Protective effect of puerarin against myocardial reperfusion injury. Myocardial metabolism and ultrastructure. To ascertain the beneficial effect of puerarin on the myocardium against reperfusion injury, studies on myocardial metabolism and ultrastructure were made. Twelve dogs divided into two equal groups were placed on moderate hypothermic cardiopulmonary bypass, and their hearts were subjected to 140 minutes cold cardioplegic arrest and 60 minute reperfusion. In the control group, the hearts were perfused with crystalloid cardioplegic solution (CPS) every 20 minutes during arrest. In the treated group, the hearts received CPS containing puerarin (2 mg/kg). Myocardial oxygen consumption, lactate production, creatine phosphokinase (CPK) release, water content and ultrastructural alterations were determined before ischemia, during cardiac arrest and at reperfusion. The results showed that intermittent infusion of CPS containing puerarin significantly decreased myocardial lactate production during ischemia, as well as myocardial oxygen consumption, CPK release and water content during reperfusion. Under electronmicroscopy, the degree of ischemic damage judged by a scoring method was less pronounced in the puerarin group than in the control. The authors conclude that puerarin has protective effects on the function of hearts that have undergone long periods of arrest and reperfusion.


Electrophysiologic follow-up in dogs receiving intracoronary ethyl alcohol injection. 96% ethyl alcohol was injected into a diagonal branch of the left anterior descending coronary artery of 10 out of 12 anesthetized dogs, and saline solution was injected in the remaining 2. After chest closure, the dogs were subjected to ambulatory monitoring (H) during days 1, 2, 3 and 7. The electrophysiologic study (EPS) and signal-averaged electrocardiography. (SA-ECG) were performed before the injection and again at day 7 after injection. H failed in one dog which died of ventricular fibrillation. In the other 9 dogs that received alcohol injection, H showed frequent ventricular premature and ventricular tachycardia (VT) after injection; six of the 9 dogs sustained VT, which was not inducible by EPS. VT was not found in 2 control dogs receiving saline solution injection. The SA-ECG showed no ventricular late potentials in dogs receiving alcohol injection. Post-ablation ventricular arrhythmia (including VT) occurred after intracoronary ethyl alcohol injection in all dogs. Arrhythmogenicity markedly declined during the first 3 days and almost completely disappeared on day 7 after ablation. There was no evidence in favor of reentry as the mechanism of these arrhythmias. Enhanced automaticity was considered as the mechanism for ventricular arrhythmia.


Potential pitfalls in using DNA probes to counsel Duchenne and Becker muscular dystrophy families. Although the accuracy in prenatal diagnosis and carrier detection with DNA probes in families with Duchenne and Becker's muscular dystrophies is very high, various factors limit the accuracy of these probes in many families. We report four potential pitfalls, ie, intragenic recombination, genetic heterogeneity, germline mosaicism, and an evolving genetic defect encountered in a population of Duchenne and Becker families, and describe a strategy to incorporate these factors into genetic counseling.


Treatment of blepharospasm, hemifacial spasm and strabismus with botulinum a toxin. Thirty patients with blepharospasm, hemifacial spasm, strabismus and entropion were treated with botulinum A toxin giving satisfactory results. Rapid spasm relief, correction of strabismus and entropion were obtained. Only mild, transient and local side-effects occurred. The patients were followed up for 4-12 weeks with no recurrence. The clinical results show that local injection of a minute dose of botulinum A toxin in treating blepharospasm, hemifacial spasm, strabismus and entropion is a safe, effective and simple method of nonsurgical therapy.


Linkage between Rh blood group and autosomal dominant retinitis pigmentosa in ten Chinese families. Since Fei et al reported a tentative linkage between ADRP and the Rhesus(Rh) blood group in the unrelated Chinese families in 1987, additional individuals of these ADRP families have been typed for Rh. Further linkage analysis with the LIPED Program on the Rh data of the ten ADRP families showed a maximum summed LOD score of 2.01 at theta = 0.12, which is a further suggestion of linkage between ADRP and Rh. However, genetic heterogeneity certainly exists in these families. It is our presumption that ADRP in those families, which do not exhibit linkage to 3q markers, may map to the short arm of chromosome 1.


CT evaluation of malformed external and middle ear and its surgical correlation. This study evaluated the diagnostic value of high resolution CT (HRCT) for atresia of the external auditory meatus (EAM) and isolated middle ear malformation. CT scan of the temporal bone was done in 33 patients with such disorders and 5 patients with otosclerosis etc. were studied in the same way for comparison. Coronal HRCT clearly showed conditions of the atresia plate and malformation of the malleus, incus, abnormal course of facial canal and changes in the vestibular window. The axial HRCT is useful to demonstrate the articulations in between the malleus, incus, stapes and the cochlear window. All the HRCT findings were confirmed during operation. Axial and coronal HRCT for external and middle ear deformation is highly valuable for decision making and surgical planning.


Location of lectin UEA-I receptors in histological diagnosis of nasopharyngeal precancerous lesion. Three kinds of lectins (UEA-I, ConA, PNA) were used to study normal, dysplastic, neoplastic nasopharyngeal epithelium by lectin affinitive histochemical method. UEA-I (ulex europeus agglutinin-I) displayed membrane distribution in normal squamous epithelium, but most of nasopharyngeal carcinoma cells were negative. Notably, severe dysplastic epithelium (precancerous lesion) exhibited a strong membranous and cytoplasmic affinity, which contrasted sharply with the normal epithelium and carcinoma cells. The statistically significant difference in the content and distribution of lectin UEA-I in these three groups suggest that UEA-I is a hopeful marker for diagnosing precancerous lesion of the nasopharynx. However, PNA and ConA are of less diagnostic value.


Comparison of serum bactericidal activity of 4 fluoroquinolones in healthy volunteers. In vitro antibacterial activities (MBCs) and serum bactericidal activities (SBAs) of fluoroquinolones were compared. The MBCs were measured by the agar disk dilution test. The 40 tested strains belonging to 8 species were isolated from hospitalized patients. Against Gram positive cocci, Ciprofloxacin (CPLX) and ofloxacin (OFLX) were the more active and nefloxacin (NFLX) was less potent than the others. Against Ps. aeruginosa, CPLX had the highest activity, enoxacin (ENX) as well as OFLX had powerful activity, whereas NFLX was less active. All the 4 agents showed high activity against Gram negative bacilli. In the self-controlled, randomized crossover study, 4 fluoroquinolones were given to 10 healthy volunteers and then SBAs were determined using microdilution method. The peak SBAs of CPLX and OFLX were significantly higher than NFLX, and those of ENX were comparatively low, but there was no difference between ENX and OFLX against most of the strains tested. The percentages of peak SBAs greater than 1:8 of the 4 fluoroquinolones suggest that in the treatment of serious infections, CPLX and OFLX are more effective and ENX can also achieve high cure rate. The trough SBAs of the 4 fluoroquinolones suggest that the time interval of administration of CPLX and OFLX should be more than 8 hours, but increase of the dosage or shortening of the time interval between the administrations is recommended for ENX and NFLX.


Characteristic changes of DNA stemlines during hepatocarcinogenesis in rats. DNA stemlines of hepatocellular carcinoma (HCC) model of adult Wistar rats established by diethylnitrosamine were quantitatively measured by flow cytometry. The cancer-inducing course was divided into four stages, eg, precirrhosis stage, cirrhosis stage, early cancer stage and cancer progression stage. The advantageous DNA stemline of hepatocytes of normal adult Wistar rats was tetraploid (4C). It was from the cirrhosis stage that atavistic proliferation of hepatocytes with diploid (2C) DNA stemline started and replaced 4C hepatocytes, resulting in a new advantageous population. The features of early cancer stage were formation of HCC with 2C DNA stemline, whereas during the cancer progression stage, HCC cells with aneuploid (AN) DNA stemline presented the advantageous growth. The study clearly shows the change pattern of DNA stemline during the course of hepatocarcinogenesis from 4C-2C-AN, which is believed to be the biokinetic mechanism of the development and progression of HCC.


Prevalence of antibodies to hepatitis C virus in Chinese patients with viral hepatitis and hepatic failure. Anti-HCV assay with ORTHO kits was done in 100 blood donors and recipients and 374 cases of viral hepatitis, including 65 cases of fulminant, subacute and chronic hepatic failure. None of the 100 blood donors and recipients showed positive anti-HCV response. Anti-HCV was positive in 7.6% of the patients with chronic persistent hepatitis, 9.7% of the patients with chronic active hepatitis and 23.1% of the patients with liver cirrhosis. High prevalence of anti-HCV was observed in subacute hepatic failure (60.8%) and chronic hepatic failure (53.9%). Fifty-two (83.9%) of 62 anti-HCV positive cases were infected concurrently with HBV. The incidence of HBV replicating marker in patients with HCV or co-infected with HBV was lower than that of those with HBV alone. It is suggested that HCV might inhibit the replication of hepatitis B virus. The mortality rate of patients with anti-HCV positive hepatic failure was higher than that of those with HBV infection. Therefore, anti-viral therapy for anti-HCV positive hepatic failure should be considered.


Myeloid and erythroid hemopoiesis supported by human bone marrow fibroblasts in vitro. The passages 0 and 3 purified human bone marrow fibroblast layers (FLs) were established by long-term liquid cultures. After 12-day coculture of stromal. cell-depleted marrow cell suspensions with passage 0 FLs, 68.33 +/- 4.04% of the hemopoietic colonies adhered to FLs was myeloid in nature and the other 31.67 +/- 4.04% was erythroid. There were still CFU-E (colony forming unit-erythroid), BFU-E (burst forming unit- erythroid), and CFU-GM (colony forming unit-granulocyte/macrophage) among the nonadherent cells. The media conditioned by passage 0 (F0-CM) and passage 3(F3-CM) FLs stimulated the growth of myeloid and erythroid (BFU-E) colonies. From these data, it is concluded that both FLs and the media conditioned by fibroblasts can stimulate myeloid and erythroid hemopoiesis.


Effect of gluco- and mineralocorticoids on gene expression of atrial natriuretic peptide by rat atria in vivo. The effect of gluco- and mineralocorticoids on atrial natriuretic peptide (ANP) gene expression as well as plasma sodium and potassium concentrations was studied in both intact and adrenalectomized Wistar rats. Atria total RNA was extracted with cold phenol method and hybridized to alpha-32P labeled rat ANP-cDNA probe. The ANP gene transcripts in intact rats receiving dexamethasone and in adrenalectomized rats receiving both dexamethasone and deoxy-corticosterone increased by twofold, while there were no significant changes in the other groups. There were no parallel correlations between the rise of corticoid hormones-induced plasma Na+ concentration and the enhancement of rANP-mRNA level. The results suggest that glucocorticoids, when used together with mineralocorticoids, may directly increase ANP gene expression, whereas administration of mineralocorticoids alone has no effect on ANP gene expression.


Surgical treatment of cervical spine tumors. Of 40 patients with cervical spine tumor treated from 1980 to 1990, 24 had a primary tumor and 16 a metastatic tumor. Thirty-four patients were operated on through anterior and 5 through posterior approach. Preoperative neck pain was relieved and muscular strength of shoulder and arm increased after operation. Total excision methylmethacrylate or metal prosthesis replacement was used. Total excision of the lesion was emphasized for preventing the recurrence of the tumor. Indications and aims of the operation are discussed.


Oculomotor neuropathy syndrome. A diagnostic challenge in nasopharyngeal carcinoma. Of 22 patients with different kinds of oculomotor neuropathy syndrome (ONS), 18 were initially suspected of suffering from nasopharyngeal carcinoma (NPC). However, in a series of evaluations, their diagnoses eventually proved to be other diseases such as cranial neuritis, aneurysm of intracranial internal carotid artery, chordoma, etc. The remaining four patients initially diagnosed as having aneurysm of skull base or pituitary lesion were actually NPC sufferers. Therefore, one should be very careful in differentiating NPC from many other diseases contributing to the similar manifestation of ONS.


Evaluation of penetrating keratoplasties with long-term cryopreserved corneas using simplified method. Since March 1986, the authors have studied and simplified the corneal cryopreservation technique using a self-made QL Bio-freezer. On the basis of successful animal experiments, penetrating keratoplasties with human cadaver corneas cryopreserved 9 to 571 days (averaging 131 days) were performed in 60 patients (61 eyes). The patients were followed up for 1.5 to 50 months. 41 of the corneal grafts remained clear, 9 became translucent and 11 cloudy. A total of 46 eyes had their visual acuity improved significantly. The results show that clinical use of corneal cryopreservation by the simplified two-step method is noteworthy.


Ultrastructures of the glial epiretinal membrane induced by activated macrophages. A rabbit model of glial epiretinal membrane was established following the injection of activated macrophages into the vitreous. The membrane was composed entirely of cells with glial characteristics, ie, abundant intermediate filaments, microvilli, junctional complexes and basement membranes. The extracellular matrix of the mature membranes contained collagen fibrils of 10 to 15 and 20 to 25 nm in diameter. Fusiform densities were seen adjacent to the cell membrane and cells with indented nuclei were found in thick membranes. These observations demonstrate that glial cells in epiretinal membranes may synthesize collagen and possess myofibroblast-like properties.


Functional compartments in the portal system. An experimental study. The pressure of the portal trunk, the left gastric vein, the left and right gastroepiploic veins, the mesenteric vein and the abdominal aorta was measured experimentally at the same time in 10 normal dogs. The levels of the raised pressure in the different compartments with their outflow tracts obstructed respectively were observed and the potentials of the functional barriers between these compartments were quantitatively evaluated. The findings showed that the average power of the functional barriers was 45.1 +/- 5.7% between the different compartments in the gastrosplenic area and 74.3% in the mesenteric region. The weakness of the functional barriers' capacity limiting the collateral diversion between the lesser and greater splanchnic systems may be one of the causes making the distal splenorenal shunt lose its selectivity.


Syndrome of endemic arsenism and fluorosis. A clinical study. Sixty-five patients in Xinjiang with syndrome of endemic arsenism and fluorosis (SEAF) were investigated clinically from March 1982 to August 1989. SEAF is a kind of chronic syndrome resulting from the combined, harmful effects of two trace elements, arsenic and fluorine. Peripheral neuritis and cardiovascular changes were observed in this syndrome more often than in simple arsenism or simple fluorosis. The excessive quantities of these two trace elements in blood might have a synergic, harmful effect on the nervous and circulatory systems. No definite conclusion could be reached with regard to the morbidity of skin and visceral tumors in this series. The incidence of associated skin cancer was found to be 7.7% and an associated Grade II squamous cell carcinoma of the esophagus was encountered in one patient.


Biokinetics of tritium incorporation into the tissues of rats during continuous ingestion of tritiated water or tritium-labeled food. Wistar strain male rats were continuously given tritiated water or tritiated wheat as drinking water or food for 70 days. During the ingestion, the tritium incorporation into rat tissues was examined in both wet and dry samples of liver, kidney, testis and blood. The concentration of organically bound tritium (OBT) in dry tissues of rats exposed to tritiated water (HTO) and 3H-food (tritiated wheat) attained an equilibrium within 2-3 weeks after the exposure. The concentration of OBT in dry tissues of rats exposed to HTO also reached an equilibrium within 3-4 weeks after the exposure. However, rats exposed to 3H-food, except for the liver, such an equilibrium state was not reached in other tissues and the OBT concentrations increased gradually throughout the exposure. The relative concentrations of total 3H and OBT at the end of the chronic ingestion of 3H food (70 day), expressed in percentages of the total activity were 1 and 9 times higher than those in rats exposed to HTO, respectively. In both groups, OBT as well as total 3H was almost uniformly distributed among the tissues examined.


National epidemiological survey of blindness and low vision in China. According to the National Sample Survey of Blindness and Low Vision, the prevalence of blindness in China was 0.43%, resulting chiefly from cataract (41.06%), corneal diseases (15.38%), trachoma (10.87%), and glaucoma (8.80%); and the prevalence of low vision in China was 0.58%, of which the main causes were cataract (49.83%), ametropia/amblyopia (14.98%), trachoma (9.55%), corneal diseases (8.48%), chorioretinal diseases (6.27%), etc. Among children under 14 years of age, the leading cause of blindness and low vision was heredity (48.46%). Among elderly of 60 years and over, the leading cause of blindness and low vision was cataract (73.13%).


Treatment of hemophilia A by living mother-to-son splenic transplantation. First case report in the world. A case of severe hemophilia A was treated by living mother-to-son splenic transplantation. The recipient was a 9-year-old boy. After splenic transplantation, the level of blood factor VIII:C was increased, and no spontaneous hemorrhage occurred. The boy was followed up for over two years. This is a case of spleen allotransplantation with the longest function in the world.


Electrophysiological analyses of serotonergic actions on neurons in hypothalamic ventromedial nucleus in vitro: receptor subtypes involved and implications for regulation of feeding and lordosis behaviors. Previously, we have shown that serotonin (5-HT) can inhibit, excite, or biphasically inhibit and excite individual neurons of the ventromedial nucleus (VMN) in hypothalamic slices from female rats. In the present study, similar in vitro methods were used to further characterize VMN neurons responsive to 5-HT, and to identify the receptor subtypes involved in mediating these 5-HT actions. Results from a dose-response experiment indicate that increasing the dose of 5-HT can transform an inhibitory response into a biphasic or even an excitatory response. This indicates that modulation of 5-HT release in the VMN could alter the net response of the VMN to this transmitter. By comparing the actions of 5-HT with the effects of selective agonists and antagonists, the inhibitory action was found to be mediated predominantly, if not exclusively, by 5-HT1A receptors, while the excitatory action was mediated predominantly or exclusively through 5-HT2 receptors. There appear to be few, if any, 5-HT3 receptors in the VMN, and their functions are unclear. The inhibitory and the excitatory phases of the biphasic responses were not mediated together by a single receptor subtype but were mediated separately by 5-HT1A and 5-HT2 receptors, respectively. The presence of the biphasic response in a large proportion of neurons, therefore, indicates the coexistence of different subtypes of 5-HT receptors in many individual VMN neurons. The use of selective agonists and antagonists further indicates that the coexistence also occurs in neurons showing monophasic responses, and that the opposite actions mediated by the coexisting receptor subtypes can interact with each other. Therefore, changing the ratio of coexisting receptor subtypes could modify the net output of the VMN response to 5-HT. Together with behavioral studies by others, it emerges from our findings that the inhibitory action of 5-HT on VMN neurons is associated with, and may be responsible for, the stimulation of feeding and inhibition of lordosis, while the excitatory action is related and may lead to the opposite behavioral effects. Finally, with the coexistence in VMN neurons of two receptor subtypes that can mediate 5-HT effects on both feeding and lordosis, the VMN can serve as a substrate for 5-HT to coordinate these two behaviors.


Vasodilator and oxidant scavenger in the neurogenic pulmonary edema induced by cerebral compression. In vagotomized rats, cerebral compression (CC) produced marked increase in arterial pressure and pulmonary hemorrhagic edema (PHE). We studied the effects of a vasodilator and an oxidant scavenger to delineate the role of hemodynamic and permeability factors in this type of neurogenic PHE. Infusion of sodium nitroprusside at a dose of 5 micrograms/kg/min significantly reduced the CC-induced pressor response by 14% and the lung edema by 41%. A dose of 10 micrograms/kg/min blocked the pressor response by 51%, and completely prevented the lung injury. Dimethylthiourea (DMTU), a potent scavenger for oxidants such as hydroxyl radical and hydrogen peroxide, in doses of 300 and 600 mg/kg was pretreated 15 min before CC. Although DMTU was shown to block the permeability lung damage caused by phorbol myristate acetate (a neutrophil activator), this agent did not exert any effect on the CC-induced pressor response and lung injury. The data indicate that granulocyte-mediated oxidants such as hydroxyl radical and hydrogen peroxide do not appear to be involved in this type of neurogenic lung pathology. The results support the concept that PHE induced by intracranial hypertension is initiated by hemodynamic changes in the systemic and pulmonary circulation. Hydrostatic effect plays a major role in this type of neurogenic lung pathology.


Effects of intra-raphe injection of amino acids on cardiovascular function in rats. The effects of microinjection of an excitatory amino acid (glutamate, 10 micrograms) or several inhibitory amino acids (taurine 10 micrograms, GABA 10 ug or glycine 10 ug) into the dorsal raphe region on cardiovascular function were assessed in rats under pentobarbital sodium. Intra-raphe administration of glutamate, but not saline, caused an increase in the mean arterial pressure. By contrast, intra-raphe administration of taurine, GABA or glycine, but not saline, caused a decrease in both the mean arterial pressure and the heart rate. The glutamate-induced hypertension or both the hypotension and the bradycardia induced by taurine, GABA or glycine was antagonized by pretreatment with intra-raphe injection of a serotonergic receptor antagonist (1 ug cyproheptadine). In addition, the vasopressor and bradycardia responses to an intravenous dose of epinephrine (2.5 ug/kg) were assessed in saline-treated rats and amino acid-treated rats. Intra-raphe injection of glutamate produced a significant decrease in reflex bradycardia compared to the controls. On the other hand, administration of taurine, GABA or glycine into the dorsal raphe region led to an enhancement of epinephrine-induced bradycardia. Again, the reduction or the facilitation of the epinephrine-induced bradycardia following administration of these amino acids was antagonized by pretreatment with cyproheptadine. The results suggest that the serotonergic receptor mechanisms in the dorsal raphe region play a role in the elaboration or modulation of the cardiovascular responses to amino acids (including glutamate, taurine, GABA and glycine).


Antidiuresis and natriuresis induced by gastric distension in water-loaded rats. Antidiuretic and natriuretic responses were observed following gastric distension in anesthetized, water-loaded rats. An increase of intragastric pressure to 25, 30, 35, and 40 cm H2O of 2-minute duration respectively elicited 11, 35, 45, and 55% inhibition of water diuresis with concomitant, graded increase of sodium excretion by the kidney. These antidiuretic and natriuretic responses to gastric distension persisted and sustained the same intensity after acute bilateral cervical vagotomy, indicating that the sensory input conveyed from the gastric mechanoreceptors to central nervous system during gastric distension should involve afferent fibers other than vagus nerve. Rats with diabetes insipidus (D1) produced by hypothalamic lesions were also tested, but neither antidiuresis nor natriuresis in response to gastric distension was observed in these D1 rats whose neurohypophysis contained little antidiuretic hormone (ADH). In conclusion, gastric distension may induce liberation of ADH and probably other neurohypophysial hormone which affects renal handling of sodium and water in anesthetized, water-loaded rats.


Effects of thyroidectomy on the hematocrit and blood viscosity in ovariectomized rats. The hematocrit and blood viscosity were measured in ovariectomized (Ovx) rats following thyroidectomy or sham operation. Rats were thyroidectomized (Tx) or sham Tx immediately after ovariectomy. Three months later, Ovx, Ovx+Tx, and intact rats at proestrus stage were bled. Blood was collected by heart puncture in rats under ether anesthesia and heparinized. One month after the first bleeding, Ovx and Ovx+Tx rats were bled once again. the viscosity of whole blood and plasma was measured at various shear rates at 37 degrees C by means of a Mooney-Ewart viscometer. The hematocrit was determined by a microhematocrit centrifuge. The plasma osmolality was measured by a computerized micro-osmometer. Concentrations of plasma sodium and potassium were measured by a flame photometer. The concentration of plasma thyroid stimulating hormone (TSH) was measured by a radioimmunoassay. In Ovx+Tx rats, the plasma TSH concentration was higher, but the hematocrit and blood viscosity were lower than those in Ovx and proestrus rats. The levels of plasma sodium, osmolality and viscosity were not altered by ovariectomy and thyroidectomy. These results suggest that the lower blood viscosity in thyroidectomized rats is due to a reduction of hematocrit.


Influence of temperature on rheology of human erythrocytes. The effect of variation in temperature on the rheological properties of human red blood cells was studied by determining the cell deformation in response to aspiration pressure applied via a micropipette. The time history of the deformation was analyzed by the use of a Voigt viscoelastic model consisting of an elastic element (E) in parallel with a viscous element (eta). Viscosity values were obtained during the initial rapid deformation phase (phase I), the later slow deformation phase (phase II), and the recovery phase. With a rise in temperature from 0 to 45 degrees C, both E and eta values decreased. A thermotropic transition temperature was found at approximately 21 degrees C for phase I viscosity and at approximately 9 degrees C for viscosity in the recovery phase, but not for phase II viscosity. These responses to temperature variations provide insights into the viscoelastic properties of the erythrocyte membrane.


Cardiovascular effects of endothelin in chronic hypoxic rats. This study was undertaken to investigate the effects of endothelin (ET) on cardiovascular function in chronic hypoxic rats (HA) and sea level controls (SL). Catheters were placed in femoral and pulmonary arteries for measurements of mean systemic arterial pressure (Psa) and mean pulmonary arterial pressure (Ppa). The cardiac output was measured by thermodilution method. It is very interesting to find that intravenous injection of ET (0.2 and 1 nmol/kg) caused a decrease in pulmonary arterial pressure (from 44 +/- 2.6 to 36 +/- 1.9 mmHg, p < 0.05) which sustained for about 10 min while the systemic arterial pressure rise (1 nmol/kg) or no significant change (0.2 nmol/kg) in the HA rats. The depressor action of ET is mainly resulted from the drop of cardiac output in HA rats, since the pulmonary vascular resistance resisted to the action of ET in those chronic hypoxic rats could be due to a simple loss of reaction to ET or the release of vascular dilators which balance the ET vasoconstrictive action. This report also found indomethacin and saralasin did not involve the action of ET on pulmonary arterial depression.


[Combined use of glucose and rest period between noise exposure to reduce noise--induced hearing loss]. This paper reports the way which protects hearing from harmful effects of noise. 67 guinea pigs were divided into four groups, 8-9 guinea pigs in each group. The total time of noise exposure at 125 dB SPL white noise in each group was one hour. The control was exposed continuously for 1 hour. Group I was exposed to noise for 30 minutes after intraperitoneal injection of glucose at 2 mg/g body wt., then rest for 30 minutes and exposed for 30 minutes again. Group II was exposed continuously to noise for 1 hour after intraperitoneal injection of glucose at 2 mg and 4 mg/g body wt. Group III was exposed to noise for 30 minutes, rested for 30 minutes and exposed for 30 minutes again without glucose injection. The auditory brain stem response (ABR) was measured before, immediately, 3 days, and 7 days after noise exposure. The effect on hearing conservation was evaluated according to ABR threshold. We observed that the auditory threshold in group I was significantly lower than that of the control in the given time after noise exposure (P less than 0.05), but not so in group II and III. Combined use of glucose and rest period between noise exposure was able to decrease the threshold shift by 15 dB and 10 dB immediately and 7 days after noise exposure respectively.


[Studies on metabolism of a fungicide yekuling in rat]. The metabolism of a fungicide Yekuling was studied in rat after oral administration. Four metabolites were isolated and purified by means of reverse-phase HPLC and TLC. They were identified to be acetic acid, 1,3-dithiolan-2-ylidenehydrazide; pyruvic acid, 1,3-dithiolan-2-ylidenehydrazide; benzoic acid and hippuric acid with UV and MS. The latter was further confirmed by chemical synthesis. The experimental results showed that Yekuling was metabolized extensively in rat. Yekuling was hydrolyzed by amidase to produce benzoic acid and 1,3-dithiolan-2-ylidenehydrazide. The latter was further acetylated by N-acetyltransferase to form acetic acid, 1,3-dithiolan-2-ylidenehydrazide, or condensed spontaneously to form pyruvic acid, 1,3-dithiolan-2-ylidenehydrazide. Benzoic acid was further conjugated with glycine to produce hippuric acid.


[Biomechanics study during march with different military equipment of equal carrying load]. The purpose of the study is to select the best military equipment of equal carrying load by the index of biomechanics. Six healthy young men whose age varied between 19 and 25 years were volunteered, in the experiment. They were required to march 1.5 h, carried with different military equipment of equal weight, 25 kg, at a speed of 5 km/h. The centre of gravity of the body and its kinesic parameters were measured before and after the march. The frequency and length of pace were measured during the march. The results of the study indicated that the displacement of the body's centre of gravity carrying equipment I was the most evident, whereas the least displacement of centre of gravity of the body was that carrying equipment II. The frequency of pace during the march was reduced in all three carrying different kinds of equipment, the most evident being that carrying equipment I; the length of pace became bigger with those carrying equipment II and III; the speed of pace was decelerated using equipment I, but it was quickened with equipment II. The change of kinesic parameters have no significant difference among the three. In summary; equipment II conforms best to biomechanical principles, whereas equipment I is the worst.


[A study on lymphocyte subpopulations and immunologic status of female workers exposed to benzene]. Peripheral blood from 29 female subjects occupationally exposed to benzene for an average duration of 8.6 years were analysed with monoclonal antibodies (McAbs). Acid alpha-naphthyl acetate esterase (ANAE)-positive cells among lymphocytes and serum IgG, IgA and IgM levels were determined simultaneously. The results showed a decreased total number of lymphocytes, reduction of percentages and absolute numbers of both CD2+ and CD4+ cells, decreased CD8+ and CD57+ cell numbers without significant changes in their percentages, a decreased CD4+/CD8+ ratio, an increased CD20+ cell percentage without significant change in its absolute number and a decreased serum IgM level. ANAE(+)-cell percentages of lymphocytes were of marked positive correlation to CD2+ cell percentages. These results indicate that benzene can cause immunosuppressive effects to the exposed subjects and the analysis of lymphocyte subpopulations by means of McAbs may be useful for evaluating early adverse health effects on workers exposed to the toxicant.


[Experimental study of antagonizing effect of calcium and magnesium against fluoride toxicity in collagen]. SD rats were given fluoride 20 mg/kg for 14 days through a gastrointestinal tube, body weight growth depressed obviously, the ratio of the Liver and Kidney weight to body weight increased significantly, hemoglobin concentration dropped and morphology and metabolism of collagen all became abnormal. All these effects can be antagonised by giving calcium (100 mg/kg), magnesium (100 mg/kg) or calcium and magnesium combined (50 mg/kg each). When Ca and Mg is given combined, the antagonizing effect is even better.


[Inhibition of eight natural foods on mutagenic effect by aflatoxin B1 and extracts of fungi]. Eight natural foods were tested for their antimutagenic activities on AFB1, metabolic extracts of A. versicolor and A. ochraceus which induced mutagenic activity in TA 98 and TA 100 mutants. The tested substances were extracted repeatedly with acetone. The revertance induced by AFB1, metabolic extracts of A. versicolor and A. ochraceus were significantly decreased when the eight natural foods were added into the medium. The results showed that the eight natural foods had different degrees of anti-mutagenic effect in vitro, suggesting that anti-mutagenic substances were present in these natural foods. It is considered that these foods may be practically valuable in the chemoprophylaxis of liver cancer in men.


[The effects of kitchen air pollution on animals immune function]. The paper determined the activity of mitogenic response of T lymphocyte, NK cell activity and lymphocyte IL-2 production activity after the mice had been exposed to the polluted kitchen air. The results showed that these immune functions were inhibited in descending order in the mice exposed to pollutants from burning coal mixed with oily smoke.


[Multivariate analyses of causal factors included cooking oil fume and others in matched case-control study of lung cancer]. This paper reports the study of 1:1 matched case-control for eighty-three cases of primary lung squamo-carcinoma and seventy-one cases of primary lung adenocarcinoma from urban districts of Nanjing. The result of conditional analysis reveals that cooking oil fume is a common risk factor for lung squamo-carcinoma and adenocarcinoma. Its relative risk and population attributable risk (%) are 3.8138, 3.4466 and 51.56%, 60.99% respectively. Besides, smoking, family history of tumors, fuel types and keeping warm by coal stove in winter are selected as risk factors for lung squamo-carcinoma, while history of chronic bronchitis and family history of tumors are screened as risk factors for lung adenocarcinoma. Summary population attributable risk for all five factors of lung squamo-carcinoma and all three factors of lung adenocarcinoma are 99.47% and 76.65% respectively.


[A SOS induction test screening study for vegetables inhibiting mutagenicity caused by antineoplastic drugs]. Using mutational and anti-mutational synchronous in SOS inductest (+/- S9), We found that 7 out of 11 kinds of commonly eaten vegetables had the ability to inhibit mutagenicity caused by chemical drugs such as Mitomycin C, Bleomycinia, Fluorouracil, Cis-Diaminodichloroplatinum, Arabinosylcytosin and mustargen, They were garlic, green Chinese onion, onion, garlic bulb, tomato, cucumber and water radish. The other 4 lacking this ability were rape, chinese toon, ginger and asparagus lettuce stalk. We believe that our results can be helpful in the preparation. of cancer patients' diet, who are receiving chemotherapy and in the prevention of cancer.


[The confidence interval of micronuclei and chromosome aberration induced by radiation]. This paper proposes a revised method of determining confidence interval of population rate based on statistical theory. The confidence interval of population rate can be obtained accurately by using this method. In addition, it includes a discussion of confidence interval about some basic indexes of micronuclei and chromosome aberration induced by radiation.


Preparation and microscopic visualization of multicolor luminescent immunophosphors. The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acids. The optimal reagents were then applied to other phosphors which differ in spectral characteristics as well as in luminescence lifetime. These phosphors were ground to a size of 0.1-0.3 micron and proteins or other macromolecules were adsorbed to the phosphor particles to prepare conjugates of different physico-chemical properties. A time-resolved microscope, suitable for real time visualization of the time-delayed luminescence of the immunophosphors by the human eye, is described in detail. Since most phosphors require excitation with far UV light, a special fluorescence microscope allowing far UV excitation was developed for conventional visualization of the luminescence emitted by the phosphor. The possibility of multiple color labeling using various phosphor conjugates was demonstrated in a model system consisting of haptenized latex beads.


Quantification by laser scan microscopy of intracellular doxorubicin distribution. Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).


Image cytometric DNA analysis in human breast cancer analysis may add prognostic information in diploid cases with low S-phase fraction by flow cytometry. Measurements of DNA ploidy can be performed either with image cytometry (ICM) or flow cytometry (FCM); both methods provide independent prognostic information in primary breast cancer. The aim of the present investigation was to compare the two methods and to relate the findings to prognosis (median follow-up 42 months). Concordance in ploidy status (diploid, tetraploid, aneuploid) was obtained in 76% of the samples (168/222). When the fraction of S-phase cells (SPF) from FCM analysis was also taken into consideration, four different groups of samples were obtained (Flow I-IV), which were considered to correspond to the Auer classification (Auer I-IV) of DNA histograms obtained from image cytometry. Complete concordance between the two techniques now was 70% (155/222). Samples classified as Flow I (diploid or near-diploid with low SPF) and Auer I had a distant metastasis rate of 3/60 (5%), as compared to 62/154 (40%) for all other combinations of the Flow and Auer classifications taken together. Thus, the only findings of prognostic importance were that some samples were Flow I but not Auer I, or vice versa. These two groups represent 17 (7.7%) and 14 (6.3%), respectively, of the total number of samples, and had frequencies of distant metastasis similar to those of the other high-risk groups, namely, 7/17 and 5/14, respectively. In a multivariate analysis, flow cytometric S-phase value was a stronger prognostic factor than either the Flow and Auer classification. We conclude that when routine FCM DNA analysis is used, diploid or near-diploid samples with a low S-phase value should be reanalyzed with ICM.


Methods for topographical analysis of intra-nuclear BrdUrd-tagged fluorescence. The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k-nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd-tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience.


Eukaryotic DNA replication is a topographically ordered process. This paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd-labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature.


Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method. Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.


Differences between platelet and microparticle glycoprotein IIb/IIIa. Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).


Differential analysis of animal bone marrow by flow cytometry. A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.


Drop-delay measurement using enzyme-coated particles. A simple technique is suggested for the measurement of drop delay for flow sorting. While the flow cytometer was set to sort a fixed number of particles, the drop-delay setting was changed step by step, and at each step the HRP-coupled particles were sorted into a well of an immunoassay strip. Then the HRP activity of the sorted samples was revealed by routine methods. The maximum level of the enzyme activity shows the proper drop-delay setting. Determination of the drop-delay setting takes only a few minutes. The technique is independent of the type of flow cytometer and does not require any additional equipment.


Protooncogene expression in subpopulations of cells from leukemia patients. This report describes a method for preserving the light scatter patterns of cells in which myc and myb expression are being measured. Exposure of cells to 1% paraformaldehyde for 72 h prior to antibody staining for myc and myb proteins preserved the light scatter patterns. Using this method, myc and myb expression was found to be highest in lymphocytes and monocytes and lowest in granulocytes. The measurement of differences in the level of expression of these genes in subpopulations of leukemia cells obtained from individual patients is possible as is assessment of the levels of expression amongst normal and leukemia cells present in the same patient.


Flow cytometric analysis of peripheral blood erythrocyte chimerism in alpha-thalassemic mice. A rapid and reliable method for longitudinal studies on the degree of red cell chimerism following bone marrow transplantation of alpha-thalassemic recipient mice is presented. Blood obtained by tail clipping from transplanted mice was analyzed by measuring forward light scatter (FLS) distribution of red cells using a flow cytometer. Amplification and threshold of FLS were specifically adjusted. For flow cytometric analysis, the red cells needed to be suspended in hypotonic saline (103 mmol/l NaCl). Osmotic fragility testing showed that lysis of erythrocytes did not significantly influence the measurements. Flow cytometric measurement allowed for a rapid determination of the degree of red cell chimerism.


Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid. A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis-parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37 degrees C, with 5 microM for 60' giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37 degrees C while being examined in an Epics Elite flow cytometer equipped with a 325 nm HeCd laser, and parinaric acid fluorescence at 405 nm was measured over time. Addition of the oxidant tert-butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells with dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide. Pretreatment of the cells with the antioxidant alpha-tocopherol, 200 microM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without alpha-tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity. The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis-parinaric acid staining and could be excited by the latter using resonance energy transfer.(ABSTRACT TRUNCATED AT 250 WORDS)


Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry. The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.


Swine chromosomal DNA quantification by bivariate flow karyotyping and karyotype interpretation. Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the "DNA line" in swine identical to the human "DNA line." Estimation of the DNA content in mega-base pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D and E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H.


Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen. Five different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4 degrees C were 1) storage in buffer, 2) storage in 50% methanol, and 3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82-0.91, P less than 0.001 for all TAAs). The cell suspensions preserved at 4 degrees C all showed a significant increase in background fluorescence (P less than 0.05) and a reduction in the TAA specific fluorescence (P less than 0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week.


Quantitation of resistance of leukemic cells to cytosine arabinoside from BrdUrd/DNA bivariate histograms. The cytotoxicity of ara-C derives from an inhibition of DNA synthesis after incorporation of ara-CTP into DNA. The rate of DNA synthesis can be determined from the amount of bromodeoxy-uridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify the inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/DNA. Inhibition was evaluated in ara-C-sensitive and resistant cells after incubation with different doses of ara-C. An index of resistance to ara-C (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with ara-C to that incorporated in the absence of ara-C. In the ara-C-sensitive and resistant HL60 cells, a linear relationship between RI and log ara-C concentration was observed. Small numbers of slightly resistant cells in mixtures of ara-C-sensitive and resistant cells could be determined using this method, making it suitable for clinical use to test the resistance of leukemic cells to ara-C.


Calcein: a novel marker for lymphocytes which enter lymph nodes. Previous studies have identified unique cell surface antigens which are associated with the specific binding of lymphocytes to high endothelial venules (HEV). Evidence is presented in this paper which demonstrates that uptake of the fluorescent dye calcein by lymphocytes represents an additional marker for the lymph node homing subpopulation of lymphocytes. Calcein exhibits a characteristic ability to label lymphocytes differentially into two distinct populations, based on fluorescence intensity, that does not occur with three other structurally related, fluorescein-based dyes. In vivo lymphocyte migration studies revealed that cells displaying the "dull" fluorescence phenotype, although entering all lymphoid organs examined, preferentially homed to the lymph nodes, particularly the popliteal lymph node (PLN). By contrast, lymphocytes displaying the "bright" phenotype were essentially excluded from entering lymphoid organs, where entry is HEV dependent, but were observed entering spleen, where entry is HEV independent. Furthermore, a high proportion (76.5%) of lymphocytes displaying the dull fluorescence phenotype expressed the PLN homing receptor MEL-14. Based on these observations it is suggested that calcein uptake may be a marker for general membrane properties, such as fluidity and plasticity, essential for the passage of lymphocytes through HEV.


Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood. A flow-cytometric assay is described that can be used to determine the frequency and the DNA content of micronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes in mouse peripheral blood. Thiazole orange was used for discrimination between PCEs and NCEs, while Hoechst 33342 was used to detect micronucleated PCEs and NCEs. Up to 70,000 polychromatic erythrocytes can be analyzed in less than 10 min. This corresponds to 150-3,000 micronucleated polychromatic erythrocytes, 90-95% of which are true events as determined with a fluorescence microscope after sorting. Using X-rays as the inducing agent in dose-response experiments, a significant increase can be registered at doses of 0.02 Gy. It seems possible that the method will also allow the detection of clastogenic effects of other inducing agents at lower doses than previously possible.


Three-dimensional image processing for morphometric analysis of epithelium sections. The reproducible classification of poorly differentiated abnormal epithelium specimens is still a diagnostic problem. The computer-aided method described here improves the differentiation between benign and malignant epithelium specimens. Hematoxylin and eosin-stained sections of normal squamous epithelium, dysplasia, carcinoma in situ, and carcinoma were scanned in a TV microscope system and analyzed by means of image processing methods on a DEC 5000/200 workstation. From the 15-20 microns thick histological sections, 3-5 focus positions in steps of 1-4 microns were scanned. The segmentation of the cell nuclei was performed automatically by color analysis and geometric operations. For each nucleus the best focus level was selected and at this level the center of the cell was calculated. Graph theoretical methods were applied to analyze the morphometry of the epithelium specimens. The minimal spanning tree was computed in the three-dimensional (3D) space of the sections with the selected centers of the nuclei as vertices. The best feature found for discrimination of the specimens is the average length of all edges in a tree. In the two-dimensional (2D) analysis we had to accept an error probability of about 20% in differentiation of dysplasia and carcinoma. In contrast to this we differentiated normal squamous epithelium, dysplasia, and carcinoma with a correct classification rate of 100% in the 3D analysis.


Automated image detection and segmentation in blood smears. A simple technique which automatically detects and then segments nucleated cells in Wright's giemsa-stained blood smears is presented. Our method differs from others in 1) the simplicity of our algorithms; 2) inclusion of touching (as well as nontouching) cells; and 3) use of these algorithms to segment as well as to detect nucleated cells employing conventionally prepared smears. Our method involves: 1) acquisition of spectral images; 2) preprocessing the acquired images; 3) detection of single and touching cells in the scene; 4) segmentation of the cells into nuclear and cytoplasmic regions; and 5) postprocessing of the segmented regions. The first two steps of this algorithm are employed to obtain high-quality images, to remove random noise, and to correct aberration and shading effects. Spectral information of the image is used in step 3 to segment the nucleated cells from the rest of the scene. Using the initial cell masks, nucleated cells which are just touching are detected and separated. Simple features are then extracted and conditions applied such that single nucleated cells are finally selected. In step 4, the intensity variations of the cells are then used to segment the nucleus from the cytoplasm. The success rate in segmenting the nucleated cells is between 81 and 93%. The major errors in segmentation of the nucleus and the cytoplasm in the recognized nucleated cells are 3.5% and 2.2%, respectively.


DNA signal splitting improves detection and analysis of tetraploid populations. Detection of DNA tetraploid populations requires a high index of suspicion at the time of data acquisition and frequently requires subsequent off-line analysis for confirmation, including evaluation of the hypertetraploid region. To analyze these specimens, the flow cytometer operator must run all specimens with the G0G1 peak in low channels or rerun specimens in which tetraploidy is suspected with a lower photomultiplier tube (PMT) voltage or lower amplifier gain setting. Re-analysis may not be possible in specimens with few cells. A simple modification to the cytometer allows PMT signal splitting with simultaneous processing of the signal by two different amplifiers. This allows simultaneous acquisition of histograms optimized for both the hypotetraploid and hypertetraploid regions.


Molecular approach in spider mites (Acari: Tetranychidae): preliminary data on ribosomal DNA sequences. DNA sequence data were used to examine phylogenetic relationships between six species of economically important Tetranychidae mites: Eotetranychus carpini (Oudemans), E. pruni (Reck), Tetranychus pacificus McGregor, T. mcdanieli McGregor, T. turkestani Ugarov & Nikolski and T. urticae Koch. With primers directed toward conserved elements flanking the target region, the Polymerase Chain Reaction was used to amplify the ITS2 spacer of the ribosomal DNA molecule. The nucleotide sequence of a 300-bp fragment of the ITS2 was determined by direct sequencing and nucleotide divergence used for intra-generic comparison in mites. The resulting phylogenetic tree expressing interspecific relationships in genus Tetranychus agrees with morphological data. The study demonstrates the usefulness of the approach in the assessment of the systematics and evolution of the group.


An evaluation of the effectivity of the scrub technique in quantitative ectoparasite ecology. The effectivity of the scrub technique used in quantitative parasite ecology to collect ectoparasites from mammalian skins was evaluated. This was done by reprocessing already scrubbed skins with the alkali digestion technique to remove any remaining parasites. The skins of six adult impala (Aepyceros melampus) ewes were used in the study. The results showed that the scrub technique is variable in its effectivity, and on average removed only 30.2% ectoparasites from the skins. It was also shown that the scrub technique is unreliable and more labour intensive and time consuming than the digestion technique, and should preferably not be used in quantitative studies.


Preventing complications in the CAVH patient. Continuous arteriovenous hemofiltration (CAVH) is a recently developed extracorporeal technique for treating the fluid overloaded, critically ill patient. Severe complications can occur during this procedure, including clotting and malfunction of the filter, exsanguination, or fluid and electrolyte imbalance. The critical care nurse who understands the principles of filtration, the concepts of heparinization, and the care and maintenance of the large invasive lines required for CAVH can frequently prevent these complications.


Strategies for teaching decision making: discrepancies in cuff versus invasive blood pressures. With advanced technology the nurse has multiple assessment information to consider at once. Sometimes the data from different assessment modes conflicts and the nurse needs to evaluate the discrepancies to determine the most reliable assessment measurement. Teaching nurses clinical decision making is more difficult than before because of this increase in the amount and complexity of the data. These authors show how using an algorithm helps to teach decision making. They describe the use of an algorithm as a teaching tool for a course on evaluating the differences in cuff versus invasive blood pressure readings.


A comparison of family needs based on the presence or absence of DNR orders. This investigator found no differences between the needs of family members who selected "Do-Not-Resuscitate" (DNR) orders from those who did not. She suggests reevaluating the assumptions about families who select DNR.


Guidelines for selecting a conceptual model of nursing: focus on the individual patient. How do conceptual models help you in clinical practice? How can you select the best model to guide successful interventions for your critical care patients? These authors present three guidelines for selection of a conceptual model for your patient situation. Six case studies of patients requiring critical care nursing demonstrate the fit of the patient's problems and goals for care with particular conceptual models of nursing.


Evaluation of diabetic patients' home urine glucose testing technique and ability to interpret results. The attention of diabetes educators has focused on home blood glucose monitoring, with little attention being given to the educational needs of patients who choose to perform home urine glucose monitoring. This study assessed patients' urine glucose testing technique and ability to interpret test results. A questionnaire was developed and administered by clinic nurses to 100 adults with diabetes who perform home urine glucose monitoring. Patients selected the urine testing product they used at home and demonstrated the technique using a prepared glucose solution. The nurse recorded whether patients correctly performed each step in the procedure according to product instructions. Results indicated that 61% of patients did not perform the procedure correctly. Patients had difficulty reading package insert instructions, could not correctly read a clock, did not wait the correct amount of time before reading test results, and did not routinely keep written result diaries. This study demonstrates the importance of routine review of home urine glucose testing with the product used at home.


Adjusting insulins.

Adjusting insulins. The teaching of effective insulin adjustment is a formal process that benefits from being carried out in a standardized way. The unique methods outlined in this report have been taught to people with diabetes for over 8 years. Iterative in nature, the methods are safe and work to achieve specified blood glucose or HbA1c targets. They are designed to accommodate each individual's habits, recognizing that acceptance depends on adapting the medication to the life-style rather than vice versa. New technology was used to mediate insulin adjustments at home. Insulin adjustment of itself, however, is but one of five interdependent factors involved in successful self-management. These include (1) choosing sites of insulin injection; (2) choosing species of origin of insulins to be used; (3) reviewing life-style habits, including diet and exercise; (4) implementing dosage titration; and (5) follow-up. Lack of formalized insulin adjustment methods may be a major reason why many diabetes control programs fail to demonstrate significantly better metabolic control in their patients.


Pancreas transplantation: a University of Minnesota perspective. Because of various difficulties, transplantation is restricted to specific situations. Surgery is considered elective by many insurers, so costs are often borne by the patient. Surgical work-up includes a thorough evaluation of diabetes-related complications, tissue typing, psychological state, and social issues. Options of cadaver versus living related donors are explored. The surgical procedure is lengthy and the hospital stay long and intensive. Once a patient is ambulatory, education is begun to meet the extensive learning needs. After discharge, surgical follow-up focuses primarily on blood chemistries, medication doses, and surgical complications. Medical follow-up includes monitoring for infections and rejection as well as addressing long-term complications of diabetes. Studies of the effects of transplantation on diabetes complications are encouraging which, when coupled with improved surgical outcomes, may increase opportunities for transplant.


Comparison of first-generation and second-generation blood glucose meters for use in a hospital setting. This study evaluated and compared a first- and a second-generation blood glucose meter for precision, accuracy, and user preference. Two separate capillary blood glucose fingersticks were performed on 25 outpatients and 60 inpatients with diabetes. Samples were drawn for serum glucose determinations immediately following the capillary fingersticks. Comparison of the Accu-Chek II and Satellite G meters in the outpatient setting gave results similar to the reference laboratory's. When the meters were tested on inpatients, the blood glucose results were significantly higher than those obtained from the hospital laboratory. The Accu-Chek II was more precise than the Satellite G on both normal and high blood glucose samples. Nursing staff indicated preference for the Satellite G because of its quick testing time but not for other preference factors surveyed. Both meters provided more accurate assessments of blood glucose concentration than were obtained from the serum glucose samples routinely processed by our hospital laboratory. Use of a nonfluorinated tube and delayed separation of the sample with resultant glycolysis likely account for this difference.


A survey of patient preference for insulin jet injectors versus needle and syringe. Many studies have tested the insulin absorption rate and mechanical reliability of jet injectors. However, no published papers have dealt exclusively with patient preference for this method of administering insulin over a period of years. This paper reports the results of a survey done to determine if use of a jet injector for delivering insulin has an effect on acceptance of and adherence to a regimen of multiple doses of insulin. Over the past 15 years, the authors have instructed approximately 70 patients to use various jet injectors. A questionnaire was developed and sent to 75 patients. Of these, 42 completed and returned the questionnaire. Survey results indicate that even though some problems with the injectors were identified, 70% of those responding still preferred to take insulin by jet injector.


Photography as a tool in assessment and rapport development in clients with diabetes: research brief. Certainly no major conclusions or generalizations can be made from a pilot study of this size. However, as is the nature of pilot studies, needed modifications in methodology have been discovered. Also, it is obvious that rapport development may be evaluated with the MISS, and the use of a camera has proven to be a worthwhile assessment and intervention tool. The results of this pilot study would certainly warrant continuation with a larger study. Rapport development has not been studied in diabetes. Perhaps rapport may be at risk because of the lengthy provider-patient relationship and the need for repeated physician reinforcement of required medical regimens and some associated life-style changes. In this study, rapport appeared to increase slightly in the experimental (camera) group and decrease in the control (non-camera) group. From the patients' perspective, use of photography communicates that the health care provider cares about them as individuals, is interested in their life-styles, and is seeking methods to best integrate the treatment regimen into their individual life patterns. However, rapport development may deteriorate over time with individuals who have a chronic illness (as indicated by some decrease in pretest/posttest mean differences in both groups), thus emphasizing the responsibility of health care providers to actively work to preserve rapport. The camera may be one method to accomplish this goal. When treating an individual with a chronic illness, health care providers tend to view the patient and problem as one entity rather than as two separate components. The camera may help to enhance this separation and promote understanding of the individual by means of rapport development.


Effects of macrolide antibiotics on gastrointestinal motility in fasting and digestive states. The effect of macrolide antibiotics on gastrointestinal motility was studied in unanaesthetized dogs by using strain-gauge transducers. Midecamycin acetate, leucomycin, josamycin and acetylspiramycin, which are 16-membered macrolides, did not affect the gastrointestinal motility and no symptoms of gastrointestinal disorder were observed. On the other hand, erythromycin, roxithromycin, clarithromycin and oleandomycin, which are 14-membered macrolides, caused giant contraction and disorder symptoms. Moreover, it was found that the glycosidic linkages of the lactone ring were necessary for a structure to exert strong contractile activity.


Transfer of beta-lactam preparations created in Japan to lung tissue and the drug selection for chemotherapy of respiratory tract infections. Intraoperative transfer of eight beta-lactam preparations to lung tissues was investigated by one-gram one-hour intravenous drip infusion immediately prior to operation. The Japanese drugs used were piperacillin, cefotiam, ceftizoxime, cefuzonam, latamoxef, flomoxef, cefotetan and cefbuperazone. The serum peak level was highest with cefotetan, 104.1 micrograms/ml, followed by cefbuperazone, latamoxef, ceftizoxime, cefotiam, piperacillin, flomoxef and cefuzonam, in decreasing order. Except for cefuzonam, there was a correlation between the serum peak level and the human serum protein binding rate (r = 0.89). There was a correlation (r = 0.98) between the Cmax of normal lung tissue (alveoli) level and the serum peak level (Cmax), but no correlation between the former and the human serum protein binding rate. The tumour level was lower than that in normal lung tissue (alveoli), but the tissue level at the obstructive pneumonia area was higher. The Cmax of bronchiolar tissue level was highest with cefuzonam, followed by latamoxef. There was no correlation between the Cmax of bronchiolar tissue level and the serum peak level, human serum protein binding rate or the Cmax of lung tissue (alveoli) level. It is therefore presumed that the drug level in tissue of the acute pneumonia area can be determined from the serum peak level of the respective drug. An appropriate drug for chemotherapy may be selected from beta-lactam preparations which are effective against main causative organisms in acute respiratory tract infections. Cefuzonam and latamoxef are especially useful for chemotherapy in patients with acute bronchiolitis.


Microbiologic profile of ceftibuten, a new oral cephalosporin. The bactericidal activity of ceftibuten, alone and in combination with other drugs, has been assessed in vitro. The ability to induce a post-antibiotic effect (PAE), the rate of emergence of spontaneous resistant mutants, and the presence of Eagle's phenomenon were also investigated. Ceftibuten rapidly killed most Enterobacteriaceae, Haemophilus, Moraxella and Streptococcus species tested, including beta-lactamase-producing strains with over 90% cfu reduction after only 2 h. Using chequerboard and time-kill tests, ceftibuten was found to react synergistically with aminoglycosides, and to give an indifferent response with ofloxacin against a large number of Gram-negative aerobes and streptococci. Antagonism was never observed. Ceftibuten induced a PAE of approximately 1 h on S. pneumoniae. As expected, no PAE was observed with Gram-negative species. Spontaneous emergence of ceftibuten-resistant strains exposed to supra-MICs of the drug was rare (< or = 10(-8)) in all pathogens tested. No marked Eagle effect was detected when bacteria were treated with ceftibuten at concentrations exceeding 100-fold their MICs. This rules out the possibility that in vivo the high concentrations of ceftibuten reached in the urinary tract may hinder its excellent bactericidal activity.


Comparison of norfloxacin and pefloxacin in the prophylaxis of bacterial infection in neutropenic cancer patients. In this study the efficacy of norfloxacin and pefloxacin for the antibacterial prophylaxis of granulocytopenia was compared in cancer patients following cytostatic treatment. A total of 136 patients was randomly selected to receive either norfloxacin or pefloxacin. Nineteen patients remained afebrile in the norfloxacin group compared with thirty one in the pefloxacin group (p = 0.045). Twenty four microbiologically documented infections (twelve with and twelve without bacteraemia) occurred in sixty seven patients taking norfloxacin, and twelve in sixty nine patients taking pefloxacin (five with and seven without bacteraemia) (p = 0.015). Only one infection caused by Gram-negative bacilli was observed in the pefloxacin group compared with seven in the norfloxacin group (p = 0.019). In conclusion, both microbiological and clinical results showed pefloxacin to be a better antibacterial agent than norfloxacin for these patients.


Acute systemic and splanchnic haemodynamic effects of L-carnitine in patients with cirrhosis. L-carnitine (L-C) is a naturally occurring substance in mammalian tissues that has recently been proposed as a therapeutic agent in hepatic encephalopathy and liver steatosis. L-C also produces some acute, non-metabolic, haemodynamic effects that have not previously been studied in patients with cirrhosis. Therefore, the authors evaluated the acute effect of i.v. administration of L-C (30 mg/kg) on systemic and splanchnic haemodynamics in ten patients (L-C group) with chronic liver disease (Child-Pugh's class A: 4, B: 3, C: 3 patients) and a control group composed of ten patients with similar clinical characteristics (Child-Pugh's class A: 5, B: 2, C: 3 patients) who received placebo. Heart rate, cardiac index and pulmonary arterial pressure (measured by right heart catheterization) decreased slightly but significantly in the L-C group and the changes observed in stroke volume were highly correlated to the Pugh's score. Moreover, the hepatic venous pressure gradient (measured by hepatic vein catheterization) decreased significantly in the L-C group, whereas no changes occurred in the placebo group. The overall response to L-C was contradictory to that previously observed in animals and humans with normal liver function, and the extent seemed to depend on the severity of liver disease. The effect of the drug on cardiac index, heart rate and hepatic venous gradient could possibly be beneficial for patients with hyperdynamic circulatory condition and portal hypertension.


Phase I trial of cystemustine, a new cysteamine (2-chloroethyl) nitrosourea: an intrapatient escalation scheme. A trial of cystemustine, a cysteamine nitrosourea, was carried out on 34 patients with advanced malignancies at increasing dosage of the drug over a period of up to 190 days in seven or eight cycles. A partial response to treatment was obtained in three patients. A degree of haematological toxicity developed in a number of the patients.


[The effect of acute cellular rejection on liver function following orthoptic liver transplantation. Quantitative functional studies with the 14C-aminopyrine breath test]. To test the effect of acute cellular rejection on liver function as represented by cytochrome-P-450 enzyme activity, the 14C-aminopyrine breath test (ABT) was performed prospectively in 46 patients (31 men, 15 women; mean age 48 [15-66] years) who had undergone a total of 50 orthotopic liver transplantations. Routine biochemical tests were performed daily until the 30th postoperative day, while the ABT was done daily on days 1-10 and three times weekly on days 11-30, and liver puncture biopsies were obtained once weekly or more often if there was clinical suspicion of rejection. Histologically confirmed cellular rejection occurred within the stated period of observation in eight patients (five women, three men; median age 45 [18-59] years). Results of routine laboratory tests (transaminases, bilirubin, thromboplastin time), as well as bile-flow and body temperature, did not vary uniformly. On the other hand, results of ABT at the time of rejection showed a decrease in all patients by an average of 65% (P < 0.01). Changes in the ABT preceded those in other tests by 1-2 days in four patients, being the only measurable functional abnormality in one. All rejection episodes responded to glucocorticoid pulse-treatment (three times 1 g methyl-prednisolone). Using ABT results as criterion, liver function became normal after the glucocorticoid injection within 4-11 days. These data indicate that the ABT is suitable in the routine monitoring of transplant function, thus facilitating early diagnosis and controlled treatment of acute cellular rejection.


[Intra-arterial calcium provocation for the preoperative diagnosis of the location of an occult insulinoma]. Blood-sugar levels below 40 mg/dl were measured during syncope in two female patients (aged 59 and 73 years). Suspected organic hyperinsulinism was confirmed by a fasting test. Ultrasound examination and computed tomography failed to demonstrate an insulinoma. Coeliacomesentericography was then undertaken together with a selective intra-arterial calcium provocation test of the pancreas (0.4 or 0.5 mmol calcium in physiological saline was injected into the pancreas-supplying arteries--proximal and distal splenic, superior mesenteric and gastroduodenal). The insulin level was determined in simultaneously obtained hepatic venous blood. In case 1, the insulin level rose tenfold after calcium injection into the proximal splenic artery, indicating a process in the body of the pancreas. In case 2, a steep rise in insulin occurred after injection into the truncus coeliacus and the proximal and distal splenic artery, suggesting an insulinoma in the tail of the pancreas. The site of the insulinoma was confirmed in both cases at surgery. The adenoma was enucleated in case 1, removed by partial resection of the tail of the pancreas in case 2.--These observations show that occult insulinomas can be localized preoperatively by intraarterial calcium injection with measurement of insulin concentration in simultaneously obtained hepatic venous blood.


[The therapy of the heparin-induced thrombosis-thrombocytopenia syndrome with immunoglobulins]. A 51-year-old obese woman who had just undergone a second osteotomy for arthrosis of the hip joint was given unfractionated heparin, 7,500 IU subcutaneously three times daily, as thrombosis prophylaxis. Signs of fulminant pulmonary embolism occurred on the 16th postoperative day with a platelet count of 33,000/microliters. Suspected heparin-induced thrombocytopenia and thrombosis (HITT) was confirmed by platelet tests. When heparin had been discontinued immunoglobulin G was administered, seven times 5 g intravenously, in view of the immunological genesis of HITT. In addition thrombolysis treatment with streptokinase combined with phenprocoumon was undertaken, until satisfactory anticoagulation was achieved after 4 days. Platelet count rose to 136,000/microliters within 20 hours of the first immunoglobulin dose. Complete clinical normality was restored, scintigraphy showed no perfusion deficit in the lungs.


Imprinting of phosphoribosyltransferases during preimplantation development of the mouse mutant, Hprtb-m3. The measurement of the activity of the X-linked enzyme HPRT has been widely used as an indicator of X-chromosome activity during preimplantation development in the mouse. More recently, the concomitant measurement of the activity of the autosomally-encoded enzyme APRT has been used in an attempt to decrease the variability inherent in the measurement of enzyme activity from minute samples such as preimplantation embryos. In this study the use of the HPRT-deficient mouse mutant, Hprtb-m3, allowed the unequivocal identification of the parental origin of HPRT activity measured in embryos derived from crosses between wild-type mice, and mice which were homozygous or hemizygous for the Hprtb-m3 allele. Results were similar to those of a previous study, where oocyte-encoded HPRT activity accounted for about 10% of total HPRT activity at 76 hours post human chorionic gonadotrophin injection and the paternally-derived Hprt allele was shown to be transcriptionally active by the late 2-cell stage. In contrast to other studies, differential expression of the two Hprt alleles was detected during the preimplantation period, in embryos derived from crosses between wild-type and HPRT-deficient mice. Evidence was also found for the existence of an X-linked locus which influences the amount of APRT activity in the unfertilized oocyte. We propose that the expression pattern of this locus may be influenced by its parental origin.


Platelet-derived growth factor-A and its receptor are expressed in separate, but adjacent cell layers of the mouse embryo. The localized developmental expression of murine platelet-derived growth factor A (PDGF-A) was compared to that of its receptor (Pdgfra). Our in situ hybridization study included germ layers of primitive streak embryos, early axial structures (dermatome, myotome, sclerotome, floor plate), the skin and some of its derivatives (hair and mammary gland), the developing forelimb, the branchial arches and various sense organs (otic vesicle, olfactory epithelium and the eye). We report that PDGF-A and Pdgfra are expressed in separate, but adjacent cell layers in these structures and that in most, the ligand is expressed in the epithelium, whereas the receptor in the mesenchyme. This localization corresponds to classical experimental evidence for developmental interactions across cell layers. We suggest that the spatio-temporal regulation of PDGF-A and Pdgfra, and other related systems, represents one model for the spatial regulation of receptor-ligand interactions.


BHK-21-derived cell lines that produce basic fibroblast growth factor, but not parental BHK-21 cells, initiate neuronal differentiation of neural crest progenitors. We present evidence that basic fibroblast growth factor (bFGF)-producing cells stimulate primary differentiation of neurons from neural crest progenitors. Baby hamster kidney (BHK-21) cells were stably cotransfected with plasmid pSV2/neo, which contains the gene conferring resistance to the neomycin analog G418 and expression vectors containing the human bFGF cDNA. Various clones, which differed in their bFGF production levels, were isolated. Homogeneous neural crest cells were cultured on monolayers of bFGF-producing, BHK-21-derived cell lines. While the parental BHK-21 cells, which do not produce detectable bFGF, had poor neurogenic ability, the various bFGF-producing clones promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced by the different transfected clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental BHK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secretes high amounts of recombinant vascular endothelial growth factor (VEGF). Furthermore, the neuron-promoting ability of bFGF-producing cells could be mimicked by addition of exogenous bFGF to neural crest cells grown on the parental BHK line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neuronal cells, but not of neurons (see also Kalcheim, C. 1989, Dev. Biol. 134, 1-10). Taken together, these results suggest that bFGF stimulates neuronal differentiation of neural crest cells by a cell-mediated signalling mechanism.


Development of left/right handedness in the chick heart. The chick heart tube develops from the fusion of the right and left areas of precardiac mesoderm and in almost all cases loops to the embryo's right-hand side. We have investigated whether any intrinsic difference exists in the right and left areas of precardiac mesoderm, that influences the direction of looping of the heart tube. Chick embryos incubated to stages 4,5 and 6 were cultured by the New method. Areas of precardiac mesoderm were exchanged between donor and host embryos of the same stage and different stages to form control, double-right and double-left sided embryos. Overall, double-right sided embryos formed many more left-hand loops than double-left sided embryos. At stages 4 and 5 a small percentage of double-right embryos formed left-hand loops (13%) whereas at stage 6 almost 50% of hearts had left-hand loops. Control embryos formed right-hand loops in 97% of cases. The stability of right-hand heart looping by double-left sided embryos, may be related to the process of 'conversion', whereas the direction of looping by double-right sided embryos has become randomised. There is some indication that an intrinsic change occurred in the precardiac mesoderm between stages 5 and 6 that later influenced the direction of looping of the heart tube. The direction of body turning is suggested to be linked to the direction of heart looping.


Inhibin alpha- and beta A-subunit immunoreactivity in the chicken embryo during morphogenesis. Antibodies against synthetic peptides selected from the amino acid sequences of human inhibin alpha- and beta A-subunits were used to examine the distribution of inhibin subunit immunoreactivity in chicken embryos during the first week of development. Inhibin alpha-subunit immunoreactivity was localized in skeletal and smooth muscle myoblasts as well as developing cardiac muscle cells. In somites, immunostaining was seen exclusively in myotomes. The appearance of alpha-subunit immunoreactivity was correlated with myogenic differentiation; immunoreactivity was not seen in non-differentiated mesenchymal cells or in terminally differentiated adult muscle cells. In cardiac muscle, some immunopositive myocytes were seen also in the adult. In the adult heart, the Purkinje fibers were strongly immunoreactive, suggesting a possible role of the immunoreactive protein in the impulse-conducting function of these specialized cells. Inhibin alpha-subunit immunoreactivity was also seen in the visceral and parietal cells of the Bowman's capsule in both mesonephric and metanephric kidneys. In addition to mesodermal derivatives, alpha-subunit immunoreactivity was localized in neuroepithelial cells and axons in the developing central nervous system. Immunoblotting with anti-alpha(1-32) revealed two protein bands with M(r) values of 50,000 and 32,000 in cytosol samples of whole embryos under nonreducing conditions. In reduced samples an approximately 14,000 M(r) protein species was detected. Inhibin beta A-subunit immunoreactivity was detected only in chondrocytes, suggesting that the immunoreactive protein might represent a chicken homologue of the various cartilage and bone morphogenetic proteins expressed in mammals.


Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds: vibrissa-type fibres are specified. Adult vibrissa follicle dermal papillae have the capacity to induce hair growth and follicle formation when associated with epidermis from various sources. However, the range of conditions under which hair follicle induction will take place has not been established. The question of whether or not the adult papilla carries information to impose fibre-type specificity has also not been fully answered. This study describes how the implantation of isolated papillae into small incisional cuts on the rat ear pinna resulted in the subsequent emergence of abnormally large hair fibres from the wound sites. Many of these hairs were found to display vibrissa-type characteristics. Histological observations indicated that the papillae had interacted with the edges of the wound epidermis to produce new, and particularly large follicles, while immunohistochemical staining revealed that early follicle construction was accompanied by a profusion of the basement membrane constituents laminin and type IV collagen in the subjacent dermis. These findings show that adult rat papillae retain the capacity, as displayed by embryonic dermis, to determine vibrissa specificity in induced follicles.


Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle. It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.