Sequence analysis of the 5' flanking region of the human estrogen receptor gene.

We present the sequence of 2770 nucleotides of 5' flanking sequence of the human estrogen receptor (hER) gene. The positions of potential binding sites for a number of trans-acting factors including Sp1, OTF-1, INR, TATA and CAAT box factors as well as several half palindromic hormone responsive elements (HREs) have been mapped by comparison with the consensus binding sequences. A long alternating purine/pyrimidine (APP) tract which has the potential for structural diversity as indicated by site-specific cleavage with S1 nuclease is another feature of this region. The organization of this promoter region is compared to that of other cloned members of this family. The potential roles that these sequences may play in the transcriptional regulation of this gene are discussed.


Biphasic effect of ethanol on noradrenaline release in the frontal cortex of awake rats.

Ethanol elicited a biphasic effects on the extracellular noradrenaline (NA) concentrations in the rat frontal cortex, as assessed by microdialysis in awake animals. A low dose of ethanol (0.2 g/kg i.p.) raised NA output to about 160% of baseline levels. In contrast, a dose of 2 g/kg inhibited NA output to about 70% of pre-drug levels. These results suggest that the decrease in cortical NA output may reflect the sedative-hypnotic properties of ethanol at high doses, whereas the stimulation of extraneuronal NA may represent a biochemical correlate of the arousal and increased alertness elicited by low doses of ethanol.


Reports of physical symptoms and alcohol use: findings from a primary health care sample.

The relationship between alcohol consumption and physical health was examined in a primary health care sample of 366 adults. Unlike many previous studies that relied on static measures of medical diagnoses, the data reported here are repeated assessments of self-reported symptoms and alcohol use over 12 months. The results suggest, first, that drinking patterns in non-alcoholic samples fluctuate over time, and, second, that abstainers who have more prior illnesses or worse current health consistently report the greatest number of physical symptoms. The data highlight the importance of accounting for the health status of abstainers before comparing them with users of alcohol, and suggest that the presence of physical symptoms in addition to objective indices of health (e.g. the need for medication) may play a role in the initiation or maintenance of abstinence.


Ethanol-induced chronic myopathy in the young rat: a light and electron microscopic study in type I or type II fibre-rich skeletal muscles.

An investigation was made into the characteristics of an experimental chronic alcoholic myopathy in the young rat. Male Wistar rats were fed a diet for 6 weeks in which ethanol comprised 36% of total energy. Controls were pair-fed the same diet except ethanol was substituted by isoenergic glucose. Soleus (type I fibre-rich) and plantaris (type II fibre-rich) muscles were examined by light microscopy, histochemistry and electron microscopy. Muscles from ethanol-fed rats showed a low-grade myopathy and the diameters of individual type II fibres were reduced. Infrequent atrophic fibres were necrotic and undergoing phagocytosis. Ultrastructurally, there were no observable differences in the subcellular organelles of the alcohol-fed and control rats. It was concluded that alcohol causes a specific myopathic process in the rat, selectively affecting type II fibres. These changes correlate well with the abnormalities seen in human chronic alcoholic myopathy.


Metadoxine (pyrrolidone carboxylate of pyridoxine) antagonizes the locomotor-stimulatory effect of ethanol in mice.

The effects of metadoxine (pyrrolidone-carboxylic acid; PCA) and pyridoxine on the locomotor responses to ethanol in mice were compared. Metadoxine (200 and 400 mg/kg i.p.) and PCA (86 and 172 mg/kg) prevented, in a dose-related manner, the locomotor stimulant effect of a low dose of ethanol (1.5 g/kg i.p.), whereas pyridoxine (228 mg/kg i.p.) was completely ineffective. Neither compound modified the sedative effect of a high ethanol dose (2.5 g/kg i.p.). The results indicate that the antagonistic effect of metadoxine on ethanol stimulant response is due to the PCA component of the molecule.


The concentration of thiamin and thiamin phosphate esters in patients with alcoholic liver cirrhosis.

The blood and plasma concentrations of thiamin and thiamin phosphate esters were determined concomitantly by high-performance liquid chromatography (HPLC) in 22 patients with alcoholic liver cirrhosis, and also in 10 of them 24 hr after a 100 mg thiamin i.m. injection. Sixteen patients were abstaining from alcohol at the time of the study, 6 were currently misusing alcohol. The control group included 30 healthy volunteers, of whom 10 were given the same thiamin injection as the patients. Blood thiamin diphosphate was the only compound decreased in the abstaining patients compared to controls (70.9 +/- 21.9 nmol/l vs. 84.4 +/- 19.0 nmol/l), but all thiamin compounds in blood and plasma were decreased in the misusing patients. All thiamin compounds (except blood monophosphate) were also significantly lower in the misusing than in the abstaining patients (plasma thiamin: 5.3 +/- 1.3 vs. 11.7 +/- 8.3 nmol/l; plasma monophosphate: 1.0 +/- 1.1 vs. 4.1 +/- 2.9 nmol/l; blood diphosphate: 45.7 +/- 18.3 vs. 70.9 +/- 21.9 nmol/l). Thiamin phosphorylation ratio was decreased in the patients after thiamin administration compared to controls (2.83 +/- 0.74 vs. 3.68 +/- 0.58). Plasma thiamin was higher in the abstaining patients than in the controls (11.7 +/- 8.4 nmol/l vs. 7.3 +/- 2.5 nmol/l), and above the mean + 2 SD of the controls in 31% of the abstaining patients. In conclusion, current ethanol misuse is associated with low thiamin concentrations, and liver cirrhosis is associated with a decreased thiamin diphosphate concentration and thiamin phosphorylation.


TRH test in alcoholics: relationship of the endocrine results with neuroradiological and neuropsychological findings.

Neuroradiological, neuropsychological and neuroendocrine parameters were evaluated in 20 non-depressed alcoholic men after 4 weeks (N = 11) or after at least 1 year (N = 9) of abstinence from alcohol and in normal men (N = 9). With regard to normal controls, 4-week abstinent alcoholics showed larger lateral and third ventricles, without modification in the number of cerebral sulci, and altered scores of tests evaluating subcortical and frontal function. Furthermore, in these patients the TSH (thyroid stimulating hormone) and PRL (prolactin) responses to thyrotropin releasing hormone were higher than in controls, suggesting a reduced hypothalamic control of TSH and PRL secretion. Taken together, these findings suggest the presence of a frontal-subcortical disorder in alcoholics. Patients who had been abstinent from alcohol for at least 1 year were not distinguishable from controls for neuroradiological, neuropsychological and neuroendocrine findings, suggesting that the alcohol-related brain alterations are reversible after a long period of abstinence.


Intestinal iron absorption in chronic alcoholics.

Chronic alcohol misusers frequently accumulate significant amounts of excess iron, but the mechanism of this loading is unknown. In vivo whole-body retention studies demonstrated, on average, a two-fold increase in intestinal iron absorption in six male chronic alcoholics. Degrees of iron loading as assessed by serum ferritin or hepatic iron levels did not correlate with alcohol consumption or liver function tests. In vitro studies of iron uptake at varying medium iron concentrations by duodenal mucosa biopsies showed increased iron uptake by tissue from the chronic alcoholics, particularly at the highest medium iron concentration used. Analysis of the uptake data showed similar Michaelis-Menten kinetic constants for uptake by tissue from control subjects and alcoholics. The analysis showed, in addition, a linear component for 59Fe uptake. This component was five-fold greater for the tissue from the chronic alcoholics compared to the controls at the highest medium iron concentration. 57Co-cyanocobalamin was included in the incubation medium as a tissue extracellular fluid marker (ECF). It was found that the apparent distribution volume of the ECF marker, reflecting tissue permeability, was 75% higher for the biopsies from the alcoholics compared to control subjects. These results, together with the previous reports of enhanced in vitro and in vivo intestinal permeability to 51Cr-EDTA in chronic alcoholics, indicate that unregulated increased iron absorption via the non-carrier-mediated paracellular route contributes to the iron overload in chronic alcoholics.


Evaluation of plasma cholesteryl ester transfer protein (CETP) activity as a marker of alcoholism.

Plasma cholesteryl ester transfer protein (CETP) activity was measured in 52 alcoholics and 38 controls and compared with conventional laboratory markers of alcoholism. Mean daily alcohol intake was 180 g/day among the alcoholics and 10 g/day among the controls. Plasma CETP activity was 26% lower in the alcoholics (P < 0.001) and was inversely correlated with daily alcohol intake (r = -0.288, P < 0.05). CETP activity detected 63% of the alcoholics, and its specificity was 82% if the cut-off point was set at the mean CETP activity of the controls -1 SD. The mean -2 SD gave a very low sensitivity for CETP (8%) and cannot be used as its cut-off point. The sensitivities and specificities of gamma glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, mean corpuscular volume and high-density lipoprotein cholesterol were similar to those of CETP activity when the cut-off point for CETP was mean -1 SD. The results thus indicate that plasma CETP activity is not sufficient as a single marker of alcoholism but could be used as an additional method to detect alcohol misuse, although its wide variation in normal population and the elaborate analysis limit its usefulness.


Serum trypsin-like activity in chronic alcoholized men: possible relationship with lipids, apoA-1 and apoB lipoproteins.

Chronic alcoholization is known to increase plasma trypsin levels. One-hundred and forty-six male chronic alcohol users were tested for serum trypsin-like activity (STA), total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-/cholesterol (HDL-C), triglycerides (TG), apoA-1 and apoB lipoproteins. STA was positively correlated to LDL-C, TG and apoB rates and the CT/HDL-C index and negatively correlated to HDL-C and apoA-1 rates and the apoA-1/apoB index. Eighty-four patients with high STA (group B) compared to 62 patients with normal STA (group A) showed significantly higher LDL-C, TG, apoB rates and TC/HDL-C index contrasting with significantly lower HDL-C and apoA-1 rates and the apoA-1/apoB index. The two groups were matched for age, overweight, cigarette smoking and glycemia. Hepatic dysfunction does not explain the differences in the lipoproteic parameters. Such results would suggest that there may be a tryptic alteration of apoproteins in vivo as already demonstrated in vitro and experimentally suspected in vivo in some other studies. Competition by the trypsin-activated alpha 2 macroglobulin for the chylomicron-remnant LDL receptor-related protein may be evoked.


[Physiological evaluation of clothing made of new material for protection against the solar heat load].

The purpose of this study was to evaluate the effects of clothing made of a new material that is, the polyester staple containing the ceramics and to reflect the solar heat load on physiological responses during rest, exercise (50% VO2max) and recovery on a cycle ergometer. Six young female subjects exposed their back to an artificial solar heat load of an intensity of 680 kcal/m2/h with an air temperature of 30 degrees C. The data were compared to those obtained by wearing clothing made of cotton material. The results were as follows. The cardiac output and oxygen consumption obtained at the end of recovery were increased by solar heat load when the subjects wore cotton material. However, these values showed no significant increase when the subjects wore solar heat reflecting clothing. Furthermore, the cardiac output at the end of submaximal work and recovery were higher for the cotton material compared to the heat reflecting clothing in the solar heat load. The increase of cardiac output for the cotton material may show the increase of skin blood flow for the body heat dissipation. Those results suggest that the solar heat reflecting clothing may decrease the physiological strain like a blood redistribution for the body heat dissipation during exercise in summer sunlight.


Distribution of evaporation rate on human body surface.

Relative humidity (R. H), vapour pressure (Psk) and evaporation rate (Esk) of the human skin surface were measured at 29 points by an evaporimeter at 25 degrees C, 28 degrees C, 31 degrees C, 34 degrees C and 37 degrees C of air temperature (Ta), with 35 +/- 10 % R. H and air velocity of less than 0.2 m/s. The skin temperatures and the body weight loss were also measured by thermography and electronic balance. Ten healthy female subjects aged 22-34 years, wearing brassiere and shorts, took a supine and a prone posture during experiment. Comparisons of the obtained Esk with the previous results of other studies showed that there were no definite differences among the measuring methods of Esk, while it is ascertained that the calibration of the results to some standard values such as weight loss were more important to get accurate values of Esk. Distribution patterns of Esk were almost the same in the insensible zone but they remarkably changed in the sweating zone. According to the regression analysis of Esk and Ta, 29 regions were classified into the following 9 groups which showed similar values and changes of Esk with the air temperature: 1) face, 2) front upper trunk, 3) back upper trunk, 4) front lower trunk, 5) back lower trunk, 6) arm, 7) leg, 8) hand and foot and 9) palm, sole and axilla. These results were discussed in relation to the contributing factors such as the number and the capacity of the sweat gland, the regional skin temperature and the pressure-sweat response and also in relation to the skin wettedness.


On the visual recognition of moving letter.

The visual recognition (i.e., legibility) of moving object was studied. The numerical letters were mainly used as the objects. The influences of (1) direction of the movement, (2) thickness of line of letter (i.e., stroke width), and (3) design of letter to the visual recognition were measured. From these measurements, the optimal numerical letters were proposed in the first step of the study, which were called as IMAL(initial most optimal arabic letters). The visual recognition of both moving letters IMAL and letters JIS (Japan industrial standard) employed for motionless-state was compared for both the moving and the motionless states. In the presentation for motionless state, letters JIS showed lower error rate, but the statistical significance was not proved, while in the presentation for horizontally moving state letters IMAL showed better visual recognition than letters JIS was the significant level of 5%. Therefore, the useful numerical letters for moving state could be designed in the study. Moreover, the visual recognition of letters IMAL was evaluated for various moving velocities. The optimal moving velocities for both letters IMAL and JIS were found.


Effect of chronic hypoxia on skeletal muscle fiber type in adult male rats.

The effects of chronic hypoxia on muscle fiber distribution were investigated in 10-week-old male Wistar rats. Samples of the soleus and the plantaris muscles were extracted after the animals were exposed to normobaric hypoxia (10% O2) for 4 weeks. Histochemical myosin-based classification of skeletal muscle fibers was used. There was no evidence of transformation between different fiber types in either muscles. These results differ from those of previous studies. The rats used in those studies were younger, and it is difficult to distinguish the conversion of fiber types that occurs during normal development from other factors. These data suggest that chronic hypoxia does not affect muscle fiber type in adult rats.


The physical mechanisms of the perception of dampness in fabrics.

The detection of dampness in hygroscopic materials has been investigated both by subjective tests and by the application of a model of the physical mechanisms involved. Subjects were asked to rate the degree of dampness of a range of materials of different moisture contents after a short period of contact with the inner forearm. Skin and fabric inner surface temperatures were recorded. It was found that highly hygroscopic wool fabrics were perceived as being dryer and maintained a higher temperature at the skin surface than polyester, a less hygroscopic fabric, during fabric-skin contact. A physical model of the sorption/desorption process in hygroscopic materials has been developed from knowledge of fibre sorption kinetics and used to study the physical processes which take place at the skin-fabric interface during transient contact. These predictions agree well with the subjective responses and the measured temperatures.


[Comparison of submaximal front crawl and breast stroke swimming in relation to energy expenditure].

The purpose of this study was to compare the energy expenditure during submaximal front crawl (Fr) and breast stroke (Br) swimming. Six male college swimmers performed submaximal and maximal exercise tests in both styles in a swimming flume. In submaximal exercise tests, they swam at the following given velocities for 5 min, Br: 0.3, 0.5 and 0.7 m/sec; Fr: 0.3, 0.5, 0.7 and 0.9 m/sec. In maximal exercise tests, following submaximal swimming at 0.9 m/sec in Br and 1.1 m/sec in Fr, swimming velocity was increased progressively by 0.1 m/sec every 1 min until the subjects reached to voluntary exhaustion. VO2max obtained from the maximal swimming tests in Br and Fr were 4.27 and 4.18 l/min, respectively. And there was no significant difference between these two values. VO2 during Br and Fr swimming at four and five submaximal velocities were 1.06, 1.30, 1.79, 2.65 l/min and 1.17, 1.34, 1.63, 2.04, 3.05 l/min, respectively. And, it was found that VO2 at 0.3 and 0.9 m/sec were significantly different (p < 0.05 and p < 0.01) between Fr and Br. VO2 in both styles curvilinearly increased with swimming velocity, and these relationships were well fitted for the regression equation of the second order (Br: y = 3.84625x2 - 1.95914x + 1.310463,r2 = 0.999 (p < 0.05), Fr: y = 3.233446x2 - 2.28136x + 1.611524, r2 = 0.979 (p < 0.05)). It was calculated that the two curves crossed at a point on 0.49 m/sec, and that VO2 at this point was 1.27 l/min. This value equivalented to 30.4% VO2max in Br and 29.7% VO2max in Fr.(ABSTRACT TRUNCATED AT 250 WORDS)


Thresholds for decrease in intracellular pH and increase in blood lactate during progressive exercise: 31P-MRS study.

The purpose of this study was to compare the intramuscular and the intravascular events in relation to energy metabolism during progressive arm exercise. Twelve healthy untrained Japanese males participated in this study as subjects. They performed wrist flexion in a ramp incremental load of 0.14 W/min until exhaustion. 31P-MR spectra were obtained from wrist flexor muscle before and throughout the exercise. Venous blood was also sampled from antecubital vein with one minute interval during the exercise, and a change in plasma lactate concentration (La) was observed. Intracellular pH (pH) was calculated from a chemical shift between phosphocreatine (PCr) and inorganic phosphate (Pi) of the 31P-MR spectra. Change in pH showed a threshold behavior during exercise. Threshold points at decline in pH (pHT), increase in Pi/PCr (PT), and increase in La (LT) were determined by piecewise linear regression analysis of minutes-by-minutes data. Mean values of pHT, PT and LT were 43.0, 42.5, and 24.8% of maximal work rate, respectively. LT was significantly smaller than pHT and PT. This result suggests that lactic acidosis has already existed when pH is kept at resting level, and pHT reflects the capacity of remaining intracellular biochemical homeostasis, which might be one of the physiological indices of muscle fatigue.


[The effects of the menstrual cycle on temporary threshold shift (TTS) and sex differences in TTS].

The aim of this research was to investigate the influences of the menstrual cycles of women on temporary threshold shift (TTS) and to compare the results with TTS of men. The subjects were, after 5 min rest, exposed to 100dB of continuous white noise for 8 min. The audiometry with a 4000 Hz test tone was done in the 45-min recovery process. In the three phases of the menstrual cycle, TTS tended to be lower in postovulatory phase than in preovulatory phase and during menstruation. The male TTS was higher than that of the female in all phases of menstrual cycle.


Prognostic value of EEG in neonatal meningitis: retrospective study of 29 infants.

Neonatal meningitis is associated with significant neurologic sequelae. Previous studies from our laboratory and others demonstrated electroencephalography (EEG) to be a useful tool in predicting long-term neurologic outcome in at-risk neonates. We, therefore, retrospectively studied 29 infants with culture-proved neonatal meningitis who died in the neonatal period or survived to follow-up at a mean of 34.4 months. Seventy-five EEGs were obtained during the acute phase of infection; the degree of EEG background abnormality proved to be an accurate predictor of outcome. Infants who had normal or mildly abnormal backgrounds had normal outcomes, whereas those with markedly abnormal EEGs died or manifested severe neurologic sequelae at follow-up. When the EEG was considered with the presence or absence of seizures and the level of consciousness, an accurate prediction of neurologic outcome was obtained in 27 infants (93%). Although the EEG patterns were generally nonspecific, some abnormalities, such as positive rolandic sharp waves, persistent hemispheric or focal voltage attenuation, suggested more specific pathology (i.e., deep white matter necrosis, large-vessel infarction and abscess, respectively). EEG was also valuable for the recognition of subtle and subclinical seizures. Therefore, we conclude that EEG is a valuable tool for predicting the long-term prognoses of infants with neonatal meningitis.


Head-upright tilt table testing in evaluation of recurrent, unexplained syncope.

Recurrent syncope is one of the most common problems referred to the pediatric neurologist for evaluation. Traditional evaluations are time consuming and expensive, and leave 40% of patients without a precise diagnosis. Vasovagal syncope has been believed to be a common cause of syncope; however, no reliable diagnostic modality has been available to confirm this theory. Head-upright tilt table testing has recently emerged as a useful tool in the evaluation and management of recurrent, unexplained syncope. In this review, we present the pathophysiologic mechanisms of vasovagal syncope and relate them to the reflexes triggered during head-upright tilt table testing. Additionally, we review the clinical data on the uses of this test in unexplained syncope, suggest a practical testing protocol, and elaborate potential therapeutic modalities that can be employed to prevent further episodes. Head-upright tilt table testing will likely become a standard test employed by both adult and child neurologists.


MRI findings in children infected by Borrelia burgdorferi.

Cranial magnetic resonance imaging abnormalities were observed in 8 children (5 boys, 3 girls; ages 4-14 years) with neurologic problems following infection by Borrelia burgdorferi, the etiologic agent of Lyme disease. Neurologic features included headache (6), behavioral changes (5), facial palsy (2), papilledema (2), papilledema with diplopia (1), disturbance of sleep pattern (2), and carpal tunnel syndrome (1). Two MRI studies demonstrated multiple focal areas of increased signal intensity in white matter on long TR (both proton-density and T2-weighted) images.


Prediction of dystrophin phenotype by DNA analysis in Duchenne/Becker muscular dystrophy.

Allele-specific molecular diagnosis of Duchenne and Becker muscular dystrophies (DMD and BMD) has been largely dependent upon muscle biopsy for dystrophin protein assay. We performed lymphocyte DNA mutation analysis by polymerase chain reaction on 14 boys presenting with a clinical picture compatible with DMD or BMD. DNA analysis revealed that 12 of 14 boys had a deletion of the dystrophin gene, thus establishing the diagnosis of DMD/BMD. Furthermore, genotypes for 9 of 12 deletion patients permitted prediction of the specific allelic disorder (i.e., DMD or BMD). Subsequent dystrophin testing confirmed all of the DNA-based diagnoses. We propose that DNA mutation analysis be included in the initial evaluation of patients suspected of having DMD/BMD, thus potentially eliminating the need for muscle biopsy in the majority of patients.


Oral glucose lactate stimulation test in mitochondrial disease.

We selected 23 patients with neurologic diseases, including 15 boys and 8 girls from 1 month to 10 years of age, who were divided into mitochondrial and nonmitochondrial disease groups. All patients were required to fast and rest for at least 4-10 hours before the test. Glucose was administered orally using a 50% glucose:water solution at a dose of 1.75 mg/kg. Blood samples then were drawn from a retained heparinized venous line at 0, 30, 60, 120, and 180 min and tested for lactate and glucose levels. Of the mitochondrial disease group, 10 of 11 patients had an upward sloping curve of lactate metabolism; the one who had a flat line response was a patient who suffered from a free-fatty-acid metabolic defect presenting with a recurrent Reye-like syndrome. There was a relatively flat line response in the nonmitochondrial disease group except in a patient with alternating hemiplegia whose symptoms responded well to flunarizine therapy. A significant increase in blood lactate content at 60 min after glucose loading occurred in the mitochondrial disease group, in contrast to that of the nonmitochondrial group. Sensitivity and specificity of a 5 mg/dl increase in blood lactate concentration at 60 min was 72.7% vs. 91.7% Moreover, all 4 patients whose blood lactate increased by 13 mg/dl at 60 min were in the mitochondrial disease group.


Postnatal maturation of phrenic nerve in children.

The phrenic nerve at the pericardial level was examined postmortem in 17 children, ages 3 days to 8 years. Detailed macroscopic and histologic examination of the central nervous system in all patients disclosed no abnormalities. Quantitative developmental studies demonstrated that myelinated axons doubled in number from birth to age 1 year when a plateau was reached. The main period of growth in diameter of myelinated axons also corresponded to the first year when median diameters increased from 1.75 microns at 3 days of age to 3.0 microns by 8 months of age. Unmyelinated axons also grew significantly in the first 11 months when median diameters reached 1.4 microns. There was no significant increase in axonal diameter at later ages. The slope of the regression line for the number of myelin lamellae on axonal diameters increased with age until 6 months of age, whereas the dispersion around the regression lines decreased in the same period. This finding suggests a direct relationship between myelination and axonal growth. Significant maturation of the phrenic nerve occurs during the first year of life.


Auditory brainstem responses in congenital heart disease.

To evaluate the effect of chronic hypoxemia on brainstem maturation, auditory brainstem responses were examined in 70 children (32 with and 38 without cyanosis) who had congenital heart disease. Ninety-one age-matched normal children served as controls. At 1-3 months of age, the I-V interpeak latencies of cyanotic infants (mean +/- S.D.; 5.17 +/- 0.17 ms) were more prolonged than were those of controls (4.95 +/- 0.11 ms) and those without cyanosis (4.84 +/- 0.22 ms; P < .05; P < .01). At 4-11 months of age, the I-V interpeak latencies of cyanotic infants (4.85 +/- 0.13 ms) were more prolonged than were those of controls (4.67 +/- 0.19 ms) and those not experiencing cyanosis (4.5 +/- 0.17 ms; P < .05; P < .01). In the cyanotic children, there was a significant negative correlation between the I-V interpeak latency and oxygen partial pressure (P < .01) or oxygen saturation (P < .05). Three of the 70 patients (4.3%) with congenital heart disease had absent auditory brainstem response. These data indicate that chronic hypoxemia may be one of the factors in retarded brainstem maturation.


Clinicopathologic studies on leptomeningeal glioneuronal heterotopia in congenital anomalies.

Leptomeningeal glioneuronal heterotopia was observed in 40 of 129 autopsied infants (31%). It was present in 49% of patients who had congenital anomalies in general and in 65% of patients who had central nervous system malformations. Most of the leptomeningeal glioneuronal heterotopias appeared in the base of the brain (62.5%), midbrain (40%), frontal lobe (37%), and pons (35%). Leptomeningeal glioneuronal heterotopia is closely related to migration disorders on the basis of frequent association with polymicrogyria or neuronal heterotopias.


[Characterization of murine monoclonal antibodies against fetal erythrocytes].

Balb/c mice were immunized against papain-treated fetal erythrocytes and splenocytes were fused with Sp2/0-Ag-14 myeloma cells. Several hybrids secreting antibodies directed against antigenic determinants predominantly exposed on fetal and cord cells were selected and cloned twice. Antibodies NaM61-1A2 and NaM61-768 (IgM class) were shown to be specific for an endo-beta-galactosidase-sensitive oligosaccharide chain. The antigen, strongly expressed on fetal and cord cells, was identified as the i blood group antigen. The antibodies represent powerful blood group reagents to be use in conventional agglutination techniques as well as in the gel typing system and in indirect flow cytometry. The antibody NaM46-4A8 (IgG class) is specific for an antigenic structure expressed on fetal cells and accessible only after papain, ficin, bromelin and endo-beta galactosidase treatment. The antigen was not identified.


[Blood donors and anemia].

Biological examinations of 2,630 blood donors showed that 8.6% of them were suffering with anemia. In 4% of the cases we discovered iron deficit and an hemoglobinopathy was found in 0.64% of all the cases.


Autosomal recessive neurodegenerative disorder with trichorrhexis invaginata and ectodermal dysplasia.

Two siblings are reported with an autosomal recessive syndrome characterized by hair and skin abnormalities, hypoplastic nails, generalized hypotonia, absent reflexes, and progressive neurologic deterioration. Although this disorder shares clinical features with an ectodermal dysplasia syndrome with neurodegenerative changes, no specific neuropathologic findings were present. Instead, trichorrhexis invaginata was found in some hair shafts. Hair analysis may be helpful in classifying clinically confusing neurologic conditions.


Serial MRI and CT findings in infantile Krabbe disease.

Krabbe disease (globoid cell leukodystrophy) is an autosomal recessive childhood disorder characterized by severe motor and mental deterioration. The disease has been divided into 3 main types and further subdivided into several subtypes based on age of onset and symptoms. Initial clinical findings and magnetic resonance imaging (MRI) with several follow-up scans are presented to identify the order and extent of white matter involvement and developing brain atrophy in a child with the floppy infant variant of Krabbe disease. When the patient's clinical condition proceeded to stage 2, MRI disclosed severe involvement of the deep white matter around the atria and posterior limbs of the internal capsules. At the same time there was progression of normal myelination around the frontal horns. At age 32 months, the patient's clinical condition proceeded to stage 3; she did not interact with her environment. MRI revealed a significant decrease of white matter volume, generalized atrophy, and abnormal high signal in all white matter areas except the anterior limbs of the internal capsules. At the same time the volume of the central gray nuclei was decreased and also demonstrated abnormal high signal. Despite its sensitivity, MRI could not differentiate the findings of this variant of Krabbe disease from the classic form; therefore, subclassifications of Krabbe disease should be made on clinical grounds because they cannot be distinguished by biochemical or radiologic (MRI) criteria.


[Method for studying anti-complement reagents: use of freeze-thawed sensitized erythrocytes].

The use of red blood cells coated with complement fractions using low ionic strength saline conditions is part of the evaluation of anti-complement antibodies, specially monoclonal antibodies used for antiglobulin reagents. This work shows that such cells stored in liquid nitrogen allow satisfactory long-term studies except for the anti-C3g antibodies.


Eastern equine encephalitis presenting with a focal brain lesion.

Eastern equine encephalitis (EEE) virus causes a severe meningoencephalitis with high morbidity and mortality. Despite numerous clinical reports of EEE, there are only 11 patients in whom cranial computed tomographic (CT) findings are described. In 6 patients, CT was normal and in 5 patients diffuse edema was present; none had a focal brain lesion. Based on these reports, it has been suggested that focal findings on CT support the diagnosis of herpes simplex encephalitis rather than EEE. The first patient with serologically-confirmed EEE and a focal lesion demonstrated by cranial CT and magnetic resonance imaging is described; these findings underscore the importance of including EEE in the differential diagnosis of encephalitides that can cause focal brain lesions on neuroimaging.


Chorea, eosinophilia, and lupus anticoagulant associated with acute lymphoblastic leukemia.

A child is reported with chorea as the initial presentation of acute lymphoblastic leukemia. Subsequent laboratory studies revealed marked eosinophilia and a lupus anticoagulant. No peripheral or central nervous system lymphoblasts were observed. The chorea, eosinophilia, and lupus anticoagulant all resolved once remission of the acute lymphoblastic leukemia was induced. It is suggested that acute lymphoblastic leukemia be included in the differential diagnosis of chorea and eosinophilia in childhood.


Sjögren-Larsson-like syndrome with bone dysplasia and normal fatty alcohol NAD+ oxidoreductase activity.

We report a boy and girl with a "new" multiple congenital anomalies/mental retardation syndrome which resemblances Sjögren-Larsson syndrome. Both patients had a concordant pattern of anomalies consisting of congenital lamellar ichthyosis with spontaneous improvement, moderate mental retardation, mild pyramidal involvement, telecanthus, flat facies, stubby long bones, and coxa valga. Severe myopia, ventriculoseptal defect, and postaxial polydactyly were present in the girl who had more severe bone involvement with dense, enlarged metaphyses, vertebral dysplasia, and advanced skeletal maturation of the lower limbs. Long-chain fatty alcohol NAD+ oxidoreductase (FAO) and steroid sulfatase were normal.


[Healthy aging: a holistic nursing theory].

"Healthy aging" is the theme of the International Nurses Day 1992, announced by the International Council of Nurses. This article discusses briefly critical questions, among which the following: How is the world aging? What are the most important health issues? What does "Healthy aging" really mean? What is the Greek philosophical view for aging? And what are the problems and perspectives of the nursing care of the elderly?


Coadministration of haloperidol and SCH-23390 prevents the increase in "perforated" synapses due to either drug alone.

Perforated synapses, which have a discontinuous density along the postsynaptic membrane, undergo changes in numbers under various experimental conditions. We have previously shown that 14-day administration of haloperidol, a typical neuroleptic which induces extrapyramidal side effects (EPS) and tardive dyskinesia (TD) in patients, causes an increase in the percentage of perforated synapses within the caudate nucleus. This increase was reversed if the animals were taken off the drug for an equal period of time (14 days). There was no effect within the nucleus accumbens. The atypical antipsychotic drug, clozapine, which when administered precipitates a very much lower incidence of EPS and TD, had no effect on the percentage of perforated synapses within either the caudate or nucleus accumbens. Because clozapine binds to both dopamine (DA) D1 and D2 receptors, it was of interest to determine if any changes in perforated synapses occurred following administration of the specific D1 antagonist, SCH-23390. Furthermore, because the action of D2 agonist may be dependent on the activation of the D1 receptor, we asked whether concomitant blockade of the D1 receptor could prevent the increase in perforated synapses due to the action of haloperidol, a drug which upregulates D2 receptors. We found that 14-day treatment with SCH-23390 (1.0 mg/kg per day) or haloperidol (0.5 mg/kg per day) caused an increase in the percentage of perforated synapses within the caudate but not the nucleus accumbens. There was a corresponding increase in DA D1 and D2 receptors in the caudate following administration of SCH-23390 or haloperidol, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


[Aged patients and chronic illness].

Nursing personnel taking care of aged patients should thoroughly recognize respect and protect these special patients' rights. The nurse should also interpret and cope with the various problems arising from both functional and psychological diseases of the elderly, being also responsible for application of specially elaborated educational programmes on long-term care of chronic illness.


Effects of alcohol on E2 beta-stimulated luteinizing hormone in ovariectomized rhesus monkeys.

Anovulation is a frequent concomitant of alcohol abuse, but it has been difficult to assess the acute effects of alcohol on ovulation. Estradiol benzoate (E2 beta) can stimulate a luteinizing hormone (LH) surge in ovariectomized monkeys that appears to be associated with increased luteinizing hormone-releasing hormone (LHRH) pulse frequency and amplitude. The acute effects of alcohol (2.5 and 3.5 g/kg) and an isocaloric sucrose control solution on LH and follicle-stimulating hormone (FSH) secretory activity were studied in five ovariectomized monkeys 41 to 51 hours after administration of E2 beta (42 micrograms/kg, IM). Integrated plasma samples were collected at 20-minute intervals over 10 hours. Under sucrose control conditions, LH increased to 445 and 584 ng/ml within 46 to 49.3 hours after E2 beta administration in two monkeys and high-amplitude LH pulses were evident in three monkeys. Alcohol (2.5 and 3.5 g/kg) significantly decreased the number of LH peaks and valleys (p < 0.01). Peak blood alcohol levels averaged 195 and 291 mg/dl. After 2.5 g/kg alcohol, there was no LH surge or LH pulses in four of five monkeys. A delayed LH surge occurred in one monkey 48 to 50.6 hours after E2 beta when blood alcohol levels decreased to 62 mg/dl. After 3.5 g/kg alcohol, no monkey had an LH surge and pulsatile LH release was significantly reduced in comparison to control conditions (p < 0.01). FSH levels remained stable across alcohol and control conditions. These data suggest that alcohol attenuates pituitary release of LH in response to E2 beta stimulation. These findings are consistent with menstrual cycle disruptions observed in alcohol-dependent women, social drinkers, and in a primate model of alcoholism.


[Healthy elderly--healthy aging].

Human statistics show an extraordinary increase in the proportion of old people in the world, in Europe, and in Greece. The role of the nurse in covering the health needs of this group of the population is analyzed. Emphasis is placed in keeping old people active and independent.


Effect of repeated administration of antidepressants on serotonin uptake sites in limbic and neocortical structures of rat brain determined by quantitative autoradiography.

The binding of 3H-cyanoimipramine, a selective radioligand for the serotonin (5-HT) transporter, was measured by quantitative autoradiography on sections of rat brain to determine if 5-HT uptake sites are regulated by repeated administration of antidepressants. The drugs studied included selective inhibitors of the uptake of 5-HT (citalopram, sertraline) or norepinephrine (protriptyline). Also, effects of inhibitors of monoamine oxidase (MAO) that inhibit both type A and type B MAO (phenelzine), or just type B MAO (deprenyl), were investigated. In addition, the atypical antidepressant mianserin, which has antagonist properties at both alpha 2 adrenoceptors and 5-HT2 receptors, was studied. A total of 19 limbic areas and 4 regions of the parietal cortex were quantitated. The binding of 3H-cyanoimipramine was increased (14% to 31%) by phenelzine and deprenyl in a total of 3 brain areas and decreased (15% to 21%) by sertraline in 4 brain areas. Citalopram, protriptyline, and mianserin produced no statistically significant effect in any brain region examined. The results indicate that different types of antidepressants do not exert consistent or substantial regulatory effect on the density of uptake sites for 5-HT in the limbic system or parietal cortex.


[Outpatient care and social rehabilitation of psychiatric patients in a halfway house].

This paper deals with the foundation and the function of a half-way house, whose intention is the social and professional restoration of psychiatric patients. The half-way house is one of the latest developments of social psychiatry.


Candidate natural killer cell receptors.

Among the high points of immunological discovery has been the identification of antigen-recognizing receptors on B and T cells. Of the lymphocyte populations, only the NK cell receptor remains unknown. Consequently, any newly-recognized, cell-surface molecules expressed selectively on NK cells, especially ones that can transmit a signal to the cell upon appropriate ligand interaction, are possible candidates. This article describes such candidates.


The evolution of immune memory and germinal centers.

Antibody responses in homoiothermic and poikilothermic vertebrates are significantly different in their heterogeneity and affinity range, and in the speed of the secondary response following repeated antigenic stimulation. This article presents the hypothesis that the evolutionary development of unique lymphoid structures, the germinal centers, in combination with the development of a distinct B-cell lineage, is a determining feature of these differences.


The cognitive principle challenges clonal selection.

Here, Irun Cohen argues that the clonal selection paradigm is no longer a convenient paradigm for organizing thinking about the immune system. He contends that most immunologists now investigate questions for which the clonal selection paradigm makes no provision and that one of its major tenets is contradicted by the prevalence of natural autoimmunity. Instead, he proposes a cognitive paradigm.


The extrathymic T-cell development pathway.

In normal mice, not all T-lineage cells are generated and selected in the thymus; an alternative, extrathymic, development pathway exists. Extrathymic T cells are rare in the spleen and lymph nodes, but are abundant in some tissues, such as the gut. Here, Benedita Rocha, Pierre Vassalli and Delphine Guy-Grand discuss the rules of selection of extrathymic T cells, assess the possible role of these cells in the defence of epithelial integrity and their potential role in autoimmune disease.


Thymic selection: a matter of life and death.

Mice transgenic for the T-cell receptor (TCR) were instrumental to the understanding that developing alpha beta T cells undergo programmed cell death unless the TCR beta gene properly rearranges and produces receptors of appropriate specificity. The delineation of developmental pathways for 'conventional' alpha beta T cells has resulted in the recognition of a different lineage of alpha beta T cells that develop in the thymus, the function of which remains to be elucidated.


Rat granulosa cells express the gonadotrophin-releasing hormone gene: evidence from in-situ hybridization histochemistry.

There is still debate as to whether natural sequence gonadotrophin-releasing hormone (GnRH) is produced in the mammalian gonads and concerning its potential role as a paracrine modulator of gonadal function. To address this question, we have used in-situ hybridization histochemistry with an oligonucleotide probe complementary to the GnRH decapeptide coding sequence, to determine the cellular site(s) of expression of the GnRH gene in rodent ovaries. GnRH mRNA was detected in granulosa and thecal cells from ovarian follicles at all stages of development (primary-->Graafian), with no significant change in grain density during follicular development. The granulosa cell compartment always contained more mRNA than the thecal cell compartment. Corpora lutea expressed the GnRH gene to the same extent as thecal cells. These results indicate that preproGnRH mRNA is detectable under physiological conditions in the mammalian ovary, though whether this produces authentic GnRH decapeptide or an alternative protein product is not known. The physiological significance of these findings remains to be determined.


Transforming growth factor-beta 1 production in porcine thyroid follicular cells: regulation by intrathyroidal organic iodine.

The present studies have demonstrated the production of transforming growth factor-beta 1 (TGF-beta 1) by porcine thyroid follicular cells (TFCs) maintained in vitro as subconfluent monolayers, and have confirmed a stimulatory effect of iodide on thyroidal TGF-beta 1 mRNA and peptide release. RNA extracted from TFCs maintained in the absence of iodide contained a 2.5 kb transcript which hybridized specifically with a cDNA probe for human TGF-beta 1, and which showed an approximate doubling in intensity in cells exposed to 10 mumol NaI/1. In the presence of the anti-thyroid thionamide drug methimazole (MMI; 1 mmol/l), the action of iodide on TGF-beta 1 mRNA was attenuated, although MMI alone had no effect on the control level of TGF-beta 1 mRNA. The TGF-beta 1 peptide content of TFC-conditioned media (TFC-CM) was assessed using the fetal mink lung cell line Mv1Lu, in which activated TGF-beta 1 specifically suppresses trichloroacetic acid-precipitable [methyl-3H]thymidine incorporation. Newly conditioned TFC-CM stimulated [methyl-3H]thymidine incorporation into Mv1Lu cells, but after heat treatment to inactivate growth stimulators and activate the latent TGF-beta 1 component this medium inhibited [methyl-3H]thymidine incorporation. This inhibitory effect was prevented by immunoadsorption of TFC-CM with a TGF-beta 1-neutralizing antiserum, confirming the specificity of the inhibitory response. The inhibitory activity of TFC-CM was increased when the TFCs were preincubated with 10 mumol NaI/1, and lost when TFCs were exposed to MMI. In conclusion, TFCs produce TGF-beta 1 mRNA and TGF-beta 1 peptide, which are both increased by iodide treatment in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of the clusterin gene in the tissues of Booroola sheep which were homozygotes or non-carriers of the fecundity gene FecB.

Clusterin or sulphated glycoprotein-2 is a major component of the rete testis fluid, synthesized by the rete testis epithelial cells and Sertoli cells. Differences in the two-dimensional polyacrylamide gel electrophoresis pattern of clusterin-like proteins have been reported in rete testis fluid from Booroola rams carrying the fecundity gene FecB, when compared with that from non-carrier rams. In order to determine whether the FecB gene influences the expression of the clusterin gene, we used a rat clusterin cRNA probe to investigate mRNA species in the tissues of homozygous (BB) or non-carrier (++) Booroola sheep. Northern blots of polyadenylated RNA showed hybridization to the cRNA probe in the testis, ovarian follicles, corpora lutea and stroma, pituitary and liver. A major mRNA transcript was observed at 2.3 kb and a minor transcript in some tissues at 0.8 kb. Densitometry of the autoradiographs revealed no FecB-specific differences in the densities of the hybridization signals from ++ and BB testis or ovarian follicle, corpora lutea or stromal RNA. We conclude that the gene for ovine clusterin is expressed widely in the tissues of sheep and that its expression is not affected by the presence of the FecB gene.


The first disulphide loop of the rabbit growth hormone receptor is required for binding to the hormone.

Residues within the first disulphide loop of the GH receptor are highly conserved, and the two cysteines forming this motif are conserved across many cytokine receptors. We have used site-directed mutagenesis and the polymerase chain reaction with splicing by overlap extension to show that these residues are essential for binding of bovine (b)GH and human (h)GH to the rabbit GH receptor. When all residues within this loop were replaced with an equivalent polyalanine sequence, hormone binding was abolished. Conversion of Arg 39 within the loop to aspartate (R39D) decreased affinity for bGH by up to 20-fold. Conversion of Glu 42 to lysine (E42K) also resulted in a fivefold loss of affinity for bGH. However, charge reversals at Lys 37, Glu 44 and the conversion of Leu 43 to an arginine (as in the human receptor) were without a major effect on bGH binding. The lack of effect of the L43R mutation on bGH affinity, despite a significant (threefold) decrease in hGH affinity, argues against a role for Arg 43 as a residue conferring primate GH-binding specificity on the human receptor. Examination of the affinities of poly Ala, R39D and E42K mutants for a variety of hormone-binding-site directed and other monoclonal antibodies (MAbs) to the GH receptor revealed that these mutations were without a major effect on tertiary structure. It is of interest that the epitopes for the hormone-binding inhibitory MAbs 263 and 7 are located within this first loop, since the poly Ala mutation abolished the binding of both MAbs, and the R39D mutation abolished binding of MAb 7.(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo.

An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human beta-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived beta-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehyde-reactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat beta-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.


Characterization of the extracellular region of the human thyrotrophin receptor expressed as a recombinant protein.

DNA encoding the N-terminal 415 residues of the human thyrotrophin receptor (predicted to code for the large extracellular region) was introduced into Chinese hamster ovary (CHO) cells using the glutamine synthetase/cytomegalovirus amplifiable expression system, and into E. coli using the pGEX-3X expression vector. Substantial quantities of insoluble fusion protein product resulted from bacterial expression; by Western blot analysis, this was shown to be reactive with anti-receptor antibodies raised against a peptide corresponding to residues 313-330. Immunoreactivity was not retained by the solubilized protein. In eukaryotic expression, several successful CHO transfectants were observed and one (ExG2) was characterized thoroughly. Using agarose-bound Concanavalin A, a glycoprotein with an M(r) of approximately 60,000 was detected in a detergent extract of metabolically labelled ExG2 cells, agreeing with the predicted molecular size of 45,000, plus carbohydrate. The same protein could also be detected by immunoprecipitation using the experimental anti-peptide antisera and also sera from patients with Graves' disease. The protein was immunoreactive in Western blot analyses of ExG2 cells using the experimental antisera but not the pathological sera, supporting the view that linear sequences are not sufficient for autoantibody binding. These are the first studies in which visualization of eukaryotically expressed recombinant receptor by such immunological techniques has been possible, presumably because of the higher expression of the glutamine synthetase system. Surprisingly, the recombinant protein was retained within the cells rather than being secreted. The recombinant protein was very effective at absorbing the adenylate cyclase-stimulating activity of the sera from patients with Graves' disease, but not that of thyrotrophin. This suggests that the large N-terminal extracellular region contains epitopes for stimulatory autoantibodies, but that high affinity thyrotrophin binding requires additional components.


Microheterogeneity of thyroid-stimulating hormone from the pituitaries of euthyroid, hypothyroid and hyperthyroid rats.

The microheterogeneity of pituitary thyroid-stimulating hormone (TSH) is dependent on variations in the hormone's carbohydrate moieties. In this study, changes in the pattern of heterogeneity have been assessed by chromatofocusing, which separates the isospecies on the basis of their isoelectric points (pI). Rats (n = 6 per group) were either untreated or rendered hypo- or hyperthyroid by including in the drinking water either propylthiouracil (0.05% for 8 weeks) or thyroxine (T4; 4 mg/l for 6 weeks) before they were killed at 16 weeks. On autopsy, serum TSH and total T4 were (means +/- S.E.M.): 2 +/- 0.3 micrograms TSH/l and 64 +/- 5 nmol T4/l (control); < 1 microgram TSH/l and 133 +/- 6 nmol T4/l (hyperthyroid); 58 +/- 6 micrograms TSH/l and 32 +/- 6 nmol T4/l (hypothyroid). The pituitaries were individually homogenized and the TSH isoforms separated by chromatofocusing over a pH range of 7-4. Fractions were assayed for TSH by radioimmunoassay. TSH from the control group was distributed into seven major peaks with pI values of (means +/- S.E.M., n = 6) 6.9 +/- 0.1, 6.6 +/- 0.1, 6.2 +/- 0.1, 5.8 +/- 0.1, 5.5 +/- 0.1, 5.2 +/- 0.1 and 4.8 +/- 0.1; 7 +/- 3% of the TSH had a pI of < 4.0. Six peaks of TSH were conserved in the hypothyroid group (with pI values of 6.8 +/- 0.1, 6.5 +/- 0.1, 6.2 +/- 0.1, 5.8 +/- 0.1, 5.4 +/- 0.1 and 5.2 +/- 0.1), and 11 +/- 4% of the hormone had a pI of < 4.0.(ABSTRACT TRUNCATED AT 250 WORDS)


Different isoforms of human pituitary thyroid-stimulating hormone have different relative biological activities.

The relationship between the immunological and biological activities of thyroid-stimulating hormone (TSH) isoforms present in the three human pituitary preparations 68/38 (1st IRP), 80/558 (2nd IRP) and 63/14 (MRC Research Standard A) was investigated. The isoforms were separated by chromatofocusing. Six peaks of immunoactivity were detected in 80/558, with pI values (means +/- S.E.M.) of 6.6 +/- 0.1, 6.2 +/- 0.1, 5.9 +/- 0.1, 5.5 +/- 0.1, 5.2 +/- 0.1 and 4.9 +/- 0.1. Four peaks, with pI values of 6.8 +/- 0.1, 5.9 +/- 0.1, 5.5 +/- 0.1 and 5.2 +/- 0.1, were observed for 68/38. Standard 63/14 had five peaks, with pI values of 6.9 +/- 0.1, 6.4 +/- 0.1, 5.9 +/- 0.1, 5.4 +/- 0.1 and 4.9 +/- 0.1. For each standard, six fractions around the peak areas and at the top and bottom of the gradient were pooled and microconcentrated to < 1.0 ml. Microconcentrated TSH samples were assayed in three TSH bioassays based upon FRTL-5 thyroid cells, utilizing cyclic AMP accumulation, iodide and thymidine uptake as end-points and standard 80/558 as reference preparation. The more acidic forms of TSH showed a higher biological:immunological (B:I) ratio for cyclic AMP accumulation with, for example, 63/14 having a maximum of 3.7 (pI 4.9) and a minimum of < 0.7 (pI 6.9). In contrast, the maximum and minimum B:I ratios for iodide uptake for 63/14 were 3.8 (pI 6.9) and < 0.8 (pI 4.6), and for thymidine uptake, maximum and minimum ratios were 7.2 (pI 6.9) and 1.1 (pI 4.6) respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Intraperitoneal insulin is more potent than subcutaneous insulin at restoring hepatic insulin-like growth factor-I mRNA levels in the diabetic rat: a functional role for the portal vascular link.

There is evidence that the hormonal control of hepatic IGF-I production is mediated by GH and insulin. To elucidate the role of these hormones further we administered s.c. or i.p. insulin (at 2.5 and 5.0 IU/day) and/or GH (0.8 IU/day) to rats made diabetic with streptozotocin 16 days previously. Hepatic IGF-I production was then assessed by quantifying hepatic IGF-I mRNA levels by autoradiography of Northern blots. Diabetes resulted in a fivefold reduction in hepatic IGF-I mRNA levels (optical density (OD) of the 0.7-1.1 kb band: controls, 1.3 +/- 0.09; diabetics, 0.28 +/- 0.08; P < 0.01), which was not significantly changed by treatment with s.c. insulin (OD: low dose, 0.55 +/- 0.05; high dose, 0.58 +/- 0.05) or low dose i.p. insulin (OD: 0.40 +/- 0.03). High dose i.p. insulin enhanced hepatic IGF-I mRNA levels (OD: 0.93 +/- 0.23) compared with diabetic rats (P < 0.01) and those given high dose s.c. insulin (P < 0.04), despite the blood glucose values being similar in the treated groups (i.p., 4.72 +/- 0.29 mmol/l; s.c., 3.32 +/- 0.03 mmol/l). Administration of GH alone partially restored the hepatic IGF-I mRNA level (OD: GH-treated, 1.00 +/- 0.05; diabetic, 0.28 +/- 0.08; P < 0.01), whilst having no effect on blood glucose values (diabetic, 36.35 +/- 0.45 mmol/l; GH-treated, 38.65 +/- 2.39 mmol/l). Additional administration of s.c. insulin completely restored IGF-I mRNA levels to those of controls (OD: low dose, 1.35 +/- 0.14; high dose, 1.27 +/- 0.18).(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of rat gastric inhibitory peptide cDNA.

Gastric inhibitory peptide (GIP) is a 42 amino acid gastrointestinal peptide which inhibits gastric acid secretion and stimulates pancreatic insulin secretion in the presence of glucose. Here we report the sequence of the cDNA encoding the rat GIP precursor. PreproGIP was 144 amino acids in length and comprised the GIP peptide itself, N- and C-terminal flanking peptides of 22 and 59 amino acids respectively and a typical hydrophobic signal peptide. The sequence indicated that GIP is released from its precursor by cleavage at single arginine residues. The C-terminal flanking peptide may have an important function since it was well conserved and contained a region of 16 amino acids with only a single, conservative replacement. Rat GIP mRNA was found in the duodenum and jejunum. Levels of GIP mRNA in the duodenum were increased twofold after a period of 2 days of starvation. There was no detectable expression of the GIP gene in other parts of the gastrointestinal tract or in other endocrine tissues. However, in pancreatic mRNA preparations, a larger mRNA was detected after low stringency hybridization. This could represent a further member of this gene family.


Cloning of a bullfrog growth hormone cDNA: expression of growth hormone mRNA in larval and adult bullfrog pituitaries.

A GH cDNA was specifically amplified from cDNAs constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses employing the DNA polymerase chain reaction. Sequencing analysis revealed that the cDNA clone thus obtained was 654 bp in length, and included an open reading frame encoding the entire sequence of mature GH, with its signal peptide. Slight discrepancies were noted between the deduced amino acid sequence and that determined by direct protein sequencing of purified bullfrog GH or that deduced from the nucleotide sequence reported previously. The length of the bullfrog GH mRNA was estimated to be about 1.2 kb by Northern blot analysis. Homologies of nucleotide and amino acid sequences between GH and prolactin of bullfrog origin were 48% and 26% respectively. Using the cDNA as a probe, the content of GH mRNA in the pituitary of larval and adult bullfrogs was measured. GH mRNA levels were relatively low at the preclimax stage, and rose markedly during climax. In juvenile frogs, GH mRNA levels in the pituitary were extremely high and declined towards adulthood. This finding suggests that the increase in plasma and pituitary GH levels reported previously accompanies the increase in GH synthesis.


Cell-specific expression of aromatase and LH receptor mRNAs in rat ovary.

Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstitial cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.


Rod-cone interaction in carp retina: an analysis of electroretinographic B-waves.

Changes in electroretinographic b-wave in the presence of background lights of increasing intensity were examined in isolated, superfused carp retinas. Within a certain range of ambient illumination b-waves elicited by red (695 nm) test flashes against dimmer background lights were smaller in size than against brighter ones. Accordingly, incremental thresholds for red flashes decreased as the intensity of background lights increased within this intensity range. The results suggest that cone pathways may be suppressed by rods in darkness and dim illumination. The phenomenon was absent for photoreceptor potentials (PIII components), indicating that the rod-cone interaction occurs in second-order retinal neurons.


The formation and crystal structure of dihydronitidine and discussion of anticancer mechanism of nitidine cation.

The paper reports the formation and crystal structure of dihydronitidine, expounds the reasons and conditions of easily formed oxynitidine, and discusses anticancer mechanism of nitidine (cation). The crystallographic parameters of dihydronitidine are: space group P(2)1/n, a = 12.54(1), b = 9.148(5), c = 14.748(8) A, beta = 92.12(6)degrees, Z = 4. 4108 independent reflections were collected within the range of 3 degrees < or = 2 theta < or = 54 degrees, of which 2137 intensity data with I > or = 3 sigma (I) were used in the structural determination. The crystal structure has been refined by full matrix least-square method to a final R of 0.050.


Bispecific antibodies.

Bispecific antibodies--molecules combining two different antigenic specificities--are currently being developed as new agents for immunotherapy and for basic studies in cell biology. Bispecific antibodies (BsAb) are prepared by chemically linking two different monoclonal antibodies or by fusing two hybridoma cell lines to produce a hybrid-hybridoma. Both of these approaches present challenges with respect to yield and purity that should eventually be solved through newer molecular genetic approaches. BsAb have been used to demonstrate that specific surface molecules can trigger leukocytes to either phagocytose or kill tumor cells, viruses, parasites, and infected cells. Such trigger molecules include CD3 on T lymphocytes and Fc receptors for IgG on monocytes, macrophages, and natural killer cells. BsAb have also been used experimentally to localize toxins to tumor sites and fibrinolytic agents to areas of thrombosis, to study the molecular specificity of particular receptors, and as adjuvants in in vitro models of vaccines for infectious disease. The limited clinical trials that have occurred to date, primarily for therapy of tumors, suggest that BsAb may offer considerable promise for therapeutic applications, including cancer, heart disease, infectious disease, allergy, and autoimmunity.


The immunology of the eye and its systemic interactions.

Advances in immunology have provided us with new insights about the ocular inflammatory response. Still, the etiology and pathophysiology of many uveitides remain unclear. Factors such as the blood-ocular barrier, sequestration of retinal antigens, local immunomodulators in the aqueous humor, and anterior chamber-associated immune deviation (ACAID) interact and render the eye an immunologically "privileged" site. On the other hand, human leukocyte antigen complex (HLA) interactions, the immunopathology of hypersensitivity reactions, T-cell mediated disease, and autoimmunity are associated pathogenic mechanisms of immune-related ocular disease. In this paper, we review some of the immunologic characteristics of the eye and their relationship to intraocular inflammation. Clinocopathologic correlations are presented. Also, perspectives of current and future research, as well as diagnostic and treatment strategies, are discussed.


Genetically engineered antibodies: progress and prospects.

Techniques of genetic engineering and expression have been applied to the production of antibodies in a variety of expression systems. Novel antibodies have been produced with a variety of modifications: as chimeric antibodies, as "humanized" antibodies, with catalytic groups, as bifunctional or fusion proteins, and as functional fragments such as Fabs or Fvs. The domain structure of the antibody is favorable to such manipulation; the novel proteins often retain their antibody-derived activity and acquire new properties as well. Chimeric and complementarity-determining region (CDR)-grafted antibodies have been effective in immunotherapy, but problems of immunogenicity remain. Combinatorial libraries produced in bacteriophage may present an alternative to animal immunization as a source of antigen-binding specificities. Structural and mutational analysis of variable regions is providing useful information about the requirements of the variable region for antigen binding. Careful analysis and comparison of effector functions among immunoglobulin isotypes may be applied to the design of effective therapeutic antibodies.


Magnetic resonance imaging of osteomyelitis.

Early diagnosis and detection of osteomyelitis and differentiation of soft-tissue infection from bone involvement is a difficult clinical and imaging problem. Magnetic resonance imaging has proven to be as sensitive as bone scintigraphy in the early detection of osteomyelitis, and, with its superior spatial resolution, MR is often more specific than planar scintigraphy in differentiating bone from soft-tissue infection and separating arthritis, cellulitis, and soft-tissue abscess from osteomyelitis. In several comparative studies, MR has been more advantageous in detecting the presence and determining the extent of osteomyelitis over scintigraphy, CT scan, and conventional radiography. MRI may facilitate differentiation of acute from chronic osteomyelitis and may help to detect foci of active infection in the presence of chronic inflammation or posttraumatic lesions. MRI has a large role in evaluating the presence and extent of spondylitis and epidural abscess and certain distribution features may help recognize tuberculous spondylitis. Gadolinium-enhanced MR could be helpful in delineating the meninges and demonstrating the border and extent of epidural abscesses.


Immunomodulating activity of 1,2-difattyacyl-3-mercaptoglycerol adducts.

Adducts of 1,2-diacyl-(2RS)-3-mercaptoglycerol to 3-maleimidopropionic acid and to 3-maleimidopropionyl-L-alanyl-D-isoglutamine as well as a new lipophilic MTP derivative have been analysed for their immunomodulating activity in comparison to the well known adjuvants CFA and IFA using a suboptimal concentration of BSA as antigen. A concentration-dependent immunostimulating activity was established for these lipophilic molecules when applied in water/oil (IFA) emulsions. The results clearly indicate that the new series of compounds behave as competent adjuvants, a fact which is further supported by the immunoadjuvant effect triggered by two of these diacylthioglyceryl adducts as examples, even when applied in aqueous systems. In the light of these findings it seems promising to use these thiol-functionalized molecules to build in immunoadjuvancy via the maleimide-thiol reaction principle. Attachment of such "sticky fingers" to peptides might enhance interaction of haptens and immunogens with cell membranes and thus, may represent a useful tool for inducing or increasing immunological responses to synthetic antigen presenting systems.


An effective method for the synthesis of neoglycoproteins and neogangliosideproteins by use of reductively aminated sulfhydryl-containing carbohydrate conjugates.

A highly effective method for the synthesis of neoglycoconjugates has been developed. The technique is based on two steps. First, a sulfhydryl group is introduced into the carbohydrate by reductive amination with cysteamine, and secondly the sulfhydryl group carrying carbohydrate is coupled to a protein or carrier via a disulfide bridge. The method was used to couple sialic acid-containing carbohydrates such as N-acetylneuraminyllactose and the carbohydrate moieties of gangliosides (GM1, GM3 and GD1b) to bovine serum albumin. Biological functionality of the generated GM1- and GD1b-neoglycoprotein was demonstrated by a choleratoxin-binding assay and an immunoassay, respectively. The applicability of neoglycoconjugates for the quantification of gangliosides in solution by a competitive assay is shown.


Determination of lectin-cell-binding parameters by a new agglutination technique.

We applied a recently described technique which is based on a light transmission/scattering method to determine the association characteristics of the Geodia lectin to sheep erythrocytes. The agglutination assays were performed in a total volume of 3 ml with 5.4 x 10(6) erythrocytes/ml. At a concentration of 360 ng/ml 50% of the lectin molecules were bound to the cells within the first 10 s of incubation. Scatchard analyses revealed an association constant (K(a)) of 0.9 +/- 0.1 x 10(8) M-1 and a number of 3.8 +/- 0.6 x 10(6) lectin binding sites on one erythrocyte. The method was also successfully applied to determine quantitatively the inhibitory potential of sugars competing with cell surface glycoproteins for the lectin binding site. At a lectin concentration of 360 ng/ml 1 mM of lactose or 70 mM of D-N-acetylgalactosamine are required to inhibit lectin-mediated agglutination by 50%. These analyses confirm the potential of the novel light absorption agglutination technique to evaluate lectin characteristics.


Ascorbate-dependent capacity of dialysed rat liver cytosol to prevent nonenzymatic lipid peroxidation.

The capacity of rat liver cytosol to decrease Fe/ADP ascorbate-induced lipid peroxidation in microsomes was evaluated using the chemiluminescence technique and measuring the formation of thiobarbituric acid-reactive substances (TBARS). The inhibiting effect of dialysed cytosol depended on the ascorbate concentration and was highest in the range of 1.0mM. Precipitation of cytosolic proteins with ammonium sulfate at 53% saturation yielded an active antioxidative fraction. Gel-filtration on Sephadex G-200 led to the separation of at least two cytosolic compounds of approximate molecular masses of 60 kDa and > 400 kDa. Both factors were active at 1.0mM ascorbate in the presence of freshly prepared microsomes. No inhibition of lipid peroxidation was observed using microsomes stored for one month at -70 degrees C.


Approaches to the preparation of 6- and 7-methyl-8-substituted pterins: Part 1. The effect of reaction conditions on isomer distribution and a novel approach to isomer separation.

A new approach to the preparation of 6- and 7-methyl-8-alkylpterins is reported. This procedure relies primarily on the separation of the isomers due to their different propensities to form adducts with hydrogensulfite. The primary synthesis of 6-methyl-8-propylpterin was achieved by the condensation of 2,5-diamino-6-(propylamino)pyrimidin-4(3H)- one with pyruvic aldehyde in the presence of NaHSO3 at pH 4. This results in a solid product which was found to be > 98% pure 6-isomer in the form of the HSO3- adduct. Further treatment by a simple procedure gave the final product as the pterin hydrochloride greater than 99.5% isomer pure. The 7-methyl isomer was obtained from the filtrate of the primary synthesis by a cation exchange chromatographic procedure, again exploiting the different propensities of the two isomers to form adduct. The effect of reaction conditions and the nature of the 8-substituent on the proportions of the isomeric products produced by the above condensation reaction was investigated by a series of small scale syntheses. It was found that the proportion of the two isomeric products was dependent on the size of the 8-substituent and, to a lesser extent, the pH of the reaction mixture. For large 8-substituents such as n-propyl, isopropyl and 2-hydroxyethyl, 6-methyl isomer was the predominant product under all conditions. The presence of HSO3 [symbol: see text] did not generate any significant regioselective effect toward further formation of 6-isomer but had either negligible effect on the product distribution or increased the amount of 7-isomer formed. For the small methyl 8-substituent, the major product in the absence of HSO3 [symbol: see text] was found to be 7-isomer at all pH values. However, in the presence of HSO3 [symbol: see text] the major product became the 6-isomer. A 1H-NMR study of the nature of pyruvic aldehyde in acidic water solution is reported which shows that pyruvic aldehyde exists primarily as the monohydrated geminal ketodiol with a lesser proportion of the bishydrated vicinal diol. No free aldehyde was observed by 1H-NMR. The implications of these findings and the product distribution experiments are discussed in relation to the mechanism of the reaction and the role of HSO3 [symbol: see text] within this mechanism.


pH-dependence of the structure and degradation of 8-alkyl-substituted pterins.

Structures of 8-(2-hydroxyethyl)pterins (8-11) investigated using 1H- and 13C-NMR and UV/vis spectroscopies showed a complex dependence on pH, solvent and 6,7-substitution pattern. In acid, only one cation was observed for all the pterins. In neutral aqueous solution, only one neutral form, the normal quinonoid tautomer, was observed for 7-unsubstituted pterins 9 & 11, but two neutral tautomeric forms, quinonoid and 7-exo-methylene, were observed for 7-CH3 substituted pterins 8 & 10 with 70% and 92%, respectively, of quinonoid. The neutral pterins in MeOH, however, showed a different distribution of structural forms: quinonoid and a five-membered intramolecular ether forms were observed for 7-unsubstituted pterins 9 & 11 as 60% and 25%, respectively, of quinonoid, while quinonoid and 7-exomethylene forms were observed for 7-CH3 substituted pterins 8 & 10 as 10% and 50%, respectively, of quinonoid. In base, for 7-unsubstituted pterins 9 & 11 only the intramolecular ether forms were observed, while for the 7-CH3 substituted pterins 8 & 10 two anion forms, the 7-exo-methylene and intramolecular ether, were observed in the ratio 2:1. Investigation of the distinctive proton-resonance pattern of the ethanomoiety of the intramolecular ether anion of 9 using 600 MHz NMR and spectrum simulation, indicated all four protons have different chemical environments. One conformation of the cyclic-ether ring satisfying the experimental data has been deduced, and the conformational energetics of the ring studied further using AM1 semiempirical quantum chemical calculations. Structural distributions of 8-methylpterins 12-15 were also studied in base only. These showed the 7-unsubstituted pterins 13 & 15 existed solely as the hydrated anion forms, while the 7-CH3 substituted pterins 12 & 14 existed predominantly as the 7-exo-methylene anions. Spectroscopic investigations of the degradation processes of 8-(2-hydroxyethyl)pterins and 8-methylpterins in base indicated a complex pattern of oxidation, ring opening and elimination reactions as a function of time. Using authentic samples, the 7-oxo compounds 16 & 17 and ethanolamine were identified, and evidence for ring-opened forms was obtained by comparison with the relevant 2,5-diamino-6-alkyl-aminopyrimidin-4(3H)-ones 1 & 2. Characteristically different degradation pathways for 7-CH3 and 7-unsubstituted compounds were established.


Human immunoglobulin genes of the kappa type. The long-range map of an orphon V kappa gene region.

As was previously shown by Zimmer et al. (EMBO J. 9, 1535-1542, 1990 and Biol. Chem. Hoppe-Seyler 371, 939-951, 1990), the so-called W regions comprising 11 V kappa pseudogenes are located on the long arm of chromosome 2, very closely to the centromere. They are probably derived by a pericentric inversion and amplification events from gene regions of the kappa locus, which is located on the short arm of chromosome 2 also very closely to the centromere. The restriction map of the W regions was now extended from the previous 1.2 Mb to 4.3 Mb and, at the same time, revised with respect to certain features. This was made possible by a new hybridization probe specific for the Wc region and by the improved resolution and extended range of pulsed field gel electrophoresis. On the basis of the long-range maps of the W regions and the kappa locus the V kappa genes of the kappa locus have to be at least 2.5 Mb apart. This distance can be taken also as a minimal estimate for the size of the centromere DNA of chromosome 2; it is quite possible that the size is much larger.


Isolation and structure elucidation of neuropeptides of the AKH/RPCH family in long-horned grasshoppers (Ensifera).

An identical neuropeptide was isolated by reversed-phase high-performance liquid chromatography from the corpora cardiaca of the king cricket, Libanasidus vittatus, and the two armoured ground crickets, Heterodes namaqua and Acanthoproctus cervinus. The crude gland extracts had adipokinetic activity in migratory locusts, hypertrehalosaemic activity in American cockroaches and a slight hypertrehalosaemic, but no adipokinetic, effect in armoured ground crickets. The primary structure of this neuropeptide was determined by pulsed-liquid phase sequencing employing Edman chemistry after enzymically deblocking the N-terminal 5-oxopyrrolidine-2-carboxylic acid residue. The C-terminus was also blocked, as indicated by the lack of digestion by carboxypeptidase A. The peptide was assigned the structure [symbol: see text]Glu-Leu-Asn-Phe-Ser-Thr-Gly-TrpNH2, previously designated Scg-AKH-II. The corpora cardiaca of the cricket Gryllodes sigillatus contained a neuropeptide which differed in retention time from the one isolated from the king and armoured ground crickets. The structure was assigned as [symbol: see text]Glu-Val-Asn-Phe-Ser-Thr-Gly-TrpNH2, previously designated Grb-AKH. This octapeptide caused hyperlipaemia in its donor species. The presence of the same peptide, Scg-AKH-II, in the two primitive infraorders of Ensifera, and the different peptide, Grb-AKH, in the most advanced infraorder of Ensifera, supports the evolutionary trends assigned formerly from morphological and physiological evidence.


Effects of T-2 toxin on ethanol production by Saccharomyces cerevisiae.

A trichothecene mycotoxin, T-2 toxin, inhibits several aspects of cellular physiology in Saccharomyces cerevisiae, including protein synthesis and mitochondrial functions. We have studied growth of, glucose utilization by, and ethanol production by S. cerevisiae and show that they are inhibited by T-2 toxin between 20 and 200 micrograms/ml in a dose-dependent manner. At 200 micrograms/ml, T-2 toxin causes cell death. This apparent inhibition of ethanol production was found to be the result of growth inhibition. On the basis of biomass or glucose consumption, T-2 toxin increased the amount of ethanol present in the culture. This suggests that T-2 inhibits oxidative but not fermentative energy metabolism by inhibiting mitochondrial function and shifting glucose catabolism toward ethanol formation. As T-2 toxin does not directly inhibit ethanol production by S. cerevisiae, this system could be used for ethanol production from trichothecene-contaminated grain products.


Oral or percutaneous administration of lipopolysaccharide of small molecular size may cure various intractable diseases: a new version of Coley's toxin.

Based on our new finding that an inflammation in which tumor necrosis factor (TNF) is primed or triggered (ontogenic inflammation) can regulate the homeostasis in ontogenesis, we have identified a new lipopolysaccharide from wheat flour (LPSw) that can induce ontogenic inflammation in adult mice. LPSw can prime adult mice to produce TNF when given orally or percutaneously, suggesting that it may maintain homeostasis in adults. LPSw can cure experimental animals of diabetes, hyperlipidemia, ulcer, and herpes. It can also stimulate bone resorption and egg-laying, and shows a strong analgesic effect that is blocked by naloxone. This effect even allows a release from drug addiction. Suppression of serum cholesterol level by oral uptake of LPSw in Watanabe heritable hyperlipidemic (WHHL) rabbit was also observed. Infection of toxoplasma was prevented by oral uptake of LPSw. The realization that a single oral or percutaneous administration of LPSw may be a cure for multiple intractable diseases may lead to the presentation of a nontoxic type of Coley's toxin, which is known to be an efficient cancer treatment, but has high toxicity.


Construction and characterization of a high-affinity chimeric anti-colorectal carcinoma antibody ccM4.

We have produced a high-affinity chimeric anti-colorectal carcinoma antibody, ccM4, chimerized in both heavy and light chains by the construction of two expression vectors, the chimeric heavy-chain expression vector mpSV2neo-EP1-Vm4Cr1 and chimeric light-chain vector mpSV2gpt-EP1-VKCK. These vectors contained the neo or gpt gene as a selection marker, the murine immunoglobulin promoter and enhancer (EP1), the genomic DNA fragments of human immunoglobulin constant region (CK and C gamma 1), and murine cDNA fragments of VH and VK region amplified and cloned directly from the B72.3 hybridoma RNA by the polymer chain reaction technique. These two vector DNAs were sequentially transfected into the SP2/0Ag14 cell line. Transfectants were selected in media containing both G418 and mycophenolic acid. The ccM4 antibody was purified from transfectant supernatants with positive binding reactivity for the TAG72 antigen on a protein A column. We demonstrated that ccM4 antibody retained the same high binding reactivity for the TAG72 antigen as its counterpart, the high-affinity chimeric heavy-chain cB72.3m4 antibody. The ccM4 antibody bound specifically to human colon cancer cells, displayed biodistribution patterns similar to cB72.3m4 antibody, and mediated effective antibody-dependent cellular cytotoxicity to human OVCAR3 tumor cells. Therefore, the high-affinity chimeric ccM4 antibody should be useful in cancer immunotherapy.


Distribution of vasotocin- and mesotocin-like immunoreactivities in the brain of the South African clawed frog Xenopus-laevis.

In order to obtain more insight into primitive and derived conditions of neuropeptidergic systems in vertebrates, in particular amphibians, we have studied immunohistochemically the distribution of vasotocin (AVT) and mesotocin (MST) neuronal elements in the brain of the South African clawed frog Xenopus laevis. Apart from a well-developed hypothalamohypophysial system, the antibodies revealed the existence of extrahypothalamic AVT- and MST-immunoreactive cell groups as well as extensive extrahypothalamic networks of immunoreactive fibres, thus confirming the phylogenetic constancy of this condition in vertebrates. The wide distribution of AVT- and MST-immunoreactive fibres throughout the brains of amphibians suggests that the two neuropeptidergic systems are involved not only in hypothalamohypophysial interactions, but also, as in mammals, in a variety of other brain functions. In particular, the relationship of AVT- and MST-immunoreactive fibres with catecholaminergic cell bodies was noted. The present study has underscored once again that considerable differences in relative densities of AVT- and MST-immunoreactive fibres occur between species, even within a single order of vertebrates.


Relation between the biologic activities and chemical structures of synthetic microbial lipopeptide analogs in mice.

Mitogenicity, lethal toxicity, and antitumor activity against Meth A fibrosarcoma of chemically synthesized lipopeptide analogs, S-[2,3-bis(palmitoyloxy)-2R-propyl]-N-[(2,2,2)-tri- chloroethoxycarbonyl: Troc group]-cysteinyl-seryl-seryl-asparaginyl-alanine (compound KAB-2), which contain the amino acid sequence of lipopeptide in Escherichia coli, S-[2,3-bis(palmitoyloxy)- 2R-propyl]-N-(Troc- or amino-group)-cysteinyl-asparaginyl-seryl-glycyl-glycine (compound KAB-14 or -20), which is found in the amino acid sequence of lipopeptide in Streptomyces, and the compounds binding one to six amino acids, were examined. The analogs showed the mitogenic activity toward splenocytes of C3H/He mice. Low concentrations (0.4 and 2.0 micrograms/ml) of compounds KAB-20 and -21, which have five and six amino acids, respectively, increased the incorporation of [3H]thymidine better than a high concentration (50 micrograms/ml), suggesting that KAB compounds carrying amino groups exert better mitogenicity than KAB compounds carrying Troc group. The decrease of amino acid number in lipopeptide analogs appears to result in a lowering of mitogenicity at low concentrations. KAB-14 and KAB-2 did not exhibit the lethality at a high dose of 50 micrograms/mouse in galactosamine-loaded C57BL/6 mice. By twice intravenous injections of 50 micrograms against Meth A fibrosarcoma in BALB/c mice, KAB-2 showed a higher inhibitory effect than KAB-14. Based on these results, we concluded that the difference of amino acid sequence in the synthetic lipopeptides affects the potency of biologic activities.


Production and immunohistochemical application of monoclonal antibodies against delta sleep-inducing peptide.

Monoclonal antibodies were produced following immunization of rats with delta sleep-including peptide (DSIP). The spleen cells of the rats were fused with the myeloma cell line SP2/0. The supernatants of hybridomas were screened on a solid-phase immunoassay using dot-immunobinding of DSIP and some DSIP fragments. The supernatants of six stable producer clones were found to react with DSIP. From this procedure it was also deduced that all these monoclonal antibodies recognized epitope(s) of the penta carboxy-terminal region of DSIP (DSIP5-9). Application of these monoclonal antibodies to rat median eminence sections gave a strong immunolabelling of a large population of fibres and terminal-like structures, mainly localized through the lateral areas. Elution-restaining experiments using a monoclonal antibody to DSIP and a polyclonal antiserum to luteinizing hormone-releasing hormone (LHRH) showed that the patterns of immunoreactivity respectively visualized overlap almost completely. Although numerous LHRH-immunoreactive neuronal elements were also easily demonstrated in the median eminence of the mouse, the hamster and the gerbil species, incubation of sections with monoclonal antibodies to DSIP failed to give any immunoreaction. Taken together these data argue for the independence of the DSIP/LHRH immunolabelling systems. Furthermore, it was demonstrated that DSIP5-9-related epitopes detected in the rat median eminence have no counterpart in the three other rodent species investigated. These species differences may reflect the fact that the carboxy-terminal sequence of the nonapeptide DSIP originally discovered in the rabbit is not conserved in all rodent species.


Distribution of histochemically reactive zinc in the forebrain of the rat.

The major cytoarchitectonic regions of the rat brain that stain with the Timm-Danscher metal stain were tested with the fluorescent probe for zinc, 6-methoxy 8-para toluene sulfonamide quinoline (TSQ). Throughout most of the striatum, cerebral cortex and limbic system, the diffuse, even neuropil staining produced by the Timm-Danscher method was mirrored by comparable fluorescence in TSQ-stained sections. Blockade of the TSQ fluorescence by prior treatment with sulphide indicated that the Timm-Danscher and the TSQ procedures both labeled the same pool of endogenous metal, which is inferred to be the zinc that is in axonal boutons. It is concluded that the Timm-Danscher staining generally indicates zinc-containing axonal boutons. The distribution of the zinc-containing axonal boutons throughout the forebrain is described.


Antitumor effect of recombinant human tumor necrosis factor-alpha analog combined with desmuramyl dipeptides LK-409 or LK-410 on sarcoma in mice.

Antitumor effect of recombinant human tumor necrosis factor (TNF)-alpha lacking one to three amino acids from the N terminal part (TNFNv3) was tested for its antitumor effect on subcutaneous fibrosarcoma SA-1 tumors. Peritumoral treatment with 5 x 10(4) U TNFNv3 three times every second day significantly delayed tumor growth. Treatment with 10 times higher dose (5 x 10(5) U) produced 6.0 +/- 1.0 days tumor growth delay, but had side effects such as weight loss. The two new desmuramyl N-acyl dipeptides, LK-409 and LK-410, also exhibited such effect; however, the tumor growth delay was barely significant. The treatment was performed with two concentrations (2.5 micrograms and 25.0 micrograms) applied intraperitoneally for 5 consecutive days, without a dose-dependent effect. Combined treatment with TNFNv3 and desmuramyl dipeptides augmented the antitumor effect of treatments. The effect was additive and significant in the combination of 2.5 micrograms LK-410 with 5 x 10(5) U TNFNv3. LK-410 treatment also reduced the side effects of TNFNv3. The results indicate that combined treatment with both biological response modifiers is effective in tumor treatment.


Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes.

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.


Regenerating neurons. Changes in protein phosphorylation.

We have been studying the phosphorylation of proteins of both normal and regenerating superior cervical ganglia of the rat. Here we report the incorporation of radioactive phosphate into proteins of ganglia homogenates incubated with 32P-labeled ATP under various conditions at day 3 after postganglionic axotomy. The proteins were analyzed by two-dimensional electrophoresis followed by autoradiography. Incubation in the presence of Ca2+ or Ca2+ plus cyclic AMP produced only about 20 spots corresponding to distinctly labeled proteins. This number was reduced to about five under EGTA plus cyclic AMP conditions, whereas the presence of EGTA alone suppressed the phosphorylation reaction almost totally. All these proteins fell within the narrow pI range of 4-6, whereby no qualitative differences between regenerating and control cases were observed. However, the growth-associated protein, variously designated GAP-43, B-50, F-1, and pp-46, had enhanced levels of phosphate incorporation in regenerating ganglia compared to controls. Injury also caused consistently higher levels of phosphorylation of proteins running in the position of alpha- and beta-tubulin. Since these three proteins are major constituents of regenerating axons, these results suggest that the changes in their phosphorylation induced by injury may be involved in the regulation of their transport.


Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration.

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injury-induced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.