BMY-14802, a sigma ligand and potential antipsychotic drug, reverses amphetamine-induced changes in neostriatal single-unit activity in freely moving rats. The effects of BMY-14802 (5, 10, or 20 mg/kg), a sigma-receptor ligand showing preclinical evidence of antipsychotic efficacy, were tested on single-unit activity in the neostriatum of freely moving rats with or without pretreatment with 1.0 mg/kg D-amphetamine. Relative to resting baseline, amphetamine activated the large majority of neurons that changed firing rate in close temporal association with movement. All doses of BMY-14802 reversed this neuronal response, but the effect was most pronounced at 20 mg/kg. This dose, however, was equally likely to reverse or to induce a haloperidol-like potentiation of those neurons inhibited by amphetamine. In contrast, 10 mg/kg BMY-14802 consistently reversed amphetamine-induced neuronal inhibitions. All doses of BMY-14802 attenuated the locomotor effects of amphetamine, but only the higher doses also blocked other aspects of the amphetamine behavioral response. By itself, BMY-14802 dose dependently inhibited motor-related neurons, but elicited less behavioral activation than amphetamine. BMY-14802 (20 mg/kg) also induced hindlimb ataxia and occasional backwards locomotion. Haloperidol (1.0 mg/kg) reliably suppressed both behavior and neuronal activity when injected 30 min after BMY-14802, whether or not amphetamine pretreatment was given. Thus, BMY-14802 shares with other neuroleptics the capacity to reverse amphetamine-induced excitations of neostriatal motor-related neurons, whereas other effects of BMY-14802 reveal some haloperidol-like actions at 20 mg/kg that do not occur at lower doses.


Failure of salmeterol to inhibit circulating white cell responses and bronchoconstriction induced by platelet activating factor. Platelet activating factor (PAF) is a potent mediator of inflammation. Inhalation of PAF causes acute bronchoconstriction and a transient fall in white blood cell count in humans. Salmeterol inhibits pulmonary inflammation induced by PAF in guinea pigs. The effect of salmeterol on effects induced by PAF was investigated in eight normal subjects who inhaled salmeterol (50 micrograms) twice daily or a matched placebo for one week before challenge with PAF. Blood samples were taken from a forearm catheter for total white cell and neutrophil counts before and for 30 minutes after administration of PAF (48 micrograms) through a Mefar dosimeter. Blood films were stained for unsegmented neutrophils before and after treatment with PAF on a placebo day. Mean baseline total white cell counts and neutrophil counts did not differ on the two days. Mean baseline sGaw was significantly higher after inhaled salmeterol (1.84 (95% C1 1.45-2.23) s-1kPa-1) than after placebo (1.53 (1.24-1.82)). After placebo mean total white cell counts, neutrophil counts, and sGaw were reduced to 60 (43-78)%, 39 (14-64)%, and 82 (71-93)% of baseline respectively five minutes after inhaled PAF. After salmeterol treatment mean reductions five minutes after inhaled PAF were 59 (45-73)%, 40 (19-61)%, and 82 (71-93)% of baseline respectively. At 30 minutes after treatment with PAF the neutrophil count rebounded to 143 (82-204)% of baseline after placebo and to 127 (93-161)% after inhaled salmeterol. There was no significant difference in the percentage of immature neutrophils before and after treatment with PAF (2.0 (0.5-2.6)% compared with 3.9 (2.2-5.6)%. Treatment with salmeterol did not inhibit reduction in total white cell count or neutrophil count, rebound neutrophilia, acute bronchoconstriction, or transient flushing after inhalation of PAF. These results conflict with the inhibitory effect of salmeterol on lung inflammation in guinea pigs but are consistent with the lack of effect of salbutamol in humans. Salmeterol does not have an anti-PAF effect in vivo in humans.


Evidence that lymphocyte traffic into rejecting cardiac allografts is CD11a- and CD49d-dependent. Acute cardiac allograft rejection is characterized by infiltration of leukocytes into tissue parenchyma, but the site of entry and endothelial adhesion molecules involved are not yet defined. Lymphocyte binding to frozen sections prepared from day-3 rejecting cardiac allografts was significantly increased compared with sections made from normal hearts (number of bound lymphocytes, 983 +/- 216 per mm2 vs. 309 +/- 121, respectively, P < 0.001) or syngeneic grafts. The bound lymphocytes were located exclusively only on the top of the capillary structures and not on any other sites on the heart vasculature. We further wanted to analyze which of the cloned endothelial adhesion molecules and their counterreceptors would be involved in the increased lymphocyte binding. Lymphocyte pretreatment with mAb anti-CD11a or anti-CD49d inhibited this binding more than 50%. This inhibition on lymphocyte binding could not be increased by combining these two antibodies. Lymphocyte binding to endothelium has been shown to be at least partly organ specific; therefore, we asked whether increased lymphocytes adhere to cardiac allografts could be organ specific. Lymphocyte binding to lymph node high endothelial venules (HEV) has been shown to be inhibited by mannose-6-phosphate (M6P) and to kidney peritubular capillaries by mannose-1-phosphate (M1P). In the present study neither of these carbohydrates had any effect on lymphocyte binding to cardiac allograft endothelium. Monosaccharide inhibition studies demonstrate that the mechanism of lymphocyte adhesion to cardiac capillary endothelium differs from adhesion to kidney allografts or peripheral lymph node high endothelium.


Novel immunohistochemical markers of human renal allograft dysfunction--antithrombin III, Thy-1, urokinase, and alpha-smooth muscle actin. We have studied the expression of alpha-smooth muscle actin (alpha sm-1) by mesangial cells, and the expression of Thy-1 glycoprotein, antithrombin III (ATIII), and urokinase by tubular epithelial cells in normal kidneys and dysfunctional renal allografts. Kidney biopsies were studied immunocytochemically for changes in each of these markers and the findings were classified into two groups and compared with creatinine plasma levels at the time the biopsies were taken. In dysfunctional grafts, mesangial alpha sm-1 and tubular epithelial Thy-1 reactivities were greatly diminished, and urokinase and ATIII were missing from proximal renal tubular epithelial cells. Urokinase, which was absent from normal renal glomeruli, appeared in glomeruli of some dysfunctional allografts. The possible usefulness of these markers in patient evaluations was supported by our finding that the distribution of vinculin, fibronectin, myosin, actin B4, desmin, glomerular HLA-DR, and the tubular expression of CD15 remained unchanged. These data prompt us to suggest that the immunocytochemical localization and evaluation of alpha sm-1, Thy-1, ATIII, and urokinase in kidney allografts may be useful adjuncts in the assessment of function in renal allografts.


Immobilization of mink (Mustela vison) with medetomidine-ketamine and remobilization with atipamezole. Four groups of mink were immobilized with medetomidine-HCl (MED) 0.1 mg/kg + ketamine (KET) 5 or 7.5 mg/kg at different ambient temperatures. The induction time, degree of immobilization and analgesia, rectal temperature, heart and respiration rates were recorded at intervals throughout the immobilization period. The animals were then given atipamezole-HCl (ATI) 0.5 mg/kg for reversal at different times after injection of MED/KET and the effects of the antagonist were evaluated. Subcutaneous administration of MED/KET induced complete immobilization in all 20 animals, and the highest dose was considered suitable for major surgery. Prolonged immobilization at low ambient temperatures (-10 to +5 degrees C) caused severe hypothermia in all animals. The mean rectal temperature had dropped to 37.8 degrees C and 32.1 degrees C at 15 and 85 min, respectively, after injection of MED/KET, significantly lower than the corresponding values for animals immobilized at room temperature. Intramuscular administration of ATI 20 or 40 min after injection of MED/KET rapidly remobilized the animals without apparent side-effects. Administration of ATI to animals recovering spontaneously 90 min after injection of MED/KET induced thermogenesis (shivering) in animals immobilized at a low ambient temperature, while no such effect was seen in animals immobilized at room temperature. One hour after injection of ATI, the rectal temperatures of all treated animals had returned to normal and there were no signs of abnormal behaviour.


Value of ambulatory blood pressure recordings. In the present paper, the value of ambulatory blood pressure recordings is discussed. Such recordings can be very helpful in the management of high and low blood pressure. The fact that more readings are obtained and that they are made in normal life conditions are the strongest arguments in favour of those recordings. Whether ambulatory recordings predict long term prognosis better than office blood pressure is still open for discussion; the long term European study OvA should help clarifying this important question. Data coming from these techniques are especially useful when conflicting elements are present such as high pressure with no organ damage or blood pressure resistant to all drugs and side effects occurring with even minor doses of antihypertensive drugs. Short term episodes of high or low pressure are best documented with ambulatory blood pressure recordings and this holds particularly true in patients with arterial hypotension. Finally several research aspects can be approached by ambulatory recordings of pressure such as the study of pharmacokinetics of drugs and analysis of blood pressure variability.


The increase in skin hydration after application of emollients with different amounts of lipids. Emollients can increase the water content in the stratum corneum by delivery of their water to the skin, and by occlusion. These two mechanisms were studied using three preparations with different concentrations of lipids. The products were applied to the skin and then removed by cleaning the surface after 5 and 40 min. The increase in skin water loss following removal of product residue was considered as a release of excess water in the skin. Exposure of the skin to pure petrolatum for 5 min gave no increase in the water loss from the skin surface following removal of the product residue. A lipid rich cream (66% lipids) gave a significant increase, but the highest increase was found after removal of an ordinary cream (27% lipids). Release of water from the skin indicates that water in the creams had previously been absorbed into the skin. The occluding properties of the products were determined after 40 min of exposure. Petrolatum reduced the water loss by approximately 50% and the other products by 16%. The occlusion caused an increase of water in the skin, which resulted in a release of water following removal of the products. The release was related to the reduction of water loss. Thus petrolatum gave a higher release of water than the other emollients.


Interleukin-8 receptors in normal and psoriatic polymorphonuclear leukocytes. Polymorphonuclear leukocyte (PMNL) infiltration is an important characteristic in psoriatic lesions. The proinflammatory 8-kD peptide interleukin-8 (IL-8) is present in psoriatic scales and possesses a high chemotactic activity on human neutrophils, which may relate to its role in psoriasis. Its chemotactic activity is mediated via specific receptors on PMNL. The goal of our work was to ascertain whether PMNL infiltration in psoriasis can be accounted for by functional abnormalities of the circulating PMNL due to alterations in the IL-8 receptor density or affinity (or both). Results of radioligand binding studies performed in 10 psoriatic patients, 10 patients with atopic eczema and 11 normal controls showed no difference in receptor affinity (Kd) between the groups. However, a slight but significant elevation in IL-8 receptor density was seen on PMNL from psoriatic individuals (31,230 +/- 3,237 binding sites per cell) compared to those from normal volunteers (24,152 +/- 2,643) and atopic eczema patients (24,092 +/- 2,743). Increased number of IL-8 receptors may, besides elevated cutaneous IL-8 concentrations, contribute to the intraepidermal accumulation of PMNL in psoriasis.


A new marker of epidermal differentiation associated with the membrane coating granules: characterization and applications to pathology. A murine monoclonal antibody, BC12, was obtained after immunization against suprabasal human keratinocytes. In the epidermis of normal human skin, the antigen recognized by BC12 (BC12 antigen) is located at the apex of keratinocytes in the upper stratum spinosum and stratum granulosum but is absent in other layers. The BC12 antigen is also present in hair follicles. Immunoblotting performed on keratinocyte subpopulations confirmed the presence of the BC12 antigen in differentiated keratinocytes only. Two-dimensional immunoblotting showed that the BC12 antigen corresponds to a set of polypeptides with an apparent molecular weight of approximately 33kD. In keratinocyte cultures, the antigen is present only in stratified areas. The distribution of the BC12 antigen, as studied by indirect immunofluorescence and immunoelectron microscopy, and its presence in certain subcellular fractions of epidermal cells suggest that it is a component of membrane coating granules (MCGs) or that it is associated with these structures. Strikingly, in psoriasis, eczema and many other diseases, the BC12 antibody does not label the epidermis, but vessels in dermal papillae. The BC12 antibody may thus be a useful tool in the study of keratinocyte differentiation and MCG physiology, and, also, in pathology.


Immunophenotypical characterization of inflammatory cellular infiltrates in tinea. In order to elucidate the still poorly understood pathogenetic pathways of acute tinea, the inflammatory cellular infiltrates in this infection were analyzed. Lesional punch biopsies were cryostat-sectioned and stained with monoclonal antibodies for immunophenotypization of T cells, B cells, macrophages and activation markers. For each antibody the positively stained inflammatory cells in the dermis and in the epidermis were quantified separately. Most of the dermal mononuclear cells in acute tinea were identified as T helper lymphocytes of the memory type. Furthermore, considerable amounts of Langerhans' cells and macrophages were found, but virtually no B cells. A high proportion of cells expressed markers of activation. Within the epidermis, accumulations of Langerhans' cells and LeuM5+ dendritic macrophages were detected near fungal element. In view of the otherwise rather similar cellular infiltrates in acute tinea and different non-infectious dermatoses, acute tinea may be particularly suitable to study the functional relationship of Langerhans cells and LeuM5+ macrophages.


Quantitative analysis of Langerhans' cells in epidermis at irritant contact reactions using confocal laser scanning microscopy. Confocal laser scanning microscopy (CLSM) was used for quantitative analysis of CD1a+ cells in epidermis at irritant reactions. Sodium lauryl sulphate (2% and 4%) or non-anoic acid (20% and 80%) were applied to the skin of healthy volunteers under occlusion for 24 h. Skin biopsy specimens were taken after additional 24 h and were snap frozen. Freeze-sections, 25 microns thick, were stained with anti-CD1a antibodies (Leu-6) followed by FITC-labelled rabbit anti-mouse IgG. The sections were viewed and optically sectioned in the CLSM at four depth levels. The data was analysed using a threshold value for the fluorescence. The obtained result is presented as the proportion of specimen area having a fluorescence intensity above the threshold. The result demonstrates that the CLSM is a useful tool for obtaining not only structural information but also quantitative information from a defined tissue volume. In the present investigation it was possible to demonstrate variations in CD1a+ reactivity in epidermis at detergent-induced irritant reactions with a marked decrease in CD1a+ after 80% non-anoic acid exposure and only minor differences in the CD1a+ after 2% and 4% sodium lauryl sulphate exposure.


Comparison of muscle-derived serum carbonic anhydrase III and myoglobin in dermatological patients: effects of isotretinoin treatment. The serum levels of muscle-specific serum carbonic anhydrase III (S-CAIII) and myoglobin (S-Myo) were analyzed in various male dermatological patients of the same age. The mean levels of S-CAIII and S-Myo were essentially similar in patients with acne, psoriasis vulgaris, atopic eczema and tinea, suggesting that common dermatological diseases do not affect the serum levels of the muscle markers. Increased levels of S-CAIII, which is specific for skeletal muscle cells, were found in the acne patients who had been treated with isotretinoin. However, when S-CAIII and S-Myo were studied in 24 patients (16 males, 8 females) before and during isotretinoin treatment, no constant increases in these markers could be observed. When individual patients were followed for several months, transient increases or decreases could be observed. The changes in S-CAIII, or S-Myo, did not correlate with the dose of isotretinoin, nor with the duration of the treatment. The results suggest that systemic isotretinoin does not specifically affect skeletal or myocardial muscles. The increases in these markers observed in the course of dermatological diseases and isotretinoin treatment are obviously due to other factors, such as exercise.


Sequential concentration of chloroquine in human hair correlates with ingested dose and duration of therapy. Human scalp hair was analyzed for chloroquine using gas-chromatography. In 5 patients it was demonstrated that the amount of uptake of chloroquine into the hair varied proportionally with the dosage (from 500 mg/week to 10 g single dose) and with the time of administration. The chloroquine concentrations ranged from 8 to 1100 micrograms/g hair. Chloroquine could be determined quantitatively after a single toxic dosage used in a suicidal attempt and also after low therapeutic doses. The sequential examination of the hair shaft allows an assessment of the chloroquine amount taken over time, the individual dosage, the initiation and termination of therapy. As hairs can be collected easily, they are a unique specimen for investigation, and it is suggested that they can virtually be used as a "tachogram" of chloroquine drug-therapy or intoxication.


Lichenoid eruption induced by low dose captopril. A patient with congestive cardiac failure developed a rash following captopril treatment. The clinical and histological features were consistent with a lichenoid eruption. The rash spontaneously resolved without any treatment three months after captopril was discontinued.


Pityriasis alba in a psoriatic location. Three patients with pityriasis alba whose lesions were confined to the knees only are reported. Such cases can be misdiagnosed as psoriasis. The key to the correct diagnosis lies in the physician's awareness of the existence of this variant of pityriasis alba.


Methotrexate hepatotoxicity in psoriatic patients submitted to long-term therapy. Eighty-four patients with severe psoriasis who required methotrexate (MTX) therapy have been reviewed. A total of 134 liver biopsies were performed. The lack of correlation between alcohol intake and the use of potential hepatotoxic drugs with pretreatment liver biopsies is noted. A review of 30 patients who had liver biopsies performed before and after MTX treatment showed no statistically significant difference between the histological grades before and after treatment. Nor was there any absolute correlation between the cumulative MTX doses and the histological changes of follow-up biopsies. In this group of patients, 15 (50%) developed fibrosis, which was severe in 2 patients, after 3,431 mg MTX average dose. Cirrhosis was observed in 3 patients (10%) after 1,667 mg MTX average dose. Follow-up liver biopsies are recommended for patients treated with MTX.


Elimination diet in young children with atopic dermatitis. Thirteen children with severe current atopic dermatitis unresponsive to topical treatment were started on an elimination diet. One child was excluded because she could only keep to the diet for 3 days. Twelve children aged 0.8-4.1 years maintained the diet for 2-4 weeks. In six children the dermatologist's score showed a clear improvement while on diet, in 2 children there was a minor improvement and in 4 children the dermatologist's score did not change during elimination diet. Challenges were performed with egg, milk and wheat in 6 children and with milk and wheat in 2 children. The challenges were done in an open way except for the dermatologist, who was unaware of which food the child had received. No child in the study had an immediate reaction but 3 children had late reactions, one after egg, one after milk and one child after challenge with wheat.


Interferon-alpha therapy in atopic dermatitis. Thirteen patients with a severe adult form of atopic dermatitis (AD) received 3.0 x 10(6) IU of recombinant interferon-alpha 2a (rIFN-alpha 2a) 3 times a week. A satisfactory response was obtained in 5 of them. Serum IgE levels in all 13 patients remained unchanged throughout the study. Flu-like symptoms were common, but clinical or laboratory adverse effects were otherwise slight. The moderately beneficial therapeutic effects observed in this study support a possible role for IFN-alpha in controlling immunologic deficiencies in atopic dermatitis.


The genetic risk for alopecia areata in first degree relatives of severely affected patients. An estimate. Substantial evidence indicates that genetic factors may have a role in the etiology of alopecia areata (AA). Most studies, however, provide only general information on the familial incidence but fail to specify family relationships. We therefore obtained information on the incidence of AA in first degree relatives of 348 severely affected patients. In 7% one of the parents was affected. Among the siblings of the patients 3% had developed AA, while AA was present in 2% of the children. Taking into account the age of the children, their lifetime risk was calculated to approach 6%. However, a severe type of AA is to be expected only in about 2% of the children. The degree of involvement observed in the patients did not influence the frequency and type of AA present in their first degree relatives.


A new case of Zimmermann-Laband syndrome with atypical retinitis pigmentosa. This paper reports a case study of a 10-year-old girl exhibiting symptoms of a Zimmermann-Laband syndrome (ZLS), including an ocular involvement not previously observed. In addition to the case reported, we have also discovered 21 patients described in the literature. Major clinical findings, defined as being present in more than 75% of the cases under discussion, are presented.


The relation between lichen planus and hepatitis C: a case report. A case of simultaneous occurrence of lichen planus (LP) and hepatitis C in the same patient is presented. The patient had received treatment with interferon alpha for her chronic liver disease, and the association between LP, hepatitis C and interferon alpha treatment is discussed.


Adjuvant treatment of recalcitrant genitoanal warts with systemic recombinant interferon-alpha-2c. Seventeen male patients with recalcitrant genitoanal warts, who had been unsuccessfully treated with classical destructive modalities for 16 months on average, were included in an open uncontrolled trial. The treatment regimen consisted of caustic and/or surgical measures as judged optimally suited in the individual cases, combined with an intermittent systemic low-dose adjuvant interferon-alpha-2c regimen (3 or 6 5-day-courses with intervals of 2 weeks) followed by a 1-year-observation period. At the end of interferon treatment, no patient had clinically visible warts but 10 still had subclinical acetic acid positive lesions. At the end of the 1-year-observation period, clearance of both warts and acetowhite lesions was observed in 4 patients (23.5%), whereas acetowhite lesions persisted in 4 others (23.5%). Recurrence of clinically visible lesions, always within the acetowhite areas, was observed in 9 (53%) patients. Interferon may thus have been effective in suppressing clinical recurrences of genitoanal warts, but its potency to eradicate subclinical papillomavirus infection was disappointing.


The route of rapid access of drugs to the distal nail plate. It has recently been shown that antimycotic drugs have unexpectedly rapid access to distal nail, which we have suggested occurs through the site of continuous ventral nail formation along the nail bed. To exclude the alternative possibility of diffusion through the nail plate, we have measured the effect of topical terbinafine cream in onychomycosis. Sustained outward movement of the fungally affected distal segment was seen in only 2 of 10 measured nails, and there was no change in the mean severity of clinical involvement in 53 nails under study. This excludes significant diffusion of drugs through the nail plate, and we conclude that the route of rapid access is indeed through the nail bed.


E rosette formation in B cell chronic lymphocytic leukemia. We describe a patient with B cell chronic lymphocytic leukemia whose B lymphocytes showed the capacity to form rosettes with sheep red blood cells during prolymphocytoid transformation of the disease. In this case, rosette formation was found to be related to the presence of the classic E rosette receptor since the leukemic cells were recognized by the OKT11 monoclonal antibody. This feature was not shown at diagnosis. Evaluation of this phenomenon is discussed.


Aminopeptidase P: purification of a membrane-bound bradykininase from rat lung. Aminopeptidase P that hydrolyzes the Arg1-Pro2-bond of bradykinin was solubilized from rat lung microsomes using phosphatidylinositol-specific phospholipase C. The enzyme was purified 420-fold by chromatography on decylagarose (two steps), omega-aminodecyl-agarose and DEAE-Sephacel. A single stained band was observed following native gradient (4-15%) polyacrylamide gel electrophoresis. Dipeptidylaminopeptidase IV-like activity was also present in the final preparation and co-migrated with aminopeptidase P in the above gel system.


Immunolocalization of high molecular weight kininogen (HKg) and T kininogen (TKg) in the rat hypothalamus. Specific HKg immunostaining detected with antiserum against the light chain (LC) of HKg was restricted to SRIF neurons of the hypothalamic periventricular area projecting to median eminence (ME). Heavy chain (HC) immunoreactivity related to HKg and/or low molecular weight kininogen (LKg) was found in some other hypothalamic territories. Specific TKg was mainly associated with vasopressin in neurons of suprachiasmatic (SCN), supraoptic (SON) and paraventricular (PVN) nuclei. By direct RIA, hypothalamus was found to contain the highest level of TKg (10ng/mg protein) and after trypsin hydrolysis and HPLC separation of kinins, 10.3 pg BK and 7.3 pg T-kinin/mg protein.


Beta-blocker therapy in acute myocardial infarction: evidence for underutilization in the elderly. To assess the impact of patient age on the use of beta-blocker therapy in the management of acute myocardial infarction. The population studied consisted of 4,762 patients hospitalized with validated acute myocardial infarction in 16 hospitals in the Worcester, Massachusetts, Standard Metropolitan Statistical Area during the years 1975, 1978, 1981, 1984, 1986, and 1988. Logistic regression analysis was employed to control for relevant demographic and clinical variables in evaluating the independent effect of patient age as a determinant of receipt of beta-blocker therapy during the hospitalization. A consistent trend toward reduced use of beta-blocker therapy in older patients was demonstrated. After adjustment for demographic and clinical variables (gender; prior history of angina, hypertension, or diabetes mellitus; myocardial infarction characteristics; complications including congestive heart failure and shock; and use of digoxin and diuretics), odds ratios for receipt of beta-blocker therapy relative to patients less than 55 years of age were 0.61 for those 55 to 64; 0.52 for those 65 to 74; 0.36 for those 75 to 84; and 0.26 for those 85 or older. Analyses performed for each study year demonstrated results consistent with those for the overall study population. The results of this population-based study suggest that there are substantial opportunities for expanded use of beta-blocker therapy in elderly patients who have sustained an acute myocardial infarction.


Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data. Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle.


Rapid screening for taxanes by tandem mass spectrometry. A highly specific and sensitive method is described for determining taxol, cephalomannine, and baccatin III in crude plant extracts. Radical anions of the taxanes are formed by desorption chemical ionization, and a parent tandem mass spectrometric scan is used to recognize these compounds by their characteristic dissociations. The limit of detection of the individual taxanes in typical plant matrices is less than 500 pg when all three species are screened simultaneously. Because of the sensitivity of the method, extraction times can be shortened to 30 min and crude extracts can be examined at the rate of 6/h. Detection of all three taxanes extracted from a single Taxus cuspidata needle in a combined extraction/analysis time of less than 1 h is demonstrated.


Fiber-optic ammonia sensor for measuring synaptic glutamate and extracellular ammonia. A fiber-optic ammonia gas sensor designed for neurochemical applications is presented. Parameters evaluated in terms of effect on the steady-state and dynamic response of this sensor include the indicator dye, concentrations of indicator and total ammonia nitrogen in the internal solution, volume of the internal solution, structure of the gas-permeable membrane, and temperature. The final ammonia sensor responds over the concentration range from 7 to 3000 nM with a limit of detection of 7 nM and response times ranging from 2 to 5 min. Glutamate oxidase is immobilized at the tip of this ammonia sensor to provide a glutamate biosensor with a detection limit of 0.1 microM when operated at pH 7.8. In addition, this ammonia sensor is used to measure extracellular ammonia levels in perfused retinal and eye-cup tissue preparations. These measurements indicate a calcium-dependent, potassium-evoked release of ammonia during these depolarization conditions.


Priming doses of atracurium and vecuronium depress swallowing in humans. The administration of low doses of muscle relaxant may cause peripheral muscular weakness including difficulty in swallowing. In the present study, the effect of priming doses of atracurium and vecuronium on swallowing was studied. Sixty patients undergoing elective surgery under general anesthesia were divided randomly into four groups of 15 patients and received as a priming dose either vecuronium (10 or 15 micrograms/kg) or atracurium (50 or 75 micrograms/kg). Swallowing muscle activity was measured by electromyography using submental surface electrodes. Swallowing was initiated by administration of 0.3 ml distilled water through an oral catheter. Swallowing reflex was determined by measuring the latency time (i.e., time from water administration to start of EMG activity of glossal muscles). Swallowing activity was determined by integration of the EMG of glossal muscles during swallowing. Peripheral muscle strength was determined by hand grip strength. Swallowing reflex activity and peripheral muscle strength were measured before and 3 and 6 min after administration of vecuronium or atracurium. Latency time remained unchanged after any of the priming doses. Integrated EMG decreased significantly (P < .001) 3 and 6 min after all priming doses tested (42-75% of baseline value). Only after atracurium 75 micrograms/kg was the hand grip strength significantly decreased (P < .01). These results suggest that owing to its effect on swallowing, the priming dose should be used with caution.


Effects of intravenous dexmedetomidine in humans. I. Sedation, ventilation, and metabolic rate. Dexmedetomidine (DMED) is a highly selective centrally acting alpha 2-adrenergic agonist thought to provide significant sedation without appreciable ventilatory effects. This double-blind, placebo-controlled experiment evaluated four dose levels of DMED (0.25, 0.5, 1.0, and 2.0 micrograms/kg intravenously over 2 min) in 37 healthy male volunteers. Measurements of sedation, arterial blood gases, resting ventilation, hypercapnic ventilatory response (HVR), and metabolic rate (O2 consumption and CO2 production) were performed at baseline, 10 min after DMED infusion, and thereafter at the end of each subsequent 45-min period. DMED caused sedation resulting in loss of responsiveness in most of the subjects administered 1.0 and 2.0 micrograms/kg; sedation was evident for 195 min following 2.0 micrograms/kg (P < .05). Ten minutes following infusion of 1.0 and 2.0 micrograms/kg, PaCO2 had increased by 5.0 and 4.2 mmHg, respectively (P < .05), and 60 min following 2.0 micrograms/kg, VE had decreased by 28% (P < .05). The placebo group showed a progressive increase in the HVR slope (50% increase by 330 min following the infusion; P < .05). Overall, across all the DMED doses, the slope was decreased (P < .05) at all times after DMED. The calculated ventilation at a PaCO2 of 55 mmHg was decreased (39%; P < .05) 10 min following 1.0 and 2.0 micrograms/kg, returning to control values by 285 min following 2.0 micrograms/kg. O2 consumption increased 16% (P < .05) at 10 min following 2.0 micrograms/kg; CO2 production decreased (22% at 60 min). By 5 h postinfusion, both had returned to normal.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of intravenous dexmedetomidine in humans. II. Hemodynamic changes. Dexmedetomidine (DMED) is a novel clonidine-like compound known to have sedative, analgesic, and cardiovascular stabilizing qualities. DMED is a more highly selective alpha 2-adrenergic agonist than clonidine. This investigation examined the hemodynamic effects of four selected iv doses in consenting healthy male volunteers. In a randomized, double-blind, placebo-controlled trial subjects received 0 (n = 9), 0.25 (n = 6) 0.5 (n = 6), 1.0 (n = 6), or 2.0 (n = 10) micrograms/kg of DMED by infusion (2 min). ECG, heart rate (HR), arterial blood pressure (MABP), bioimpedance cardiac output (CO), and plasma catecholamines concentrations (CA) were monitored from 90 min before to 360 min after infusion. Plasma DMED concentrations were measured. DMED produced a maximum decrease in MABP at 60 min of 14%, 16%, 23%, and 27% for the 0.25, 0.5, 1.0, and 2.0 micrograms/kg groups, respectively (P < .05). At 330 min MABP remained below baseline by 8% and 17% at the two largest doses (P < .05). Both HR and CO decreased maximally by both 17% at 105 min. The two largest doses produced a transient (peak at 3 min lasting < 11 min) increased in MABP (16 +/- 2.5 and 24 +/- 10 mmHg, respectively; P < .05) with a concomitantly reduced CO (41%, 2 micrograms/kg; P < .05) and HR (22%, 2 micrograms/kg; P < .05), whereas systemic vascular resistance doubled. Even the lowest dose decreased CA immediately to values close to 20 pg/ml for 5 h. A 2-min iv infusion of DMED produced a transient increase in MABP and a longer lasting decrease in MABP and CA. These DMED doses were well tolerated in the healthy volunteers.


Ketamine as a probe for medetomidine stereoisomer inhibition of human liver microsomal drug metabolism. Medetomidine (MED) is a novel, selective, alpha 2 adrenergic agonist with potent sedative, hypnotic, and analgesic properties, currently undergoing evaluation as an anesthetic adjuvant. The pharmacologic effects of MED are stereospecific, due entirely to the D-isomer (DMED), whereas the L-isomer (LMED) is essentially inactive. DMED, a 4(5)substituted imidazole, has been shown to inhibit adrenal steroidogenesis and human liver microsomal alfentanil metabolism, reactions mediated by cytochrome P-450. The mechanism of MED inhibition of cytochrome P-450 is unknown. The purpose of this investigation was to determine the mechanism of DMED inhibition of human cytochrome P-450-mediated microsomal metabolism, using ketamine as a probe. Ketamine undergoes extensive hepatic biotransformation and has been used previously to characterize the effects of imidazole anesthetics on human P-450-catalyzed drug metabolism. Ketamine N-demethylation by microsomes from three human livers was measured by gas chromatography-mass spectrometry with selected-ion monitoring. DMED was a potent, competitive inhibitor of S(+) ketamine N-demethylation, with a Ki of 0.11-0.18 microM for the high affinity ketamine demethylase. The IC50 for DMED inhibition of therapeutic concentrations of racemic ketamine (10 microM) was 0.15 +/- 0.02 microM. Preincubation of DMED with microsomes and an NADPH generating system prior to ketamine addition had no additional effect on the inhibition of ketamine demethylase activity, thereby implicating the parent compound rather than a DMED metabolite as the inhibitory species. LMED, although pharmacologically inactive, had a greater inhibitory effect than DMED on racemic ketamine and ketamine enantiomer demethylation at therapeutic concentrations. Spectral studies showed that DMED interacted with microsomal cytochrome P-450 to elicit a Type II binding spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)


Non-beta-adrenergic-mediated peripheral circulatory hyperkinesia in hyperthyroidism. Systolic time intervals and brachial circulation, evaluated by pulsed Doppler in terms of arterial diameter, blood velocity and flow, and vascular resistance, were studied in 12 hyperthyroid patients and in 12 normal controls. In patients, arterial circulation was studied before and during mechanical exclusion of the hand, and hemodynamic measurements were repeated after beta-blocker treatment and after obtainment of euthyroid state. Compared with controls, patients had higher heart rate (P < 0.001), lower systolic time intervals (P < 0.05, P < 0.01), and higher blood velocity (P < 0.05). Beta blockade decreased heart rate (P < 0.05, P < 0.001) but did not change systolic time intervals and arterial circulation. Euthyroid state decreased heart rate (P < 0.01), preejection period (P < 0.01), and blood velocity (P < 0.01) and flow (P < 0.05). The decreases in velocity and flow before hand exclusion when euthyroid state was obtained were correlated with hyperthyroid values of velocity and flow respectively (r = 0.85, r = 0.90, P < 0.01, P < 0.001). Vascular resistance during hand exclusion was correlated negatively with serum T3 level during hyperthyroid and euthyroid states. Thus, thyroid hormones but not beta-adrenoreceptors participate in the peripheral hyperkinesia of hyperthyroidism.


Additive and synergistic pharmacologic inhibition of equine fibrinoligase (factor XIIIa*-like) biochemical activity. A selected group of pharmaceutical compounds were evaluated for the ability to inhibit the biochemical activity of fibrinoligase (coagulation factor XIIIa*) in pooled equine plasma. Criteria for the pharmaceuticals selected were based on the mechanism of the transglutamination biochemical reaction mediated by coagulation factor XIIa*. These criteria were complemented by recognition of the molecular configuration and chemical composition of amino acid residue side chains involved in the process of covalent fibrin monomer polymerization (cross-linking, transglutamination) mediated by this enzyme. Each pharmaceutical was evaluated individually and in combination with other potential coagulation factor XIIIa* inhibitors in an effort to detect additive and synergistic phenomenon. In this context, pharmaceuticals with a carbonylamide (eg, cefuroxime, Girard's reagent-P, prolinamide) were applied in concert with compounds with a terminal amine (eg, D-arginine, L-lysine) functional group. In concept, this method theoretically served to competitively simulate glutamine and lysine amino acid residues within strands of fibrin monomer substrate involved in phase I (carbonylamide) and phase II (terminal amine) of the transglutamination reaction (covalent fibrin monomer cross-linking). Halogen-dinitro and ethylene compounds were also evaluated because of their reported ability to inactivate enzyme systems dependent on an intact sulfhydryl group located at their biochemically active site (eg, cystine amino acid residue). This group of pharmaceutical compounds failed to inhibit the biochemical activity mediated by coagulation factor XIIIa* in equine plasma.


Graded work exposure to promote work return after severe hand trauma: a replicated study. Fifty-one patients with posttraumatic stress disorder after work-related hand injuries were placed on a graded work exposure program to facilitate return to work. These patients consisted of an initial group of 25 patients and a replication group of 26 patients. The program returned 92% of the initial group and 88% of the replicated group to work with their previous employers. At 6-month follow-up, 88% of the initial group and 80.1% of the replication group were still working full-time at the jobs to which they had returned. All of the patients not working with their previous employer at follow-up had appraisal/projected flash-backs, which have previously been associated with a 90% failure to return to work. This intervention was successful with 73% of the patients experiencing such flashbacks. In conclusion, graded work exposure was an effective treatment to promote return to work for patients experiencing significant psychological symptomatology after severe hand injury.


Polymorphism of class I alcohol dehydrogenase in French, Vietnamese and Niger populations: genotyping by PCR amplification and RFLP analysis on dried blood spots. The polymorphism of human alcohol dehydrogenase (ADH) can contribute to the explanation of the important ethnic differences towards alcohol metabolism. Its assessment at the genomic DNA level with a procedure, excluding labelled probes, consisting of PCR (Polymerase chain reaction) amplification on dried blood spots and analysis of allele-specific RFLP (Restriction fragment length polymorphism) profiles, is well adapted to extensive studies in population samples. It can emphasize the importance of ADH as a genetic marker of population. Three ethnic groups (French Caucasians, Vietnamese Orientals, Black Africans from Niger) were studied. ADH2 and ADH3 genotypes were in equilibrium according to the Hardy-Weinberg law. Important differences were noted in the distribution of ADH2 and ADH3 alleles.


Sulphasalazine inhibition of human granulocyte activation by inhibition of second messenger compounds. The effects of sulphasalazine on the production of second messenger compounds in human granulocytes have been characterised by various stimuli. The increases in cytosolic calcium, inositol trisphosphate, diacylglycerol, and phosphatidic acid (all important mediators of intracellular signal transduction) triggered by stimulation were inhibited by sulphasalazine. The metabolites 5-amino-salicylic acid and sulphapyridine were less potent inhibitors than the mother compound. It is concluded that sulphasalazine inhibits the synthesis of phosphoinositide derived second messenger compounds at the level of phospholipase C or its regulatory guanosine 5'-triphosphate (GTP) binding protein. Inhibition of phosphatidic acid synthesis was either due to the same mechanism, or to interaction with a phospholipase D regulating GTP binding protein.


Complement factor 2 deficiency: a clinical and serological family study. Inherited complement deficiencies are associated with a variety of connective tissue diseases. A family with inherited deficiency of complement factor 2 (C2) is described in which two family members with homozygous C2 deficiency developed cutaneous vasculitis and sicca syndrome. The other family members had heterozygous C2 deficiency and each member had the HLA-A25, B18, DR2 (w15) haplotype. The mother had seropositive rheumatoid arthritis. Further studies showed the presence of cryoglobulins, antibodies against endothelial cells, and anticardiolipin antibodies.


Tricyclic azaergoline analogues: synthesis, structural modifications, and pharmacological studies. As an extension of previous investigations on synthesis and dopamine autoreceptor activity of bicyclic ergoline analogues the tricyclic azaergoline analogues 9a and 9b were synthesized. Furthermore, the geometry of the aromatic beta-ethylamine moiety of 9a,b was modified by stereoselective construction of the cycloheptenyl fused pyrazolopyridine derivative 7 and the aminomethyl substituted tricycle 10. Binding affinity of these compounds at dopamine (DA) receptor sites was investigated employing rat striatum homogenate: The compounds reveal modest to weak, but selective binding to a dopamine D-2 receptor when it is labelled with the DA-autoreceptor agonist [3H]-SND 919. In vivo studies with mice showed that 7, 9a,b, and 10 affect their CNS activity.


Low density lipoprotein composition and oxidizability in coronary disease--apparent favourable effect of beta blockers. The oxidative modification of LDL in vivo may have an important role in atherogenesis. To determine whether LDL fatty acid, anti-oxidant composition and sensitivity to oxidation in vitro is different in subjects with established atherosclerosis we compared 20 men with angiogram proven coronary disease with 25 controls without clinical evidence of arterial disease. LDL-cholesterol, total triglycerides and LDL fatty acid composition did not differ significantly between the groups. LDL oxidation lag time and oxidation rate in coronary patients (132 min, 0.02 absorbance units/min) and controls (140, 0.017) were not significantly different. However coronary disease subjects taking beta-blockers had evidence for reduced LDL oxidizability (lag time 148 +/- 7 min; oxidation rate 0.017 +/- 0.002 abs units/min) compared with those not on beta-blockers (lag time 114 +/- 7 min, rate 0.025 +/- 0.003, P < 0.005). LDL beta-carotene was significantly lower in coronary patients (0.92 mumol/mmol LDL cholesterol; controls 1.58; P = 0.001). LDL alpha-tocopherol appeared lower in coronary patients (2.8 mumol/mmol LDL cholesterol; controls 3.3; P = 0.056) and was significantly lower in smokers (2.56; non-smokers 3.24; P = 0.04). LDL oxidation rate was negatively correlated with LDL alpha-tocopherol (r = -0.51, P = 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)


Carvedilol, a new antihypertensive, prevents oxidation of human low density lipoprotein by macrophages and copper. Growing evidence indicates that oxidized low-density lipoprotein (LDL) may promote atherogenesis. Therefore, inhibition of LDL oxidation may impede this process. Carvedilol is a vasodilating, beta-adrenoceptor blocking agent. As a new antihypertensive drug, carvedilol is unique by virtue of its potent antioxidant activity. Therefore, we tested the ability of carvedilol to inhibit the oxidation of LDL by either macrophages or Cu2+. Carvedilol inhibited LDL oxidation by macrophages in a dose-dependent manner, with an IC50 value of 3.8 microM, as assessed by a thiobarbituric acid reactive substance (TBARS) assay. Under the same conditions, propranolol showed only a mild inhibitory effect (IC50 > 100 microM), while pindolol, atenolol and labetalol had almost no effect. Carvedilol, at 10 microM, almost completely inhibited the macrophage-induced increase in electrophoretic mobility of LDL, while other beta-blockers at 50-300 microM had no significant effect. Carvedilol inhibited superoxide release from mouse macrophages, which correlated well with its inhibition of LDL oxidation. Carvedilol also inhibited Cu(2+)-induced LDL oxidation with an IC50 value of 17 microM, while all other beta-blockers were inactive up to 300 microM. These observations suggest that carvedilol might not only be an effective antihypertensive drug, but might also be effective in prevention of atherosclerosis.


HLA polymorphisms and evolution.

HLA polymorphisms and evolution. Polymorphisms within the human MHC are of interest to immunologists, for their functional significance, and to geneticists, as markers of populations. Here, Eleanor Riley and Olle Olerup describe how molecular analysis of the HLA complex is beginning to reveal the true extent of class II diversity, and demonstrate the use of this information to clarify the historical relationships between different human populations.


The pathobiology of HIV infection.

The pathobiology of HIV infection. The follicular dendritic cells (FDCs) of the germinal center are known to absorb antigens in the form of immune complexes and to express them on the cell surface for long periods of time. Here, Cecil Fox and Michele Cottler-Fox propose that, as a result of FDC binding of immune-complexed viruses, lymphoid organs are the major reservoirs of HIV, and that FDCs play a key role in infection of CD4+ T cells.


Sulfhydryl modification of E. coli Cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex. Differential chemical modification of E. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4'-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4'-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.


Hippocampal opioid peptides and seizures. We have employed a molecular biological approach to study the dynamic status of hippocampal opioid peptides in response to seizures elicited by different experimental models, such as electroconvulsive shocks (ECS) and amygdaloid kindling. Both ECS- and kindling-induced seizures triggered an initial large release of enkephalin and dynorphin, but produced opposite long-term effects on the biosynthesis of these two peptides, an increase of enkephalin, and a drastic decrease of dynorphin. Electrical stimulation of the perforant pathway produced differential changes of enkephalin and dynorphin, which were identical to those of ECS and kindling. This finding confirmed our hypothesis that the perforant pathway was responsible for the mediation of ECS- and kindling-induced changes in opioid peptide turnover. Strongest evidence indicating a role for opioid peptides in mediating the expression of seizure-related behaviors was found using the kainic acid model, where we saw that hippocampal enkephalin was essential to the expression of kainic acid-induced wet dog shakes (a preconvulsive shaking behavior). Furthermore, it was found that the granular-mossy fiber pathway of the ventral, but not the dorsal, hippocampus was essential for the expression of this shaking behavior. However, destruction of the granular-mossy fiber pathway potentiated the seizures and hippocampal cell loss induced by kainic acid. This unexpected, yet extremely interesting, finding not only distinguished the roles of the granular-mossy fiber pathway in mediating wet dog shakes vs. convulsive seizures, but also challenged the dogma that this granular-mossy fiber pathway is essential for the expression of limbic seizures.


Neurotransmitters and their receptors in human temporal lobe epilepsy. Patients with medically intractable temporal lobe epilepsy (TLE) undergo medial temporal lobectomy with hippocampectomy for one of two reasons. (1) A lesion (tumor or arteriovenous malformation) adjacent to, but not invasive of, the hippocampus, results in the removal of the lesion and adjacent hippocampus in order to ensure a tumor-free margin. This group will be referred to as tumor-related TLE (TTLE) patients. (2) The operation is performed when depth electrode recordings and other evaluative techniques point to the hippocampus as the focus of seizure initiation. This group will be referred to as cryptogenic TLE (CTLE) patients. Analysis of the hippocampi of these two groups of patients reveals that the TTLE hippocampus is quite similar to that of autopsy subjects in its chemical neuroanatomy. However, the dentate gyrus of the CTLE patients shows considerable morphological and cytochemical reorganization. This reorganization is characterized by a number of features. (1) There is a loss of granule cells which occurs either as a patchy loss and/or a thinning of the granule cell layer. (2) Remaining granule cells which contain dynorphin appear to produce recurrent collaterals into the inner molecular layer of the dentate gyrus. (3) In the subgranular region of the hilus (the polymorphic layer) there is a selective loss of interneurons immunoreactive for somatostatin, neuropeptide Y and substance P. (4) There appears to be an increase in fibers immunoreactive for somatostatin and neuropeptide Y which extend throughout the dentate molecular layer. Somatostatin fibers being less numerous than neuropeptide Y fibers (5). The distributions of a number of neurotransmitter receptors also show striking reorganization in the dentate gyrus of the CTLE hippocampus. (6) Second messenger systems protein kinase C and adenylate cyclase, and Na+, K(+)-ATPase activity, as determined by ouabain binding, is increased in the molecular layer of CTLE. This remodeling of the CTLE hippocampus may hold the key to the mechanisms of hyperexcitability of the granule cells in the hippocampus of this group, and consequently the generation of seizures. The removal of the hippocampus in CTLE patients results in good control of seizures, whereas removal of hippocampi that do not show such reorganization, in a group of patients classified as atypical CTLE patients, results in inadequate seizure control. These findings suggest a complex series of processes in converting the properly regulated granule cells into hyperexcitable ones.


Synaptic connections of seizure-sensitive neurons in the dentate gyrus. A selective loss of somatostatin- and neuropeptide Y-immunoreactive neurons has been reported in the dentate gyrus of rats with cerebral ischemia, following sustained electric stimulation, and in patients with non-tumor-related temporal lobe epilepsy. Three theoretical possibilities were tested that may explain why these neurons are more vulnerable than others, such as the cholecystokinin- and calcium-binding protein-containing cells: (1) the seizure-sensitive neurons are more involved in specific excitatory circuitry than are the seizure-resistant cells; (2) the somatostatin- and neuropeptide Y-immunoreactive neurons are less protected by inhibitory GABAergic inputs than cells immunoreactive for cholecystokinin; and (3) the seizure-sensitive neurons do not contain calcium-binding proteins. The present results of light and electron microscopic, single and double, immunostaining experiments and co-localization studies performed on the hippocampal formations of rats and non-human primates, support the idea that the calcium-binding protein content of a neuron defines its seizure sensitivity.


GABAergic septal and serotonergic median raphe afferents preferentially innervate inhibitory interneurons in the hippocampus and dentate gyrus. Postsynaptic targets of the GABAergic septohippocampal and the serotonergic raphe-hippocampal pathways were studied using anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with pre- and postembedding immunocytochemistry in the rat. Two types of afferents were labeled in the hippocampus and dentate gyrus from the medial septum-diagonal band of Broca complex, one with large diameter varicosities and another with smaller terminals. The former type was shown to be immunoreactive for gamma-aminobutyric acid (GABA), and to innervate predominantly GABA-immunoreactive interneurons. Subsequently, these target interneurons were demonstrated to include all subpopulations of GABAergic cells which could be visualized by antisera against parvalbumin, calbindin D28k, calretinin, cholecystokinin, somatostatin, neuropeptide Y and vasoactive intestinal polypeptide. These types of interneurons have different afferent and efferent connections, and thus participate in different inhibitory processes in the hippocampal formation. The other subcortical pathway, the serotonergic projection from the median raphe nucleus, was also shown to establish synapses predominantly with GABAergic interneurons both in the hippocampus and in the dentate gyrus. In contrast to the septohippocampal projection, this pathway did not innervate all types of GABAergic neurons. They selected a particular subpopulation, i.e. those which contain calbindin D28k, and ignored those which contained parvalbumin or the other neurochemical markers. This suggests a strong functional specialization among local inhibitory circuits, as well as among the subcortical afferents originating in the septum and raphe. These findings suggest that a mechanism by which numerically small afferent pathways may have a profound global effect on the electrical activity of the hippocampal formation is the selective innervation of local interneurons. These GABAergic inhibitory cells, in turn, control the activity of large populations of principal cells. The level of GABAergic inhibition determines the degree of population synchrony and influences N-methyl-D-aspartate receptor-mediated epileptiform burst-firing. Thus, the specific subcortical modulation of hippocampal inhibitory circuits may also have fundamental implications for epileptogenesis.


Behavioural, biochemical and histological effects of AF64A following injection into the third ventricle of the mouse. Behavioural, biochemical and histological effects were assessed following AF64A injected into the third ventricle of female NMRI mice. Doses from 3 to 7 nmol produced significant changes in behaviour, causing hyperactivity, reduced hole-board exploration, rotational behaviour in a symmetrical Y-maze corresponding to a loss of alternation, abnormal behaviour in a plus-maze task of fear/anxiety with markedly increased exploration of the open arms and finally deficits in passive avoidance responding and spatial orientation in a Morris-type water maze. In this latter test, a cue learning deficit was noted for the two highest doses only. No histological changes of consequence were observed up to 5 nmol. Beyond this dose, at 6 and particularly 7 nmol, necrosis of parts of the hippocampus and septum was apparent. ChAT and AChE activity were decreased in the hippocampus but not in the cortex although the decreases were smaller than generally reported for AF64A-treated rats. ChAT and AChE reductions correlated highly with hyperactivity in the open-field and to a lesser extent, with spatial learning deficits. Monoaminergic activity was also affected in the hippocampus, but not in the cortex, at 4 nmol and above. NE and particularly 5-HT and 5-HIAA levels were reduced although the rate of 5-HT turnover was unaltered. A highly significant correlation was obtained between 5-HT effects and the increased open arm exploration in the plus-maze task of fear/anxiety. The behavioural effects and biochemical changes lasted at least 8-9 weeks postop.


Differentiation of rat dentate neurons by morphology and electrophysiology in hippocampal slices: granule cells, spiny hilar cells and aspiny 'fast-spiking' cells. Intracellular recording and intracellular dye injection of single cells in the dentate region of rat hippocampal slices have been used to understand the different types of cells in the dentate and their possible functional organization. On the basis of combined electrophysiological and morphological data, the cells that have been sampled fall into three distinct groups: the granule cells, the spiny cells located in the hilus (the 'mossy' cell being the prototype), and the aspiny, 'fast-spiking' cells located throughout the region (many of which are likely to be GABAergic interneurons). Although there is some variability within each group, this variability is minor compared to the large differences between groups. To clarify these groups, each one is described first morphologically, at the level of the light microscope and histochemically, and then the three groups are described electrophysiologically, in terms of intrinsic electrophysiological characteristics, synaptic responses to perforant path stimulation, and possible roles in dentate circuitry. It is proposed that this apparent organization of neurons into three major classes be used as a starting point in our evolving understanding of the functional organization of the dentate region, and, in particular, the hilus. In addition, the possibility is raised that area CA3c cells of the hippocampus could be included in the dentate region as a fourth group. Together with the hilar cells, area CA3c could have the obviously important role of integrating the dentate circuitry with that of the hippocampus proper.


Purification and immunochemical studies of dipeptidyl peptidase IV from bovine kidney. DPP IV from bovine kidney has been purified and characterized by molecular mass, pI, pH optimum and sensitivity to inhibitors. Polyclonal antibodies to the enzyme protein were used to show the location of dipeptidyl peptidase IV in microvillar membranes of bovine kidney using immunogold labelling by secondary antibody. The same antibodies reacted to DPP IV in Western blots of rat, pig and bovine kidney tissue, but failed to recognise the enzyme in bovine brain. This suggests that dipeptidyl peptidase IV may exist as different organ-specific rather than species-specific proteins.


The influence of lymphocyte counts and disease progression on circulating and inducible anti-HIV-1 cytotoxic T-cell activity in HIV-1-infected subjects. To evaluate specific anti-HIV cytotoxic T-lymphocyte (CTL) activity in relation to basic clinical and laboratory parameters used to follow HIV infection. Lymphocytes from HIV-1-infected subjects with different clinical and immunologic features of HIV infection were tested for circulating and inducible anti-HIV CTL activity using autologous B-lymphoblastoid cells infected with recombinant vaccinia viruses expressing the HIV gag, pol and env genes as targets. Anti-HIV CTL were induced by stimulation with HIV-infected autologous lymphoblasts in vitro. We detected circulating anti-HIV CTL in asymptomatic subjects exclusively and found a significant association (P < 0.01) between CD8+ lymphocyte counts > or = 900/microliters blood and detectable levels of circulating anti-HIV CTL. Subjects with circulating anti-HIV CTL also had a higher mean CD8+ lymphocyte count than those without detectable circulating activity (P < 0.001), but there was no significant difference in mean CD4+ lymphocyte count. CD8+ human histocompatibility leukocyte antigen (HLA) class I-restricted anti-HIV CTL were induced in all HIV-infected subjects tested following stimulation with HIV-infected autologous lymphoblasts in vitro. In subjects without detectable circulating anti-HIV CTL, circulating HLA-DR+ cells contributed to anti-HIV CTL activity induced by stimulation with HIV or concanavalin A in vitro. Circulating anti-HIV CTL activity is associated with CD8+ lymphocyte counts > or = 900/microliters. Anti-HIV CTL retain proliferative and functional capacity following in vitro stimulation with HIV and interleukin-2 through all stages of HIV infection. Persistent inducible anti-HIV CTL activity in subjects with advanced HIV disease and without circulating CTL suggests impaired activation and/or proliferation of the CTL in vivo.


Detection of circulating p24 antigen-positive CD4+ cells during HIV infection by flow cytometry. To determine the amount of circulating CD4+ cells positive for intracellular p24 antigen during HIV infection, and to correlate the results with clinical, virological and therapeutic parameters. Data were obtained from 24 anti-HIV-negative subjects (controls) and 47 anti-HIV-positive patients classified according to clinical diagnosis, serum p24-antigen assay results, and antiretroviral treatment with zidovudine, using a modified flow cytometric assay for the detection of intracellular HIV p24 antigen (p24-FCA) in circulating CD4+ lymphocytes. The proportion of CD4+ lymphocytes positive for p24-FCA correlated well with HIV infection (1.685 +/- 1.902 versus 0.160 +/- 0.152 in controls; P < 0.001) and clinical progression [Centers for Disease Control (CDC) stage II: 1.310 +/- 1.187; CDC stage III 1.145 +/- 1.442; CDC stage IVA/C2: 2.335 +/- 2.112; CDC stage IVC1: 2.066 +/- 2.420]. The percentage of CD4+ cells positive for HIV p24-FCA was inversely correlated with an absolute peripheral blood CD4+ lymphocyte count (Spearman's rank correlation = -0.324; P < 0.05). However, there was no statistically significant difference between patients in presence (n = 27; 1.938 +/- 2.095) or absence (n = 20; 1.343 +/- 1.594) of serum p24 Ag. The variable linked most strongly to the detection of intracellular p24 in anti-HIV-positive patients was zidovudine treatment: the proportion of p24-FCA-positive CD4+ lymphocytes was significantly lower (0.825 +/- 0.910) in the treated patients (n = 25) than in the untreated patients (n = 22; 2.662 +/- 2.248; P < 0.001). Our results suggest that CD4+ p24 Ag-FCA is a rapid and easy test for the identification of the proportion of CD4+ lymphocytes with intracellular p24 Ag, and that it could be more appropriate than serum p24 Ag assay in evaluating disease progression and efficacy of antiretroviral treatment.


Determination of extracellular glutamate after local K+ stimulation in the striatum of non-anaesthetised rats after treatment with dopaminergic drugs--studies using microdialysis. The present experiments were performed in order to investigate the effects of dopamine(DA)ergic drugs on the concentrations of extracellular glutamate (GLU) in the striatum of non-anaesthetised, freely moving rats by using microdialysis and to get further information about the interactions between glutamatergic and dopaminergic pathways. GLU was determined after pre-column derivatisation with o-phthaldialdehyde by HPLC and fluorescence detection. For increasing the fraction of extracellular GLU which is of neuronal origin, an enhanced release of this neurotransmitter was evoked by 100 mM K+ administered via the dialysis probe. This stimulation was applied twice in each experiment, at the second time after administration of a subcutaneously (s.c.) given DAergic drug. For basal conditions, a perfusion fluid containing 148.2 mM Na+, 4mM K+, 1.2 mM Ca2+ was used, for conditions of stimulation with 100 mM K+ the Na+ concentration was reduced correspondingly. Activation of the D1 receptor with the selective D1 sector agonist SKF 38393 ((+/-) 1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol) 15 mg/kg) failed to influence the stimulated release of GLU, and neither a combination of the selective D2 antagonist (-)sulpiride (150 mg/kg) with the mixed D1/D2 agonist apomorphine (1 mg/kg), nor a combination of sulpiride (150 mg/kg) with SKF 38393 (15 mg/kg) were effective. Also the two selective D2 agonists quinpirole (0.5 mg/kg) or talipexole (50 micrograms/kg) had no significant influence on the release of GLU. The results suggest that DA receptor agonists have less effect on the K(+)-stimulated GLU-release than might be expected from in vitro studies or behavioral experiments (Kornhuber and Kornhuber, 1986).


Dopaminergic neurotransmission in somatodendritic and terminal areas of the rat brain: susceptibility to modulation by D1 and D2 receptors and to axotomy. We have investigated the influence of D1 and D2 dopamine receptor active drugs on dopamine (DA) release in substantia nigra (SN), striatum and limbic forebrain in intact and in hemisected rats in vivo. DA release was indirectly assessed as 3-methoxytyramine (3-MT) accumulation following monoamine oxidase inhibition by pargyline. Hemisection per se had no effect on the 3-MT accumulation in the SN. Neither, had SCH 23390, SK & F 28393, or cis-flupentixol any effect in the SN in intact animals or in the lesioned side in hemisected animals. SCH 23390 slightly increased the 3-MT accumulation both in the striatum and limbic forebrain, indicating a stimulatory action on DA release, but SK & F 38393 had no effect in these brain regions. A difference between the striatum and the limbic forebrain was that the effects of SCH 23390, and cis-FPX were almost abolished following hemisection in the limbic forebrain, but only partially reduced in the striatum. In summary, our data give further support for the concept that neither D1 nor D2 dopamine receptors have any pronounced influence on the DA release in the SN. The data also indicate operational differences in the feedback regulation of limbic versus striatal dopaminergic transmission.


Phagocyte activation in coronary artery disease. Recent studies suggest that granulocytes (PMNs) play a role in the pathogenesis of acute and chronic myocardial ischemia and extension of myocardial injury. Granulocytes can release a variety of molecules mediating tissue injury which act synergistically with other molecules and cells. The aim of our investigation was to evaluate the granulocyte function in patients affected by coronary artery disease (CAD) and during coronary angioplasty (PTCA). We studied 20 patients suffering from CAD. The PMN's aggregating activity was greater in the coronary sinus than in the aorta (P < 0.01). The increase in aggregating activity was evident in patients who were smokers: their cells release significantly lower quantities of leukotriene C4 (P < 0.025). In the 20 patients who underwent coronary angioplasty we analyzed superoxide release after stimulation with phorbol-myristate-acetate (PMA). The results showed a greater decrease of PMN's superoxide production in the coronary sinus than in the aorta (P < 0.05). In all patients affected by CAD we evaluated the PMN's expression of CD11b/CD18 membrane integrins. In these patients the increase in expression of CD11b/CD18 was statistically significant in comparison with the controls (P < 0.01). This increase in expression correlates with a higher aggregation (r = 0.87, P < 0.001). The potential role of leukocytes, oxygen radicals, leukotrienes and granulocyte enzymes in the pathophysiology of myocardial injury due to regional ischemia and reperfusion is an area of intense investigation. This paper presents studies carried out in vivo which have been instrumental in demonstrating the role of granulocytes as mediators of myocardial ischemia.


[RFLPs study of parental origin and mechanism of 3 cases with X chromosome structural abnormality]. In this study, we analysed the parental origin and mechanism of X chromosome abnormalities in 3 cases by using RFLPs on short or long arm of X chromosome as genetic markers. Their karyotypes were 46,X,dup(X)(p21); 46,X,del(X)(p11); 46,X,i(Xq). The results demonstrated that the dup(X)(p21) and the del(X)(p11) were of paternal origin and i(Xq) was of maternal origin. The dup(X)(p21) arose from an unequal sister chromatid exchange. The del(X) (p11) occurred through X chromosome breakage and deletion mechanism. The i(Xq) resulted from X chromosome centromere misdivision in oocyte.


HLA phenotype is a factor in determining rate of disease progression and outcome in HIV-1-infected individuals. HLA allele frequencies were examined for possible association(s) with the rate of disease progression and with the disease outcome (AIDS diagnosis) in a population of HIV-1-infected individuals. Certain alleles were associated with the relative rate of CD4+ T-cell decline. Association of particular alleles with several disease outcomes associated with infection was also observed. It is important to keep these two aspects (disease progression, AIDS diagnosis) separate when studying HLA in the HIV-1-infected population. Alleles that may play a role in the rate of virus speed by effecting the immune response may be different from those found to be associated with a particular disease. We feel that the only truly informative data, in this regard, can be generated from a relative precise determination of the time of infection (to study disease progression) and adequate numbers of individuals with specific diseases to study specific disease association. If such data can be generated we will have a much better understanding of the pathogenetic process(es) of HIV-1 infection.


Clonal analysis of T-cell responses to the HIV-1 envelope proteins in AIDS vaccine recipients. Both CD4+ and CD8+ CTL responses specific for the HIV-1 envelope proteins can be elicited in seronegative humans by candidate AIDS vaccines. The phenotype of the responding CTL depends upon the nature of the vaccine, with CD8+ CTL being found exclusively in recipients of live virus vaccines. Both types of CTL are active against HIV-1-infected cells in vitro. However, the potential efficacy of vaccine-induced CTL in preventing infection in vaccinated individuals exposed to HIV-1 is unknown and is likely to be dependent upon complex factors including lytic activity against divergent strains, cytokines produced, and the lysis of noninfected CD4+ T cells.


Modification of the circadian rhythm of onset of acute myocardial infarction by long-term antianginal treatment. To elucidate the mechanism of the circadian pattern of onset of acute myocardial infarction by examining the effects of prior antianginal treatment upon it. Retrospective analysis of clock time of the onset of acute myocardial infarction by linear modelling to define the circadian distribution of hourly onset rates and to examine the deviation of treated groups of patients from this distribution. Coronary care unit in a general hospital taking unselected acute admissions from a district of 0.9 million people. A series of 2231 patients with confirmed acute myocardial infarction. A major 24 h cycle and smaller 12 h and 6 h cycles were present in patients not taking antianginal medication. Onset rates varied twofold over the day, with maxima around 10.00 am and 10.00 pm. This pattern was unchanged in patients on prior treatment with regular nitrates, but in those who had been taking a beta blocker or a calcium antagonist the 24 h cycle was absent. These results are best explained by the shared property of beta blockers and calcium antagonists to reduce blood pressure and myocardial oxygen demand. The mid-morning peak of the onset of myocardial infarction is attributable to the physiological increase in sympathetic drive and cardiac work at that time. The data are not consistent with the triggering of the 24 h periodicity by fluctuations in coronary tone or haemostatic activity.


Onset and recovery of rocuronium (Org 9426) and vecuronium under enflurane anaesthesia. We have studied the onset, duration of action and recovery index of twice the ED90 of rocuronium (Org 9426) (0.6 mg kg-1) and of vecuronium (0.08 mg kg-1) in patients during enflurane anaesthesia. Rocuronium had a significantly shorter mean onset time of 1.8 (SD 0.4) min, compared with vecuronium 3.4 (0.8) min. Clinical duration (time for the first twitch in the train-of-four to recover to 25% of control) was similar for both drugs (29 (10) min vs 31 (12) min). Spontaneous recovery times (TOF ratio 70%) did not differ significantly between rocuronium (47 (10) min) and vecuronium (44 (11) min).


Effect of fentanyl on ventilatory resistances during barbiturate general anaesthesia. Fentanyl has been shown to increase the overall resistance to inspiratory flow of the ventilatory system (Rmax). Rmax is the sum of the airway resistance (Raw) and of the non-Newtonian resistance (delta R) which may result from the viscoelastic properties of the thoracic tissues, from inequalities of the regional time constants within the lung, or from both. A bronchoconstrictor challenge may increase the magnitude of variation in regional time constants. Thus, in order to describe the effect of fentanyl on the two components of Rmax, this study was performed, with the end-inflation occlusion method, during paralysis and mechanical ventilation in 10 normal men undergoing barbiturate anaesthesia for minor urological procedures. The patients were anaesthetized with methohexitone and paralysed with vecuronium. Before administration of fentanyl, delta R accounted for 56% of Rmax. Fentanyl 5 micrograms kg-1 elicited a significant increase in Rmax (+34.5%; P = 0.005) and a parallel increase in both Raw (+35.2%, P = 0.017) and delta R (+33.5%, P = 0.005). The increase in Raw, but not in delta R, was reversed by atropine, suggesting that the increase in these two components of Rmax was not linked. Thus fentanyl increased both components of Rmax, but the effects of fentanyl on Raw and delta R seemed to depend on different mechanisms.


New anticancer agents: taxol, camptothecin analogs, and anthrapyrazoles. Taxol, an agent with a unique mechanism of action, has been shown to be highly active in patients with refractory ovarian cancer and may well have significant activity in other malignancies such as breast and lung cancer. The camptothecin analogs, another unique class of agents, have demonstrated antitumor activity in phase I and II trials. Finally, the anthrapyrazoles are intercalating agents with clinical activity in breast cancer and a toxicity profile that may permit increased dose intensity using colony-stimulating factor support. While this review focuses on these three drug classes, a number of other agents with apparently unique mechanisms of antitumor activity and unusual dose-limiting toxicities are in earlier development. These include antimetabolites; inhibitors of DNA, RNA, or protein synthesis; differentiating agents; agents that inhibit tumor growth by binding to growth factors; and agents whose mechanism of action is best classified as unknown.


The calculation of molecular similarity: alternative formulas, data manipulation and graphical display. The use of electrostatic potential comparisons between molecules for the elucidation of structure activity relationships is now a well-established modeling technique. The Carbo and Hodgkin similarity indices are used extensively to make quantitative comparisons of this nature; yet their roots are found in the overlap of electron density distribution, with both formulas utilizing a product-based numerator. Two new similarity indices are suggested that calculate the electrostatic potential similarity using a difference-based numerator. The form of the new indices allows the creation of additional software functions that enhance the flexibility of similarity calculations and permit the creation of similarity maps. The general properties of these software functions and all indices are discussed and applied to a series of dopamine D2 receptor agonists.


[The role of carnosine in the function of soluble of guanylate cyclase]. The effect of carnosine on activation of human platelet soluble guanylate cyclase has been studied in 105,000 g supernatants and partially purified haem-deficient enzyme preparations. In the 105,000 g supernatant carnosine (1 mM) inhibited (by about 70%) the enzyme activation caused by sodium nitroprusside. In partially purified haem-deficient guanylate cyclase preparations the inhibition of enzyme activation by sodium nitroprusside was 86%; further addition of carnosine had no effect on the enzyme activity. The strength of the activating effect of protoporphyrin IX on partially purified haem-deficient guanylate cyclase did not differ from that for the 105,000 g supernatant; this stimulating effect did not change after carnosine addition. A conclusion is drawn that the inhibiting effect of carnosine on the ability of guanylate cyclase to be activated by sodium nitroprusside is due to the dipeptide interaction with the guanylate cyclase haem.


Ontogeny of gastric function in the pig: acid secretion and the synthesis and secretion of gastrin. Gastric acid secretion, gastrin-releasing peptide (GRP)-stimulated gastrin secretion and concentrations of somatostatin in gastric tissues were studied in sucking pigs (n = 48). In addition, gastrin concentrations in plasma and antral tissue were measured in fetal and sucking pigs (n = 66) from 22 days before birth (93 days gestation) to 36 days of age. From 3 days of age littermate pairs were treated twice a day with either saline (n = 20) or adrenocorticotropin [ACTH (1-24); n = 20]. Pentagastrin-stimulated acid secretion per unit stomach weight was 39 +/- 7 mumol H+/g/h at 0-1 day, increased to 194 +/- 15 mumol H+/g/h at 5-7 days and plateaued. Antral gastrin concentration was 0.14 nmol/g 10 days before birth and increased to 2.7 nmol/g at 5 weeks of age. Plasma gastrin was 25 +/- 2 pmol/l at 22 days before birth, increased to 102 +/- 14 pmol/l at birth and decreased during the postnatal period. Somatostatin concentrations were higher in antral than fundic tissues (p < 0.05) and remained constant during the postnatal period. Increased levels of glucocorticoids in plasma following ACTH treatment had no effect on the studied parameters except that it reduced basal (p < 0.07) and GRP-stimulated (p < 0.05) plasma gastrin concentrations at 6-7 days of age. Development of acid secretion and its gastric regulatory peptides in the pig is different from that in the rat in that it occurs at an earlier age and does not appear to be greatly influenced by elevated glucocorticoid levels from 3 days after birth.


Topographical changes in alpha power in medicated and unmedicated schizophrenics during digits span reverse matching test. The topographical distribution of alpha power reduction was compared in nine unmedicated schizophrenics (predominantly never-treated), 17 medicated schizophrenics, and 15 normal controls. The task involved four procedures: (1) listening to signal sound, (2) listening to digits for memorization, (3) after listening, and (4) listening to digits for recognition. The electroencephalograms (EEGs) during each procedure were analyzed with Fast Fourier Transformation and compared with EEGs at rest. While listening to the digits, medicated schizophrenics showed less alpha power reduction than normal controls and unmedicated schizophrenics. In addition, there were correlations found between the degree of alpha power reduction and medication dose, and score of chronic symptoms. These suggest that patients with different clinical backgrounds have differing cerebral activity.


SPEM impairment in drug-naive schizophrenic patients: evidence for a trait marker. Smooth-pursuit eye movements (SPEM) were assessed in healthy subjects and in drug-naive, chronic, and residual schizophrenic patients. SPEM gain was found to be decreased in all the schizophrenic patients who also exhibited a significant increase in the rate of saccades. The frequency of square-wave jerks was the same in schizophrenic patients and normal controls, suggesting that the primary abnormality in schizophrenic patients was a low gain rather than a defect of the saccadic system. Patients were retested 1 month later, and stability of gain was high even in formerly drug-naive subjects who had been treated for 1 month with neuroleptic drugs. Altogether these results confirm the conclusions of most previous studies, extend them to drug-naive schizophrenic patients, and favor the hypothesis that SPEM impairment is a trait marker in schizophrenia.


Origin of the ammonia used for mitochondrial citrulline synthesis as revealed by 13C-15N spin coupling patterns observed by 13C NMR. The sources of ammonia used by isolated, intact rat liver mitochondria in the production of citrulline have been investigated in situ using a novel methodology based on the analysis of 13C-15N heteronuclear couplings observed by 13C NMR. Isolated mitochondria from rat liver were incubated with ornithine, 13CO3H- and 15NH4Cl, using unlabeled glutamate or glutamine as alternative, intramitochondrial nitrogen donors. The production of (7-13C, 8-15N) or (7-13C, 8-14N) citrulline was determined in situ by 13C NMR and the relative proportions of 15N- and 14N-citrullines confirmed by high resolution 13C NMR analysis of the C-7 citrulline resonance observed in perchloric acid extracts prepared at the end of the incubations. The 15N fractional enrichment of the intramitochondrial NH3 pool was manipulated either by modifying the 15N enrichment of added 15NH4Cl, or by altering the concentration of the unlabeled nitrogen donors in the incubation medium. Fractional 15N enrichments measured in the N-8 nitrogen of the resulting (7-13C) citrulline closely paralleled those of the external 15NH4Cl with minor dilutions derived from the unlabeled nitrogen contribution from the alternative substrates. In the presence of 10 mM 15NH4Cl, 10 mM glutamate contributed 4% of the citrulline N-8 nitrogen. Under similar conditions, the contribution of nitrogen from 10 mM glutamine to N-8 citrulline was 6%. These results indicate that the primary source of ammonia used for citrulline synthesis by isolated, intact rat liver mitochondria is extramitochondrial, providing also an illustration of the use of 13C-15N spin coupling patterns observed by 13C NMR, as a new tool in the study of ammonia metabolism.


Autoimmune gastritis: tolerance and autoimmunity to the gastric H+/K+ ATPase (proton pump). The alpha and beta subunits of the gastric H+/K(+)-ATPase (proton pump) have been identified as the major molecular targets of parietal cell autoantibodies associated with pernicious anaemia and with murine experimental autoimmune gastritis (EAG) induced by neonatal thymectomy. Recent studies with EAG suggest that the mechanisms of peripheral tolerance and autoimmunity to extrathymic autoantigens are mediated by subsets of "regulator" and "effector" CD4+ T cells, respectively. The persistence of "effector" CD4+ autoreactive T cells in the periphery may be a direct consequence of the delayed developmental expression of the target autoantigen. We hypothesize that cytokines produced by the "regulator" T cells prevent the clonal expansion of the "effector" autoreactive T cells, and that neonatal thymectomy induces organ-specific autoimmunity in genetically susceptible individuals by the reduction of the "regulator" T cell population.


Inhibition of phosphatidylserine synthesis induced by a CD4 mAb, B66.6 in Jurkat T cells. A monoclonal antibody, B66.6, previously classified in the cluster of differentiation 4 (CD4), has been studied and compared with another CD4 monoclonal antibody, IOT4. It was found that B66.6 but not IOT4 was able to mobilize Ca2+ from intracellular stores in the Jurkat T cell line. Ca2+ mobilization was followed by a decrease in the extent of phosphatidylserine synthesis. In the presence of the phorbol ester, phorbol 12,13-dibutyrate, B66.6 induced interleukin-2 synthesis. Altogether, the results indicate that the CD4 monoclonal antibody, B66.6, mimics other T cell activators such as CD3 and confirm that the inhibition of phosphatidylserine synthesis in activated T cells follows the mobilization of Ca2+ from intracellular stores and is independent of the activation of the Ca(2+)-and phospholipid-dependent protein kinase C.


Transcription factors, translocations, and leukemia. The frequent occurrence of TF gene involvement in translocations associated with leukemia is remarkable, although not yet explained. The wide variety of TFs involved in these translocations and the different stages of cellular maturation argue against a unifying mechanism. Recombinases, active during B-cell and T-cell development, have been implicated in gene arrangements involving TCR genes and in the SIL/SCL rearrangement, which involves two genes not normally rearranged. However, other mechanisms must clearly be active in generating these molecular abnormalities and perhaps they relate to the multistep maturation and differentiation processes and continuous cell turnover seen in hematopoietic cells. The difficulties in obtaining human solid tumor samples may make it more difficult to identify translocations involving TF genes in solid tumors. Recently, the cytogenetic analysis of solid tumors has improved and specific cytogenetic abnormalities have been associated with specific types of tumors. With advanced techniques, such as fluorescent in situ hybridization (a technique that does not depend on cell growth) and PCR, abnormalities involving TF genes will be discovered. Abnormalities of TF genes, other than translocations, have been seen in a broad variety of nonhematopoietic malignancies. The p53 protein has been shown to bind DNA in a sequence-specific fashion and interact with a variety of DNA tumor virus oncoproteins. The broad range of cell types that harbor p53 abnormalities suggests that TF abnormalities will likely be implicated in many solid tumors. We have detailed several examples of how gene rearrangements that accompany chromosomal translocations in acute leukemia can alter the expression or activity of cellular TFs. Several translocations generate fusion RNA transcripts and fusion TF proteins with altered functional characteristics. Other translocations result in the expression of a gene not normally detectable in hematopoietic cells or alter the level of its expression, or affect the promoter usage or exon structure of the gene (Table 2). Studies are underway in many laboratories to characterize the biologic activity of these abnormal TFs and it remains to be proven that these molecular abnormalities are directly linked with leukemogenesis. The identification of abnormal fusion transcripts and proteins may allow specific therapies to be directed against "tumor-specific" DNA, mRNA, or protein targets. Therapeutic strategies based on antisense or ribozyme technology may be used to turn off expression of these genes and inhibit leukemia cell growth. Immunologic methods can also be used to direct therapy against the malignant cells.


Dyskeratosis congenita fibroblasts are abnormal and have unbalanced chromosomal rearrangements. Dyskeratosis congenita (DC) is a rare inherited disorder characterized by bone marrow failure, dystrophic changes in the skin and mucous membranes, and a predisposition to malignancy. The DC locus has been mapped to Xq28. The primary defect responsible for this disease remains unknown. We have studied four patients with this disease, three from one family and one from another. In all four patients, primary skin fibroblast cultures were abnormal both in morphology (polygonal cell shape, ballooning, and dendritic-like projections) and in growth rate (doubling time about twice normal). Fibroblast survival studies using four clastogens (bleomycin, diepoxybutane, mitomycin-c, and 4-nitroquinoline-1-oxide) and gamma radiation showed no significant difference between DC and normal fibroblasts. Cytogenetic studies performed on peripheral blood lymphocytes showed no difference between DC and normal lymphocytes with or without prior incubation with clastogens. However, bone marrow metaphases from one of three patients and fibroblasts from two of four patients (who were the eldest of the 4) showed numerous unbalanced chromosomal rearrangements (dicentrics, tricentrics, and translocations) in the absence of any clastogenic agents. Cell-specific differences and a higher rate of chromosomal rearrangements in the older patients appear to correlate with the clinical evolution of the disease. These findings suggest that the DC defect predisposes DC cells to developing chromosomal rearrangements.


Measures of tumor proliferative activity. Tumor proliferative activity, or labeling index, is of interest for gaining insight into the biologic properties of human neoplasm and for providing clinical information that might guide patient management. There has been an abundance of literature reporting experience with standard and newer techniques of measuring tumor proliferative activity. These methods are reviewed with emphasis on technical issues. Particular attention is paid to the development and use of monoclonal antibodies, Ki-67 and anti-PCNA/cyclin. These antibodies are readily available and relatively simple to use. The former has recently been shown to be of prognostic value in non-Hodgkin's large cell lymphomas. A number of studies suggest that indices using these techniques could be useful for a variety of carcinomas, soft tissue tumors, and tumors of the central nervous system.


Structural and functional analysis of oncogenes and tumor suppressor genes in adult T-cell leukemia/lymphoma shows frequent p53 mutations. The human T-cell lymphotropic virus type I (HTLV-I) is capable of inducing adult T-cell leukemia/lymphoma (ATLL). However, the long latency period between infection and development of ATLL, as well as the small fraction of the infected population that actually develops this disease, suggest that additional factors are involved in its pathogenesis. Therefore, we performed a molecular analysis of 10 cases of ATLL presenting in a nonendemic area that were shown to have HTLV-I sequences by polymerase chain reaction as well as clonal T-cell receptor beta gene rearrangements. We analyzed these cases for alterations in some of the oncogenes and tumor suppressor genes frequently involved in hematopoietic neoplasia. Specifically, we used a single-strand conformation polymorphism assay to determine the presence of mutations in the p53 tumor suppressor gene, as well as the K-RAS, N-RAS, H-RAS, and c-myc oncogenes. In addition, we studied the c-myc gene for rearrangements by Southern blotting and assessed expression of the retinoblastoma (Rb) and p53 genes by immunostaining. Analysis of the c-myc gene and the RAS family of oncogenes did not show any alterations. Also, the Rb gene was expressed in all cases analyzed. However, we found mutations of the p53 gene in 3 of the 10 cases and these results were confirmed by sequence analysis. In two of these cases, we showed by restriction fragment length polymorphism analysis of chromosome 17p sequences that the p53 mutations were accompanied by a loss of heterozygocity. Also, these mutations correlated with an altered pattern of p53 expression. Thus, mutations in the p53 locus may be a cofactor for the development of ATLL in some cases, whereas the c-myc, Rb, and RAS genes do not appear to be involved in these neoplasms.


Id expression during mouse development: a role in morphogenesis. We have characterized the spatial and temporal pattern of Id transcription during mouse embryogenesis. The Id gene encodes a helix-loop-helix (HLH) protein which can heterodimerize with the ubiquitously expressed HLH protein products of the E2A gene, and prevent them from binding DNA either alone or as a heterodimer with tissue specific HLH transcription factors such as the muscle determination gene, MyoD1 (Benezra et al., 1990: Cell 61:49-59). Since Id has been shown to be down-regulated during induced differentiation in several cell lines, it has been postulated that Id plays a general inhibitory role in cell differentiation (Benezra et al., 1990). In situ analysis of Id mRNA expression in the mouse embryo was performed in order to determine whether the pattern of Id expression is consistent with this postulate. A detailed study throughout the entirety of mouse postimplantation development reveals that Id is expressed upon gastrulation at very high levels in almost all regions of the mouse embryo and expression declines as embryogenesis proceeds. In skeletal muscle, in which the inhibitory action of Id has been established in tissue culture models (Benezra et al., 1990), Id and the HLH myogenic factors are expressed in a mutually exclusive manner suggesting that myogenic precursors do not express both types of HLH gene products. In addition, Id colocalizes both spatially and temporally with Hox-7.1, a murine homeobox gene which is associated with regions of high cell proliferation and positional fate assignment.


Neural transplantation: problems and prospects for therapeutic application. Successful demonstrations of behavioural recovery in a variety of lesion and mutant animal models have encouraged the application of neural transplantation to the alleviation of neurodegenerative disease. Apart from the continuing shortage of foetal tissue, the major problems to be resolved for successful application of neural transplantation to humans are: first, immune rejection of allograft tissue and its pathological consequences to both graft and host tissue; and second, the establishment of normal and extensive graft-host connectivity. Recent developments in transplant research are beginning successfully to apply a number of strategies to resolve these problems.