unknown. This paper hypothesises autonomic dysfunction is the abnormality underlying the tendency of the aging aortic valve to calcify, a proposition based on 2 main lines of evidence: 1. the similarity between aortic valvular calcification and medial arterial calcification, the latter a condition in whose pathogenesis autonomic dysfunction is implicated; 2. the similarity between the changes of autonomic denervation and those of natural aging in the aortic valve, of which calcific aortic stenosis is an exaggerated form. Autonomic denervation leads to withdrawal of neurotrophic influences from cells and tissues. The resulting changes in function and in structure, it is proposed, give rise to a condition of vulnerability allowing other stresses, such as haemodynamic trauma, to result in calcification.


The synergism of gamma-interferon and tumor necrosis factor in whole body hyperthermia with vitamin C to control toxicity. In a previous paper, the synergism of gamma-interferon and tumor necrosis factor was considered with whole body hyperthermia. Because of the toxic effect of TNF due to oxygen radicals, it is suggested that vitamin C be added.


Role of visuo-vestibular interaction in pathological ocular oscillation: new model and control system analysis. Control system engineering is a relatively newer field of engineering which is directly applicable to performance analysis of neuronal networks. Visuo-vestibular interaction (VVI) represents a neuronal control system which has most benefited from such analysis. In this paper, we present a new model of VVI derived from our previously reported model by adding cortical pursuit feedback. Mathematical analysis of abnormal eye oscillations and stability analysis of the VVI system provided us with a plausible hypothesis that the pathological ocular oscillations seen in oculopalatal myoclonus and see-saw nystagmus are indeed due to an unstable VVI control system. The former is due to degeneration of the inferior olivary nucleus resulting in loss of cerebellar learning effects, while the latter is due to disruption of the pathway conveying retinal error signals.


Working for patients: further implications for nurse education. The National Health Service and Community Care Act (1990) has focused attention on all aspects of health care. This paper explores the wider implications of the White Paper on nurse education. It examines the issues of student numbers; rationalisation of courses; the impact of contracts; quality control and clinical placements. It further considers the issues of capital assets; educational premises; funding and income generation. The response of schools and colleges of nursing to these challenges has yet to be evaluated. Nurse education is steering an uncertain course in turbulent and uncharted waters. If colleges of nursing are to capitalise on the changes demanded by the White Paper, the prime concern must be the establishment of an advantageous position from which to market viable courses. To this end there must be adequate resources, and control over high quality learning environments. The courses must be educationally led and respond to the learning needs of all students.


Teaching research to nurses: issues for tutor training. Research is unknown territory, in too many ways, for many nurses and nurse tutors. It is seen as the province of an elite, using obscure language and esoteric skills with no obvious connection with the world of nursing; in particular, it involves statistics. Pressure for nursing to become a research-based profession can make the fear worse; the gap between research and practice generates blame and guilt in addition to fear. It is not surprising that learning and teaching about research can be very difficult for nurses. This article explores sources of bad feelings about research and suggests ways forward in the training of nurse and midwifery tutors, considering course planning and teaching methods, and peer support in and out of the classroom. It also argues for expanding the range of nursing research used to teach about research, incorporating more qualitative methods and drawing more fully on the social science traditions.


Theory and practice and the preparation of teachers of nursing. Questions of the relationship between theory and practice frequently occur both within educational and nursing literature and they are the questions that are going to be explored in this paper. Like the double helix, this paper is going to intertwine and interlink nursing and education, since they possess so many similarities and the overall thesis of the paper is that the relationship between theory and practice presents problems because the nature of the problem has been inadequately formulated. The paper falls into four main parts: 1. Four types of relationship between theory and practice. 2. Nursing and education as fields of practice and the nature of fields of practice--implications of this for knowledge, skills, attitudes and values--skills/practical knowledge/knowledge how. 3. Nature of the fields of study and the implications of this for knowledge. 4. Theory and practice--eight possible relationships. The conclusion offers a tentative reformulation of theory and practice in terms of probability and relates this to the preparation of nurses and teachers.


An innovatory honours degree programme for nurse teacher preparation: development and evaluation. The implementation of the Project 2000 recommendations in the UK is bringing about change in the role and functions of the nurse teacher in terms of relationships with associated institutions of higher education, with colleagues in both teaching and clinical settings and in terms of the teaching and facilitating strategies employed. As a consequence there has been a reappraisal of nurse teacher preparation. This paper provides a description and an evaluation of an innovatory full-time degree programme that provides advanced study of nursing, behavioural, life and social sciences combined with the study of education and leading to a teaching qualification recordable with the United Kingdom Central Council (UKCC). The development of the BA (Hons) Nursing with Education degree programme at Portsmouth Polytechnic is briefly described and placed in context. The course has been subject to formative and summative evaluation using a range of techniques which are reported in this paper. There is a brief discussion of the action taken on the outcomes of the evaluation. The paper concludes with the identification of issues arising from the evaluation.


An exploration of issues relating to feminism and nurse education. This paper explores the issue of feminism in relation to nursing and nurse education. As a result of this exploration, the authors suggest there is a need for a move away from traditional patriarchal approaches to nurse education, towards an educational programme based on empowerment principles that maximises the potential of feminine patterns of thinking.


The nurse educator and the legal implications. This paper considers the liability of the nurse educator in her professional work. It begins by exploring the law for liability in giving negligent instructions and advice and the possibility of being liable for harm caused to a patient as a result of negligent teaching. The possibility of being liable if an incompetent student becomes registered and causes harm is also discussed. Liability for giving references on students is also considered and differences in outcomes between true but unsupportive references as against false but supportive ones are analysed.


Time management in education.

Time management in education. The topic of time management is an area that is very important to today's nurse educationalist, especially with the ever increasing demands being made upon her time. In this paper the writer examines the concept of time management, and some of the issues which should be given due consideration by anyone who is contemplating developing the skills required for success in managing time effectively. The work utilises ideas which enable one to take a problem solving approach to the subject. Here the reader will also find a selection of possible solutions that have been put forward from various sources, many of which the writer has tried herself and found to be extremely helpful in tackling her own problems in this area. One point that is reiterated throughout, is the very personal nature of time management and the fact that it is up to the individual to choose the most useful method in order to achieve an efficient and effective approach to the management of time, hopefully resulting in greater control and equally as important--'peace of mind'.


Teaching the skills of assessment through the use of an experiential workshop. Assessment is a key element of the nursing process, yet the skills required to perform a nursing assessment are poorly defined in the literature. Related to this, nurse education does not adequately address how it might be possible to develop in students the skills required to plan individualised care. As a contribution to this important area of nurse education, this paper describes the first term of an assessment course, an important component of which is an experiential workshop. This workshop introduces students to the skills of observation, measurement and testing as used when assessing physiological indicators of health status. The workshop was designed to facilitate an understanding of the connection between the students' knowledge of biological sciences and nursing practice.


An examination of the factors influencing the choice of one college of nursing in choosing an institute of higher education with which to link. This paper examines the factors influencing the choice of one College of Nursing when linking with an Institute of Higher Education, from which general principles can be drawn. The College is situated in Central London, within a Special Health Authority at a post graduate centre. The College currently offers courses in clinical nursing leading to English National Board certification, but is hoping to expand its courses in the near future. Influencing factors are examined in the light of the final decision using Roby's Deliberative Model.


Mentors, not assessors.

Mentors, not assessors. The English National Board supports the development of a mentorship scheme for Project 2000 courses. The lack of clarity regarding the role and function of mentors continues. This paper suggests that the role of mentor should be restricted to assisting, befriending, guiding, advising and counselling students, and should not incorporate the roles of supervisor, assessor, preceptor or facilitator. Some of the difficulties experienced by one college of nursing in developing such a scheme are explored.


Tutorials in nurse education: a survey of its use in district nursing training. This paper reports the findings of a survey of registered nurses undertaking the district nurse training. District nurse students were asked their views of tutorial arrangements, methods used, their effectiveness and their perceptions of the role that tutorials play in the learning process. A questionnaire was sent to 60 course tutors in five institutions within the South West Regional Health Authority area; 51 were returned completed. The results suggest that tutorial arrangements in courses preparing district nurses need to be re-examined in the light of comments from this group of students.


An integrated approach to curriculum development. This paper explores some aspects of what is meant by integration in a nursing curriculum, by looking at both integration of course content and subject matter, and a consequent move away from separated academic disciplines towards a thematic approach, and at integration of the student group, so that different groups of nurse learners study and learn together for a certain proportion of the programme. We consider reasons for adopting an integrated approach by discussion: issues related to learners and the learning context (these include theories of how adults learn and our perspective on the part to be played by experiential learning); the potential implications for client/patient care and the need for transferable skills to work as a member of a team; and staff development for the teachers, including moves away from possible isolation towards team-teaching. We then go on to describe a model for curriculum development devised in our own institution and based on these principles, which incorporates the development of learners professionally, educationally and personally. This paper discusses the topic from a mainly theoretical perspective, and many of the issues will be relevant to a wide range of courses.


Extension of the raccoon rabies epizootic--United States, 1992. Raccoon rabies, epizootic among raccoons in the southeastern and mid-Atlantic states, has become an increasingly important problem in the northeastern United States. The extension of the epizootic was largely responsible for the 43% increase in the total number of reported cases of animal rabies in the United States from 1990 (4881) to 1991 (6975). In 1991, 3079 cases of rabies in raccoons were reported, the largest number reported in the history of animal surveillance in the United States. This report summarizes the extension of the epizootic into six additional states since 1989.


Cholera associated with international travel, 1992. Approximately one case of cholera per week is being reported in the United States. Most of these cases have been acquired during international travel and involve persons who return to their homelands to visit family or foreign nationals visiting relatives in the United States. This report summarizes case reports from four states during 1992.


Cholesterol screening and awareness--behavioral risk factor surveillance system, 1990. The association between high blood cholesterol and coronary heart disease (CHD) has been well documented (1), and lowering total and low-density lipoprotein cholesterol levels can reduce the incidence of CHD. For example, a 1% decrease in serum cholesterol can result in a 2% decrease in the risk for CHD (2). To reduce the prevalence of high blood cholesterol in the United States, the National Heart, Lung, and Blood Institute initiated the National Cholesterol Education Program (NCEP) in 1985 (3) to encourage all adults to have their cholesterol levels checked at least once every 5 years, know their cholesterol levels, and if it is elevated, take steps to lower their levels. This report summarizes data on the proportion of adults who have been screened and report knowing their cholesterol levels.


Seroconversion to simian immunodeficiency virus in two laboratory workers. Simian immunodeficiency viruses (SIVs) are lentiviruses that cause acquired immunodeficiency syndrome (AIDS)-like illnesses in susceptible macaque monkeys and are used in the study of AIDS (1). In November 1988, CDC published guidelines to minimize the risk of SIV transmission to research laboratory workers (2). This report summarizes the investigation of two laboratory workers who seroconverted following occupational exposures to SIV.


[The experiment and clinical evaluation of chemosensitivity test for esophageal and gastric cancer by ATP assay using endoscopic biopsy]. The chemosensitivity test for esophageal and gastric cancer cells collected by endoscopic biopsies before operation was investigated for evaluation by ATP assay. Experimentally, ATP assay was applied in human esophageal and gastric cancer cell line transplanted in nude mice. ATP level was measured by Lumiphotometer and showed positive linear correlation with the number of cancer cells in more than 10(3). Also ATP level increased when more than 10(3) cancer cells were cultured for more than 48 hours. On the other hand, more than 10(3) cancer cells were indicated to be collected by endoscopic biopsies, experimentally. Clinically, 7 specimens collected by endoscopic biopsy and 5 anticancer agents (MMC, CDDP, 5-FU, ADM and BLM) were used for the test. Forty-nine cases, 31 cases of esophageal cancer and 18 cases of gastric cancer were subjected to the study. The evaluability rates were 93.8%, respectively. Over-all predictive accuracy for esophageal cancer between the clinical responses and results of the assay was 72.0%. These results suggested the usefulness of biopsy specimens for the chemosensitivity test of anticancer agents.


[Clinical study of 76 cases of smooth muscle tumor of the stomach]. Seventy six patients with smooth muscle tumor of the stomach (18 leiomyoma, 58 leiomyosarcoma) operated at Cancer Institute Hospital are presented. 1) Advanced age, short duration of illness, region of the middle portion of the stomach, large tumor size and ulcerative tumor were suggestive of pathological malignancy. 2) Male, symptomatic and larger tumor size were suggestive of poor prognosis. 3) The rate of curative surgery was 94.8% for fifty eight patients with leiomyosarcoma. The cumulative five-year survival rate after curative operation was 74.9% for all patients with leiomyosarcoma. Regardless of resecting method, all patients with tumors less than 5 cm in diameter lived without any indication of recurrence. 4) Four of the 58 patients with leiomyosarcoma had regional lymph node metastasis and died at less than 5 years following operation.


[A scanning electron microscopic study on experimental ischemic lesions in dogs]. Occlusion of minute vessels in the bowel wall is thought to be a cause of ischemic lesions. This study was performed to clarify the pathohistology and the morphology of these various lesions. Microbarium suspension was injected into the mesenteric vessels of twenty four dogs and the ischemic lesions were produced experimentally. The degree of the bowel damage was parallel to the amount of injected microbarium suspension. The resected specimen of the various stages of the disease was studied histologically and morphologically by a scanning electron microscopy. With the small amount of barium suspension the lesions were confined to the mucosal layers. The epithelial cells regenerated regularly and the villi normalized rather quickly, and these lesions healed within one to two weeks (transient form). With the large amount of barium suspension the lesions involved both submucosal and muscular layers. The epithelial cells regenerated irregularly and the villi rearranged to abnormal forms. These lesions healed in two to four weeks leaving the stenosis due to scar formation (stricturing form). This study suggests that scanning electron microscopic investigation can be used to assess the details of morphological changes of ischemic mucosa, special reference to the degeneration and regeneration process of epithelium of the villi.


[Radioimmunodetection of colorectal cancer, using anti-CEA monoclonal antibody CEA 102: whole IgG versus F(ab')2 fragments]. In order to improve cancer imaging with radiolabeled antibodies, three factors appeared to be of particular importance: (1) The selection of the most favorable monoclonal antibody directed to tumor-associated antigen. (2) The use of F(ab')2 or Fab fragments. (3) Selection of the most convenient isotope. Monoclonal antibody CEA 102 was produced by immunization by purified CEA, and its F(ab')2 fragments were compared with whole IgG as a radiotracer for radioimmunodetection of the colorectal cancer. Fragments were eliminated from the circulation twice as fast as whole IgG, and tumor-to-background ratio was achieved more than 1 at 2-3 days with F(ab')2, but 6-7 days with whole IgG in tumor bearing nude mice. In clinical study, F(ab')2 demonstrated clear image on the 1 at day after injection, whereas achievement of the image was possible on the 3rd day in whole IgG. These results indicated that fragments are preferred over whole IgG. Therefore fragments make it possible to preclude dual isotope subtraction methods, and omit the long delays before imaging. They also make it possible to use short half life radionuclides with excellent photon properties, such as 123I and 99mTc.


[Effect of prostaglandin E1 derivative on labilization of liver lysosomal membrane in partial liver ischemia]. The change of liver lysosomal enzymes in tissue and serum during a reperfusion period was studied in partial liver ischemic model in rats and effect of Prostaglandin E1 (PGE1) derivative on partial liver ischemia was investigated. Partial liver ischemia was induced by clamping the branches of the vessels to the right and caudate lobes of rat liver. The clamp was released after 30 minutes of ischemia. Ischemic and nonischemic lobes of the liver were separately removed and the serum was also collected immediately and two hours after the release of the clamp. Lysosomal enzyme activities from free and bound lysosomal fraction were measured separately and the fragility index (F.I.) was calculated. PGE1 derivative was administered intraperitoneally 24, 6, 0.5 hours prior to the induction of ischemia at each dose of 0.05 microgram/kg. Pretreatment with PGE1 derivative prevented lysosomal labilization in ischemic lobe, since there was a significant decrease in F.I. of cathepsin D in the PGE1-pretreated group (preischemia; 28.3 +/- 2.4%, immediately after reperfusion; 30.3 +/- 2.5%, two hours after reperfusion; 30.3 +/- 2.5%) compared to the placebo group (immediately after reperfusion; 40.9 +/- 3.4%, two hours after reperfusion; 41.7 +/- 3.4%, p less than 0.05, p less than 0.05, p less than 0.01, respectively). Pretreatment with PGE1 derivative also significantly suppressed the increase of serum lysosomal enzyme activity. These results showed that PGE1 derivative improved liver lysosomal labilization in partial liver ischemia.


[Prognostic significance of internal mammary lymph node metastases in breast cancer patients]. Examination was made of clinical, histological and biological prognostic factors in 207 patients with invasive breast cancer, and determination was made as to whether variable prognostic factors, especially internal mammary lymph node metastases, would serve as a basis for the prognosis of breast cancer. In a univariate study, overall survival was significantly corrected with tumor size, axillary lymph node status, axillary and internal mammary lymph node metastases and DNA ploidy status. In a multivariate study, however, only axillary and internal mammary lymph node metastases were recognized as important, and independent prognostic factors on survival. Neither axillary lymph node status nor DNA ploidy status appeared an important prognostic factor. Axillary and internal mammary lymph node metastases could not be predicted from their clinical assessment. Only axillary lymph node dissection and biopsy of first and second intercostal spaces were concluded to be useful for accurately indicating the status of these lymph nodes.


[The effect of prostacyclin (PGI) on denervated tissues--especially, about denervation supersensitivity]. It is well known that contractive agents of smooth muscle have denervation supersensitivity. But, the existence of denervation supersensitivity in relaxed ones does not become so clear. We investigated these supersensitivity, especially prostacyclin (PGI2). We made the denervated artery by lumbar sympathetic ganglionectomy in mongrel dogs. Namely, saphenous and dorsal pedal arteries were denervated by this ganglionectomy. And we studied the changes of these arterial tensions by magnus apparatus. Dose-response (D-R) curves of denervated arteries for KCl and noradrenaline (NE) shifted to left against control arteries. Next, we studied effects of PGI2. At first, we measured ED50 of KCl and NE contraction. And, we produced the half contraction on each artery by using ED50 of KCl and NE. Lastly, PGI2 was added on the half contractive arteries to study relaxed changes. D-R curves of denervated arteries for PGI2 shifted to left, too. These denervated arteries acquired the supersensitivity for PGI2. Clinically, if we use relaxants after organ transplantation, these drugs may be very effective for the perfusion of the grafts.


[Angioscopic prediction of late vascular patency after transluminal balloon angioplasty for arteriosclerotic obstructions of the artery of the lower extremities]. Arterial occlusion or stenosis of the lower extremity due to arteriosclerosis obliterans (ASO) was treated by transluminal balloon angioplasty (TBA). Angioscopic observation of the vascular lumen, performed immediately after TBA, provided five major findings: (1) abrasion of the intima, (2) irregularity of the lumen due to poor expansion, (3) laceration of the wall, (4) mural thrombus and (5) calcification. The degree of each of these abnormalities was scored with a 3-grade scale (0, 1 and 2), and scores for the 5 abnormalities were totaled in each patient. Analysis of the relationship between scores and postoperative vascular patency demonstrated that patients without vascular patency in the early (within 6 months) or late period (6 months or more) after the operation had higher total scores as compared to those in whom the vessel remained patient in the corresponding postoperative periods (p less than 0.01, p less than 0.05). Among others, patients with a total score over 5 showed markedly lower patency rates in the early period (10%) as well as late period (0%) after the operation (p less than 0.01). These results indicate the usefulness of post-TBA angioscopic observation of vascular lumen in predicting the long-term patency of the vessels operated on.


[Effects of surgical stress on peripheral venous functions]. Postoperative changes in peripheral venous functions were determined by Ga-In strain gauge plethysmography in 28 surgical patients, and central venous functions and urine catecholamine levels were also measured. The results were as follows: 1. The maximum venous outflow (MVO) and arterial blood inflow volume (AF) of the healthy arms tended to increase with age, but showed no sex or right-to-left difference. 2. MVO, AF and venous distensibility were decreased postoperatively, and the decreases were greater in the patients with major surgery than in those with minor surgery. 3. MVO in the patients with high urine levels of adrenaline and noradrenaline showed a greater decrease than those in normal subjects, and the majority of these patients were found to have had major surgery. There was a negative correlation between the urine noradrenaline level and MVO. 4. The decrease in peripheral venous functions preceded the decrease in central venous functions. 5. The postoperative decrease in peripheral venous functions was considered to be the result of active vasoconstriction due to hypersecretion of catecholamines caused by surgical stress and increase in the extravascular pressure. 6. Measurement of venous functions of the arm may be a good parameter for detecting circulatory disturbances due to surgical stress.


[99mTC-HSA lymphoscintigraphy and leg edema after arterial reconstruction]. To investigate the etiology of lower limb edema after arterial reconstruction, 12 patients (16 limbs) who underwent arterial reconstruction due to atherosclerosis obliterans were observed. There was no relationship between the severity of limb edema and serum factors (serum total protein, albumin, BUN and creatinine), ankle/brachial arterial pressure ratio, peripheral venous pressure or RI lymphoscintigraphy in the supine position. The lymphatic flow in RI lymphoscintigraphy at 3-4 weeks after operation increased with statistically significant difference compared to the preoperative flow whether the patient was in the supine or upright position. Though there was no significant relationship between the severity of leg edema and postoperative lymphatic flow in the supine position, postoperative lymphatic flow in the upright position decreased as the severity of leg edema increased. Increased lymphatic flow in the follow-up period was associated with increased severity of leg edema in the upright position. It is concluded that postoperative leg edema is due to the damage to the lymphatic vessels during operation, and then the lymphatic channels cannot adapt to the increased lymphatic flow after the arterial reconstruction.


[Liposarcoma of the transverse mesocolon--a case report]. Primary mesenteric liposarcoma is a rare neoplasm. We experienced a case of liposarcoma of transverse mesocolon. In our case, the examination of ultrasonography and CT scan revealed the tumor was solid and encapsulated. Preoperatively we suspected the tumor was malignant by the angiographic findings. Laparotomy revealed that the tumor was located in the transverse mesocolon and it could be removed easily. The histopathological diagnosis was myxoid type liposarcoma. It is said generally that liposarcoma is liable to recur after simple resection, therefore, we reconsider that we should have excised more extensively for this case. The patient is doing well without evidence of recurrence for 17 months after operation.


[A case report of aneurysm in the root of splenic artery of the spleno-mesenteric type treated with reanastomosis]. A 76-year-old woman with lumbo-abdominal pain as a chief complaint went through a series of examinations including ultrasonography, CT, and angiography, the final diagnosis being aneurysm of the splenic artery congenitally arising from the superior mesenteric artery (SMA). Since the SMA had to be clamped during the excision of the aneurysm, a biballoon intraluminal shunting was utilized to avoid the visceral ischemia. The splenic artery distal to the aneurysm then underwent end-to-side anastomosis to the SMA. This shunting procedure was considered convenient and applicable to other similar situations in the vascular surgery.


Human gene S31 encodes the pharmacologically defined serotonin 5-hydroxytryptamine1E receptor. The gene encoding a human 5-hydroxytryptamine (5-HT)1 receptor subtype was isolated from a human placental genomic library by using oligonucleotide probes derived from transmembrane regions of the cloned human 5-HT1D beta receptor. The deduced amino acid sequence of the genomic clone hp75d is identical to that of the recently isolated, but uncharacterized, novel serotonin receptor gene S31. Transmembrane domain sequence comparison of clone hp75d with other guanine nucleotide-binding protein-coupled receptors revealed the highest degree of homology (64%) to the 5-HT1D alpha and 5-HT1D beta subtypes and lower degrees of homology (35-52%) to other serotonergic and catecholaminergic receptors. A stable cell line expressing this gene was established, using murine fibroblasts as the host cell, for pharmacological evaluation. High affinity (Kd = 9.7 nM), saturable (Bmax = 2.4 pmol/mg of protein) [3H]5-HT binding was detected using membranes derived from stable transfectants. Most compounds displayed low affinity (K(i) greater than 200 nM) for the expressed gene, with the exception of 5-HT (K(i) = 10 nM). The rank order of potency of ligands to compete for the [3H]5-HT-labeled site best matched the binding profile of the pharmacologically defined 5-HT1E binding site, 5-HT greater than methysergide greater than ergotamine greater than 8-hydroxy-2-(di-n-propylamino)tetralin greater than 5-carboxyamidotryptamine greater than ketanserin. 5'-Guanylylimidodiphosphate decreased high affinity agonist ([3H]5-HT) binding in a dose-dependent manner. 5-HT produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in intact cells stably expressing the 5-HT1E gene. The response was blocked by the nonselective 5-HT1 receptor antagonist methiothepin. The molecular biological and pharmacological data are consistent with the designation that clone hp75d encodes a functional 5-HT1E receptor.


Association of solubilized angiotensin II receptors with phospholipase C-alpha in murine neuroblastoma NIE-115 cells. The peptide angiotensin II (AngII) has been reported to stimulate phosphoinositide-specific phospholipase C (PLC) activity in the murine neuroblastoma cell line N1E-115. In the present study, polyclonal antibodies raised against a PLC isoenzyme, PLC-alpha, reacted with a 60-kDa protein present in both membrane and cytosolic fractions of differentiated N1E-115 cells. In order to examine the possible association of PLC-alpha with cell surface AngII receptors (AngII-Rs), membranes from differentiated N1E-115 cells were solubilized, using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CHAPS (1%) solubilized AngII-Rs, from N1E-115 cells, that maintained their high affinity for agonists. Gel filtration analysis of the solubilized membranes revealed that the majority of the specific binding of 125I-AngII eluted as a large protein complex with a molecular mass of 380 kDa and that agonist binding was partially reduced by guanosine-5'-O-(3-thio)triphosphate (GTP gamma S), within this complex. CHAPS also effectively solubilized immunoreactive PLC-alpha, from N1E-115 cell membranes, that was similarly present within the 380-kDa AngII-binding complex. Anti-PLC-alpha antisera immunoprecipitated approximately 16% of the total phosphatidylinositol-4,5-bisphosphate-specific PLC activity in the 1% CHAPS extract and 40% of cytosolic PLC activity. Moreover, a 60-kDa 35S-Trans S-labeled protein, comigrating with immunoreactive PLC-alpha, was immunoprecipitated from the 1% CHAPS extract by the antisera. In addition, anti-PLC-alpha antisera immunoprecipitated approximately 20% of solubilized AngII-Rs prebound with 125I-AngII but failed to precipitate receptors prebound with the antagonist 125I-Sarc1,Ile8-AngII. The anti-PLC-alpha antisera also immunoprecipitated AngII-Rs when intact membranes were labeled with 125I-AngII before solubilization in 1% CHAPS, suggesting that the AngII-R interaction with PLC-alpha was not the result of detergent-promoted protein-protein interaction. On the other hand, monoclonal antibodies against another PLC isozyme, PLC-gamma, did not precipitate AngII-Rs in solubilized N1E-115 membranes. Finally, the formation of the immunoprecipitated AngII-R-PLC-alpha complex was disrupted by the nonhydrolyzable guanine nucleotide analog GTP gamma S, suggesting that the interaction between AngII-Rs and PLC-alpha is likely to involve a heterotrimeric guanine nucleotide-binding protein in neuron-like cells.


Null phenotype for cytochrome P450 2B2 in the rat results from a deletion of its structural gene. The absence of phenobarbital (PB)-inducible cytochrome P450 2B2 (CYP2B2) in hepatic microsomes from Marshall 520 (M520) and Wistar Munich (WM) inbred strains of rat was previously reported [Biochem. Genet. 25:527-534 (1987)], and it was subsequently shown for M520 rats that corresponding CYP2B2 mRNA was not detected in hepatic extracts from either control or PB-treated animals [DNA 8:29-37 (1989)]. In the present study, solution hybridization was used to quantify PB-induced CYP2B2 and CYP2B1 mRNAs in livers from M520, WM, and outbred Sprague-Dawley rats, as well as additional inbred strains that express all known electrophoretic phenotypes for both of these closely related isozymes. Amounts of these mRNAs were also measured for F1 and F2 progenies of crosses involving M520 rats. The results indicated that the extent of PB induction of both isozymes appears to be independent of the electrophoretic phenotype. It was also shown that the null phenotype for CYP2B2 observed in M520 rats results from a mutation of a single autosomal gene and is inherited codominantly, regarding protein and mRNA phenotypes. Analyses of restriction digests and specific polymerase chain reaction products of hepatic DNAs revealed that the basis for the null phenotype of CYP2B2 in M520 and WM rats was a deletion of the CYP2B2 gene.


Sequence analysis, in vitro translation, and expression of the cDNA for rat liver minoxidil sulfotransferase. A cDNA encoding minoxidil sulfotransferase (Mx-ST), a rat liver cytosolic sulfotransferase that catalyzes the 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfate conjugation of minoxidil and p-nitrophenol, has been isolated from a lambda gt11 cDNA library constructed from poly(A)+ RNA isolated from female Sprague-Dawley rat liver. The largest cDNA, designated Mx-STb, consists of 1245 base pairs and contains an open reading frame of 291 amino acids. The predicted size of the protein translated by Mx-STb is 33,909 Da; however, the molecular mass of the pure protein [Biochem. J. 270:721-728 (1990)] is estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 35,000 Da. The size of the protein obtained by in vitro translation of Mx-STb is identical to that of the pure protein. Results of initial studies of the expression of Mx-STb in COS-1 cells indicate that the expressed protein displays characteristic Mx-ST and p-nitrophenol sulfotransferase activity, is recognized by rabbit polyclonal antibodies raised against pure rat liver Mx-ST, and migrates at approximately 35,000 Da during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This paper presents the cloning and expression of a rat phenol sulfotransferase for which the physical, immunological, and kinetic properties are known. Isolation of the cDNA for Mx-ST will aid in the investigation of the heterogeneity, the tissue localization, and the characterization of the kinetic properties of this important drug-metabolizing enzyme, with respect to other similar phenol sulfotransferases present in rat liver cytosol.


Cloned mouse N-acetyltransferases: enzymatic properties of expressed Nat-1 and Nat-2 gene products. N-Acetylation plays an important role in the metabolism of a wide variety of hydrazine drugs and arylamine drugs and carcinogens. Humans have genetically determined differences in their N-acetyltransferase activities and are phenotypically classified as rapid or slow acetylators. Mice have a similar genetic polymorphism in N-acetyltransferase activity and have been used as models of the human polymorphism in many studies of the toxicology and carcinogenicity of arylamines. Recently, two N-acetyltransferase genes, Nat-1 and Nat-2, were cloned from rapid (C57BL/6J) and slow (A/J) acetylator mouse strains. The genomic clone encoding NAT-1 is identical in rapid and slow acetylator mouse strains, whereas the clone encoding NAT-2 differs between rapid and slow strains by a single base pair, which changes the encoded amino acid from Asn99 in the rapid acetylator strain to Ile99 in the slow acetylator strain. In this report, the N-acetylation polymorphism in mice was investigated by transiently expressing the cloned N-acetyltransferase genes in COS-1 cells. The intronless coding regions of Nat-1 and Nat-2 showed different substrate specificities; isoniazid was a preferred substrate for NAT-1, whereas p-aminobenzoic acid was preferred for NAT-2(99asn) and NAT-2(99ile). All three enzymes acetylated 2-aminofluorene, but none of them acetylated sulfamethazine. Kinetic constants determined for the expressed enzymes with 2-aminofluorene and p-aminobenzoic acid indicated that Km values were not significantly different between the enzymes, although the Vmax value of NAT-2(99asn) was consistently 2-3-fold higher than that of NAT-1 or NAT-2(99ile). Nat-1 and Nat-2 encoded mRNAs of approximately 1.4 kilobases in livers of rapid and slow acetylators. Nat-2 mRNA was more abundant in liver than Nat-1 mRNA. The abundance of Nat-2 mRNA and Nat-1 mRNA was equivalent in both rapid and slow acetylator mouse strain livers. Incubation of transfected COS-1 cell cytosols at 37 degrees showed that the time for decline of NAT activity to 50% of its initial value was 45 hr for NAT-1, 60 hr for NAT-2(99asn), and 4 hr for NAT-2(99ile). This 15-fold difference in the heat stability of the rapid and slow isoforms of NAT activity was also observed in cytosols from rapid and slow acetylator livers. Comparison of the rates of translation of the rapid and slow isoforms of NAT-2 in an in vitro system showed that NAT-2(99asn) was translated at approximately twice the rate of NAT-2(99ile).(ABSTRACT TRUNCATED AT 400 WORDS)


Transcriptional regulation of rat microsomal epoxide hydrolase gene by imidazole antimycotic agents. The effects of imidazole antifungal agents, including ketoconazole, clotrimazole, miconazole, and econazole, on the expression and regulation of microsomal epoxide hydrolase (mEH) were examined in rat hepatic tissue (doses of agents, 150 mg/kg of body weight/day, orally). Immunoblot analyses revealed that administration of either ketoconazole or clotrimazole caused a approximately 4-5-fold increase in mEH levels, whereas either miconazole or econazole resulted in a approximately 7-fold increase in mEH at day 3 after treatment. RNA hybridization analyses, probed with a 1.3-kilobase mEH cDNA, revealed that administration of these imidazole antifungal agents caused substantial elevation of hepatic mEH mRNA in total RNA. Hepatic mEH mRNA levels in total RNA were elevated approximately 11-, 15-, and 18-fold at 12, 24, and 72 hr, respectively, after ketoconazole treatment, whereas mEH mRNA levels were increased approximately 14-, 19-, and 22-fold, respectively, relative to control, at the same time points after clotrimazole treatment. The rate of increase of mEH mRNA caused by miconazole was more rapid than the rates observed for the other agents examined, with a maximal increase in mRNA being noted at 12 hr after treatment. The degree of mEH mRNA increase after 3 consecutive days of miconazole treatment was appreciably less than that observed at 12 hr after a single treatment. Econazole caused a maximal increase at 24 hr and subsequent decline in mEH mRNA levels after 3 consecutive days of treatment. Elevation of mEH mRNA levels by these antimycotic agents was confirmed in poly(A)+ RNA, as assessed by both Northern and slot blot hybridization analyses. Nuclear run-on analyses revealed that administration of ketoconazole, clotrimazole, or miconazole stimulated the rate of mEH gene transcription at 12 hr after treatment by 11-, 8.5-, and 9-fold, respectively, compared with control, whereas econazole resulted in a 4-fold increase in the rate of mEH gene transcription at the same time point. The transcription rates of mEH mRNA at 24 hr were significantly less than those observed at 12 hr after a single treatment with either ketoconazole, miconazole, or econazole, resulting in 6.5-, 2.5-, and 2-fold increases, respectively, relative to control. Clotrimazole, however, maintained the activated mEH transcription rate at 24 hr after treatment, exhibiting a 11-fold increase, compared with control. These results provide evidence that the imidazole antimycotic agents induce mEH and that the mEH induction involves large increases in mRNA, with transcriptional activation.


Anti-liver microsomes autoantibodies and dihydralazine-induced hepatitis: specificity of autoantibodies and inductive capacity of the drug. Anti-liver microsomes (anti-LM) autoantibodies in patients with dihydralazine-induced hepatitis were found to react specifically with cytochrome P4501A2 (P4501A2) but not with P4501A1 expressed in yeast and bacteria. These results were confirmed by immunoinhibition of methoxyresorufin-O-demethylase activity (supported by the P4501A subfamily); anti-LM antibodies more strongly inhibited this activity in yeast expressing P4501A2 than in yeast expressing P4501A1. Anti-LM were shown to be specific to the disease; in three cases, these autoantibodies were present at high titers during disease, whereas the titers decreased upon recovery and became undetectable a few months after recovery. Thus, there exists a time-dependent relationship between the disease and the autoantibodies, which does not prove that the autoantibodies are causative of the hepatitis; they might only be a marker. The inductive capacity of dihydralazine toward P450 was also studied. In rats treated in vivo and in human hepatocytes treated in vitro with dihydralazine, a 2-fold increase in P4501A2- and P4501A-supported monooxygenase activities was found. The levels of the other P450 isoforms tested were unchanged during treatment, both in vivo in rats and in vitro in cultures of human hepatocytes. In human hepatocytes, dihydralazine produced a dose-dependent increase in the level of P4501A up to 0.1 mM; induction of P4501A was less strong at 0.2 mM and disappeared at 0.5 mM. The same treatment did not change the level of P4503A4, taken as control. The strong heterogeneity in the expression of P4501A enzymes in human liver and the capacity of these enzymes for induction by dihydralazine and by other compounds might be predisposing factors in this autoimmune disease.


Role of ornithine decarboxylase suppression and polyamine depletion in the antiproliferative activity of polyamine analogs. Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.


Evidence for the stereoselective inhibition of chick embryo hepatic ferrochelatase by N-alkylated porphyrins. II. N-Ethylprotoporphyrin (N-ethyl-PP) was isolated from the livers of phenobarbital-pretreated rats after the administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine, separated into its four regioisomers by high performance liquid chromatography, and quantitated. The percentage ratio, in the chromatogram, of the peak areas of the ring A-substituted (NA) and the ring B-substituted (NB) regioisomers was 80:20, compared with 50:50 for synthetic N-ethyl-PP. The NA regioisomer of N-ethyl-PP isolated from rat liver was found to be approximately 5 times more potent an inhibitor of ferrochelatase than was the NB regioisomer. Because the synthetic NA regioisomer (an equal mixture of the NA and the epi-NA enantiomers) is equipotent with the synthetic NB regioisomer (an equal mixture of the NB and the epi-NB enantiomers), epi-NB must be more potent than epi-NA. The higher potency previously observed with the NA plus NB regioisomers of N-ethyl-PP isolated from rat liver, compared with the NA plus NB regioisomers of synthetic N-ethyl-PP, is explained by the fact that the biological preparation contains 80% of the potent NA, compared with 25% of the potent NA and 25% of the potent epi-NB in the synthetic preparation. The critical features for optimal ferrochelatase-inhibitory activity are the ring A N-ethyl group facing downward in the NA isomer and the ring B N-ethyl group in the epi-NB isomer being rotated through 180 degrees to occupy the same position. According to one proposed mechanism, N-alkylprotoporphyrins inhibit ferrochelatase by serving as transition state analogues for the iron insertion step. X-ray crystallography shows that the N-alkyl group-bearing pyrrole ring and the pyrrole ring opposite to the N-alkyl group are tilted out of planarity in opposite directions. We suggest that this tilting reflects the normal conformational changes required for the insertion of iron into the protoporphyrin IX ring by ferrochelatase and that the greater inhibitory activity of NA and epi-NB isomers, compared with epi-NA and NB isomers, is due to the fact that the normal mechanism for ferrochelatase-catalyzed iron insertion has preference for an A-C ring tilt over a B-D ring tilt.


Complex allosteric modulation of cardiac muscarinic receptors by protamine: potential model for putative endogenous ligands. A large number of diverse pharmacological agents bind to a secondary domain on the muscarinic receptor, to influence allosterically the interaction of ligands at the primary binding site. Based on common structural features of these antagonists, we examined the interaction of protamine, an endogenous polycationic peptide, and of polyamines with muscarinic receptors in rat heart. Our results provide several lines of qualitative evidence that protamine allosterically modulates the conformation of muscarinic receptors, in a marked negatively cooperative manner. It decelerated the dissociation of N-[3H]methylscopolamine ([3H] NMS) initiated by atropine, in a concentration-dependent fashion. Inhibition by protamine of [3H]NMS binding at equilibrium showed a distinct plateau, which increased in magnitude at higher ligand concentrations. Scatchard analysis of saturation isotherms of [3H]NMS binding in the absence and presence of protamine indicated that protamine did not alter Bmax in a statistically significant fashion, although there was a trend of a concentration-dependent increase in this parameter. On the other hand, it caused a marked concentration-dependent decrease in the affinity of [3H]NMS, and this effect reached a ceiling limit. However, there were marked quantitative deviations of the interaction of protamine from a simple ternary allosteric model. Some of these discrepancies could be explained by the tendency of protamine to increase Bmax. The allosteric actions of protamine demonstrated in kinetic and equilibrium experiments were selective for m1 and m2 muscarinic receptors, compared with m3, m4, and m5 receptors, as studied in Chinese hamster ovary cells transfected with the genes of the different muscarinic receptors. Arginine residues play an important role in the allosteric interaction of protamine, inasmuch as poly-L-arginine qualitatively mimicked the effects of protamine. In contrast, no effects of the polyamines spermine, spermidine, and putrescine were observed on [3H]NMS binding. This is the first report on the allosteric modulation of muscarinic receptors by an endogenous peptide.


Anomeric preference of glucose utilization in rat erythrocytes. The beta-anomer of glucose relative to the alpha-anomer was more rapidly metabolized into lactate by rat erythrocytes at 37 degrees C (beta/alpha ratio = ca. 1.3): the amounts of alpha- and beta-D-glucose metabolized into lactate during 3 min were 0.21 and 0.27 mumol/gHb, respectively. Also, the transport of beta-D-glucose into erythrocytes was more rapid than that of alpha-D-glucose: the amounts of alpha- and beta-D-glucose transported into erythrocytes during 3 min were approximately 3.5 and 5.0 mumol/gHb, respectively. Glucose phosphorylation by rat erythrocyte hexokinase (i.e., a possible rate-limiting step in glycolysis) occurred at higher velocities with the beta-anomer than with the alpha-anomer (beta/alpha ratio = 1.28). The Km value of hexokinase for either anomer of glucose was 53 microM. The glucose concentrations in erythrocytes incubated with alpha- and beta-D-glucose reached about 1 mM in 1 min, indicating that hexokinase is almost completely saturated with glucose within less than 1 min. The results suggest that glucose phosphorylation and glucose transport are major and minor determinants, respectively, for the anomeric preference of glucose utilization in rat erythrocytes.


Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells. In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca(2+)-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca2+ 'storage' proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca(2+)-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca(2+)-binding proteins of muscle and nonmuscle cells, respectively. Other Ca(2+)-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca2+ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca2+ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.


Biochemical characterization of Artemia ras p21. The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.


Peroxidase-promoted oxidation and peroxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine. A new pathway for its metabolic degradation. Spectral data provide the first evidence that lactoperoxidase, a model enzyme for most mammalian peroxidases, catalyzed the one-electron oxidation and/or peroxidation of 5,7-dihydroxytryptamine. This process correlates with the production of superoxide radicals as is evident from the observed inhibitory effect of superoxide dismutase on product formation. 5,7-Dihydroxytryptamine is a classical peroxidase-oxidase substrate acting as a one-electron donor for enzyme compounds I, II and III. The one-electron peroxidatic oxidation of this serotonergic neurotoxin, responsible for the selective degeneration of central (5-hydroxytryptamine) neurons, is a fast process requiring measurement on the ms time scale. Attention is drawn to the biochemical and toxicological implications, because this fast reaction results in formation of known cell damaging species: free radicals, superoxide radicals and quinoidal products probably involved in the toxic action of 5,7-dihydroxytryptamine.


Effects of tumour necrosis factor-alpha (cachectin) on glucose metabolism in the rat. Intestinal absorption and isolated enterocyte metabolism. Intravenous administration of a single dose (20 micrograms) of recombinant tumour necrosis factor-alpha (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-14C]glucose to 14CO2 was significantly increased by the cytokine. In addition, [14C]lipid accumulation from [U-14C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of 3[H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.


The association of human coagulation factors VIII, IXa and X with phospholipid vesicles involves both electrostatic and hydrophobic interactions. Blood coagulation factor X (FX) is converted to its active form (FXa) by a membrane bound multi-protein enzyme complex, comprised of factor VIII (FVIII), factor IXa (FIXa) and FX. Characterization of the molecular forces involved in the association of these proteins with phospholipids is crucial to understanding how these proteins bind to the lipid milieux of physiological membranes. In this report, the molecular forces involved in the association of FVIII, FIXa or FX with phospholipid vesicles (PLV) were characterized by ligand affinity chromatographic analyses. Treating FVIII-affinity columns with agents that disrupt electrostatic interactions caused elution of 15.2% of the total bound PLV, while agents that disrupt hydrophobic interactions caused elution of 84.8% of the total bound PLV. These results demonstrate that the association of PLV with FVIII is primarily hydrophobic. In contrast, the association of PLV with FIXa or FX is largely the result of electrostatic forces. This was established by observing that 71.3% and 78.9% of the total bound PLV was eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt electrostatic interactions. Of the total bound PLV, 28.7% and 21.2% were eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt hydrophobic interactions. These data demonstrate that hydrophobic forces play a heretofore unrecognized role in the association of PLV with FIXa or FX.


Expression of c-myc protooncogene in rat lens cells during development, maturation and reversal of galactose cataracts. It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc mRNA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, gamma crystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose.


Specific macromolecular interactions between tau and the microtubule system. The microtubule-associated protein Tau, a major component of brain microtubules, shares common repeated C-terminal sequences with the high molecular-weight protein MAP-2. It has been shown that tau peptides V187-G204 and V218-G235, representing two main repeats, induced brain tubulin assembly in a concentration-dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides and site-directed monoclonal antibodies. Tau peptides appeared to compete with MAP-2 incorporation into assembled microtubules. The interactions of the tau fragments with beta-tubulin peptides bearing the tau binding domain on tubulin were analyzed by fluorescence spectroscopy. The specificity of the binding was further demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti-idiotypic antibody produced after immunization with the beta-II(422-434) tubulin peptide, as assessed by enzyme-linked immunoassay. Western blots confirmed the interaction of tau with the monoclonal antibody. In addition, immunoassays revealed a competition between the MAP-reacting monoclonal antibody and the tubulin peptide beta-II(422-434) for their interaction with the tau molecule.


Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol. The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca(2+)-requiring proteinase required 5-10 microM Ca2+ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25-2.0 microM) in the absence or presence of Ca2+ (5.0 microM) added. The effect of regucalcin, however, was the greater in the absence of Ca2+ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca2+ (5.0 microM). In the absence of Ca2+, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 micrograms/ml), and heavy metals (25 microM cadmium or 25 microM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca(2+)-independent neutral cysteinyl-proteinase.


Identification of toxigenic Clostridium difficile strains using a toxin B gene-specific oligonucleotide probe. We describe the use of a new specific synthetic oligonucleotide probe, deduced from the sequence of the gene for Clostridium difficile toxin B, to identify toxigenic strains of C. difficile. This probe does not hybridize to the DNA of non-toxigenic strains of C. difficile nor to DNA isolated from different Clostridium species, including C. sordellii. None of the enteric pathogenic bacteria tested were seen to hybridize with the probe. A preliminary study of direct probing of faecal specimens indicates a potential for the use of this DNA probe in the clinical laboratory for the rapid identification of toxigenic strains of C. difficile.


Quantification of the detection of Pneumocystis carinii by DNA amplification. We have developed a highly specific and sensitive technique for the detection of Pneumocystis carinii DNA using DNA amplification by the polymerase chain reaction (PCR). PCR products are detected by agarose gel electrophoresis and Southern hybridization to an oligonucleotide probe. Here we report the calibration of parasite numbers with amplification and hybridization signals and show that we can detect P. carinii to a lower limit of one to two organisms. The quantification of this diagnostic technique allows us to establish the number of organisms in a clinical sample which correspond to pneumocystis pneumonia or to sub-clinical pulmonary colonization.


Development of cell surface protein associated gene probe specific for Listeria monocytogenes and detection of the bacteria in food by PCR. A genomic library of L. monocytogenes was constructed using lambda Zap II-Eco RI and screened with a monoclonal antibody which is specific for a Listeria cell surface protein. Three positive clones each contained a 6.5 kb insert which in E. coli could express the same Listeria protein. The 6.5 kb insert was further digested with Hin dIII and the smaller fragments were subcloned into a plasmid vector (pBluescript) and screened with 32P-labelled genomic DNA from L. monocytogenes or L. innocua. Three clones which were positive with L. monocytogenes and negative with L. innocua were screened and each contained a 2.1 kb insert. The 2.1 kb insert was partly sequenced and some candidate oligomer probes from the sequences were selected and compared with sequences in a Genbank computer search. One such oligomer probe (T7-list) was confirmed to be specific for L. monocytogenes. The probe hybridized with all 28 strains of L. monocytogenes tested, but not with any of six other Listeria species nor 11 other bacteria tested. Using this probe-primer, a PCR method was developed which could detect as few as 2 cfu of L. monocytogenes in pure cultures, and as few as 4-10 cfu of L. monocytogenes when inoculated into foods.


Detection of rabies virus mRNA in mouse brain by using in situ hybridization with digoxigenin-labelled RNA probes. A non-isotopic method of in situ hybridization (ISH) was developed for the detection of rabies virus RNA in paraffin-embedded tissues. Digoxigenin-labelled RNA probes for rabies virus glycoprotein mRNA were used. The method had good sensitivity and low backgrounds, and there was excellent cellular localization of signals. ISH wih digoxigenin-labelled probes was compared with ISH with 3H-labelled probes. This non-isotopic method of ISH is more convenient than the radiolabelled method, and it is quicker because a long autoradiographic exposure is not required.


Detection of Pneumocystis carinii in a rat model of infection by polymerase chain reaction. The polymerase chain reaction (PCR) was employed to detect Pneumocystis carinii in organs of infected rats. Using a pair of oligonucleotides designed to the dihydrofolate reductase (DHFR) gene of rat P. carinii, specific amplification of an expected 415 bp region of P. carinii DHFR DNA of this organism was achieved, while no amplification occurred with the human, Candida albicans, and Mycobacterium avium and tuberculosis DNAs. Using rat P. carinii isolated from in vitro cultures and infected lung homogenates, the minimum detection level by PCR on an ethidium bromide gel was about 200 organisms and by Southern analysis with radiolabelled DHFR probe the detection level improved to 20 organisms. This level of sensitivity is sufficient to detect P. carinii specific band on the gel in infected rat lung and other organs. This PCR technique is potentially useful for detecting P. carinii in bronchoalveolar lavage (BAL) fluids of AIDS patients and for quantifying the organisms in tissues and in in vitro cultures where a high background with conventional stains makes it harder to determine the number of organisms.


Polymerase chain reaction for detection of verocytotoxigenic Escherichia coli isolated from animal and food sources. Animals and their by-products have been implicated as important sources of verocytotoxigenic Escherichia coli (VTEC) associated with disease in humans. VTEC comprise a wide range of serotypes and produce a variety of closely related verocytotoxins (VT). A pair of oligonucleotide primers, targeting conserved sequences found in VT1, VT2 and VTE genes, was used to develop a polymerase chain reaction (PCR) procedure to detect all types of VTEC. Supernatants of boiled broth cultures of VTEC (223 strains) isolated from ground beef, ground pork, raw milk, bovine faeces and porcine faeces; non-VTEC E. coli (72 strains); and other enteric and food bacteria (76 strains) were tested by PCR. The verocytotoxigenicity of these strains was verified by the Vero cell assay. All 223 VTEC isolates, comprising over 50 different serotypes, were detected by the PCR procedure. Shigella dysenteriae type 1 was the only other bacterium that was positive in this assay. As little as 1 pg of VTEC DNA and as few as 17 cfu of VTEC could be detected with this method. The results indicate that these primers detect VTEC over a wide range of serotypes. This method may be applicable as a screening procedure for the detection of VTEC in samples of foods and faeces.


Differentiation between pathogenic and non-pathogenic Yersinia enterocolitica strains by colony hybridization with a PCR-mediated digoxigenin-dUTP-labelled probe. The Polymerase Chain Reaction (PCR) method was used to generate a vector-free digoxigenin-dUTP labelled probe that targets the Yersinia enterocolitica gene encoding the heat stable enterotoxin (yst). The probe was used in DNA-DNA colony hybridization to screen 113 strains of Y. enterocolitica and related species for the presence of the enterotoxin gene. In Y. enterocolitica, the probe clearly discriminated between pathogenic and non-pathogenic strains even those belonging to the same serotype. Of the other Yersinia species, only three strains of Y. kristensenii possessed DNA sequences homologous to the yst gene. The probe was further checked for its specificity in artificially inoculated fecal samples and could easily detect the target sequence of the yst gene. The digoxigenin-labelled probe proved to be a reliable epidemiological tool to discriminate between pathogenic and non-pathogenic strains in pure and mixed culture, thus offering the advantage of using a non-radioactive detection system in clinical laboratories with the possibility of reusing the same hybridization solution several times and obtaining results within a relatively short time.


Detection of Chlamydia trachomatis by use of polymerase chain reaction. A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. One primer set was used, from the published sequence of the common C. trachomatis plasmid. Detection of amplified sequences was carried out by agarose gel electrophoresis. Analysis of 106 clinical samples tested by cell culture and PCR showed a sensitivity of 100% when PCR was compared with cell culture.