The repertoire of somatic antibody mutants accumulating in the memory compartment after primary immunization is restricted through affinity maturation and mirrors that expressed in the secondary response.

The anti-(4-hydroxy-3-nitro-phenyl)acetyl (NP) response is dominated by lambda 1 chain-bearing antibodies expressing the VH gene V186.2 in combination with the D element DFL16.1. lambda 1-positive B cells were isolated from the spleens of mice immunized with NP-chicken gamma globulin 6 wk earlier. Rearranged V186.2 genes were amplified from the genomic DNA of these cells and sequenced. In cases where the rearrangement was typical for secondary anti-NP antibodies, the VHDJH sequences were generally heavily mutated. The frequency and the nature of the nucleotide exchanges mirrored those of secondary response antibodies. V186.2 genes with other rearrangements and V186.2-related genes isolated concomitantly were essentially unmutated. These results demonstrate: (a) that somatic antibody mutants are largely restricted to a small compartment of peripheral B cells, namely, that of memory cells; (b) that the memory compartment is strongly selected for high affinity precursors and largely purged from antigen-binding loss mutants; and (c) that the repertoire of binding specificities expressed in the secondary response is established in its final form before secondary immunization.

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Hepatic function in a family with a nonsense mutation (R154X) in the hepatocyte nuclear factor-4alpha/MODY1 gene.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic disorder characterized by autosomal dominant inheritance, onset usually before 25 yr of age, and abnormal pancreatic beta-cell function. Mutations in the hepatocyte nuclear factor(HNF)-4alpha/MODY1, glucokinase/MODY2, and HNF-1alpha/MODY3 genes can cause this form of diabetes. In contrast to the glucokinase and HNF-1alpha genes, mutations in the HNF-4alpha gene are a relatively uncommon cause of MODY, and our understanding of the MODY1 form of diabetes is based on studies of only a single family, the R-W pedigree. Here we report the identification of a second family with MODY1 and the first in which there has been a detailed characterization of hepatic function. The affected members of this family, Dresden-11, have inherited a nonsense mutation, R154X, in the HNF-4alpha gene, and are predicted to have reduced levels of this transcription factor in the tissues in which it is expressed, including pancreatic islets, liver, kidney, and intestine. Subjects with the R154X mutation exhibited a diminished insulin secretory response to oral glucose. HNF-4alpha plays a central role in tissue-specific regulation of gene expression in the liver, including the control of synthesis of proteins involved in cholesterol and lipoprotein metabolism and the coagulation cascade. Subjects with the R154X mutation, however, showed no abnormalities in lipid metabolism or coagulation except for a paradoxical 3.3-fold increase in serum lipoprotein(a) levels, nor was there any evidence of renal dysfunction in these subjects. The results suggest that MODY1 is primarily a disorder of beta-cell function.

Randomized phase III study of high-dose fluorouracil given as a weekly 24-hour infusion with or without leucovorin versus bolus fluorouracil plus leucovorin in advanced colorectal cancer: European organization of Research and Treatment of Cancer Gastrointestinal Group Study 40952.

PURPOSE: This trial was conducted to determine whether high-dose fluorouracil (FU) given as a weekly 24-hour infusion is more active than bolus FU + leucovorin (LV), and whether high-dose infusional FU can be modulated by LV. PATIENTS AND METHODS: A total of 497 patients with previously untreated metastatic colorectal cancer were randomly assigned to receive bolus FU 425 mg/m2 intravenously + LV 20 mg/m2 on days 1 to 5 and repeated on day 28 (FU + LV), or FU 2600 mg/m2 as a 24-hour infusion alone (FU24h) or in combination with 500 mg/m2 LV (FU24h + LV)-all given weekly x6 followed by a 2-week rest period. Survival was the major study end point. RESULTS: With a median follow-up of more than 3 years, survival did not differ among the treatment groups (median FU + LV, 11.1 months [95% CI, 10.2 to 15.0 months]; FU24h, 13.0 months [95% CI, 10.4 to 15.4 months]; FU24h + LV, 13.7 months [95% CI, 12.0 to 16.4 months]; P =.724). Progression-free survival (PFS) was significantly longer for FU24h + LV (median FU + LV, 4.0 months [95% CI, 3.4 to 4.9]; FU24h, 4.1 months [95% CI, 3.4 to 5.0]; FU24h + LV 5.6 months [95% CI, 4.4 to 6.7]; P =.029). The response rates in the subgroup of patients with measurable disease were 12%, 10%, and 17% for FU + LV, FU24h, and FU24h + LV, respectively (not significant). Occurrence of grade 3 and 4 diarrhea was higher in the FU24h + LV arm (22%) compared with the FU24h (6%) or FU + LV (9%) arms; however, stomatitis (11% in FU + LV v 3% in FU24h v 5% in FU24h + LV arms) and hematologic toxicity were higher in the bolus FU + LV arm. Global quality of life did not differ within the three arms. CONCLUSION: Neither FU24h + LV nor FU24h prolong survival, relative to bolus FU + LV. Leucovorin increases PFS if added to FU24h, but increases toxicity.

Subcortical correlates of differential classical conditioning of aversive emotional reactions in social phobia.

BACKGROUND: Conditioning processes have been proposed to play a role in the development of anxiety disorders. As yet, the neurobiologic correlates of emotional learning have not been fully understood in these patients. Accordingly, brain activity was studied in subcortical and cortical regions involved in the processing of negative affect during differential aversive classical conditioning. METHODS: Twelve patients with social phobia and 12 healthy control subjects were presented with paired conditioned (CS; neutral facial expressions) and unconditioned stimuli (US; negative odor vs unmanipulated air). Functional magnetic resonance imaging (fMRI) was utilized to examine regional cerebral activity during habituation, acquisition,a nd extinction trials. Activity was measured with echo-planar-imaging (EPI), and signal intensity in individually defined anatomic regions were analyzed. RESULTS: Subjective ratings of emotional valence to the CS indicated that behavioral conditioning occurred in both groups. The presentation of CS associated with negative odor led to signal decreases in the amygdala and hippocampus of normal subjects, whereas an opposite increased activation in both regions was observed in patients. Regional differences were not found during habituation and extinction. CONCLUSIONS: Results suggest that conditioned aversive stimuli are processed in subcortical regions, with phobic patients differing from control subjects.

The disulfide bond in chromogranin B, which is essential for its sorting to secretory granules, is not required for its aggregation in the trans-Golgi network.

Chromogranin B (secretogranin I), a protein sorted to secretory granules in many endocrine cells and neurons, undergoes selective aggregation during the sorting process in the trans-Golgi network. Reduction of the single, highly conserved intramolecular disulfide bond of chromogranin B by exposure of intact PC12 cells to the thiol reducing agent dithiothreitol has previously been shown to cause its missorting to the constitutive pathway of secretion. Using saponin perforation of membrane vesicles in aggregative buffer mimicking the milieu in the lumen of the trans-Golgi network (pH 6.4, 10 mM calcium), we show here that treatment with dithiothreitol does not prevent the aggregation of chromogranin B in this compartment. This implies that the loop in the chromogranin B polypeptide that is formed by the disulfide bond has a critical role in the membrane recognition of aggregated chromogranin B during secretory granule formation.

Synthesis and structure-activity relationship of (lactamylvinyl)cephalosporins exhibiting activity against staphylococci, pneumococci, and enterococci.

The synthesis and structure-activity relationships of a new class of vinylcephalosporins substituted with a lactamyl residue are described. These compounds show excellent activity against enterococci and retain the broad spectrum activity of third-generation cephalosporins such as ceftriaxone.

Differential regulation of chromogranin A, chromogranin B and secretogranin II in rat brain by phencyclidine treatment.

Chromogranin A, chromogranin B and secretogranin II belong to the chromogranin family which consists of large protein molecules that are found in large dense core vesicles. Chromogranins are endoproteolytically processed to smaller peptides. This study was designed to elucidate the regulation of chromgranin expression by acute and subchronic phencyclidine administration. The behavioral syndrome produced by phencyclidine represents a pharmacological model for some aspects of schizophrenia [Jentsch and Roth (1999) Neuropsychopharmacology 20, 201-225]. Tissue concentrations of chromogranins were measured with specific radioimmunoassays. Alterations in secretogranin II gene expression were investigated by in situ hybridization. A single dose of phencyclidine (10mg/kg) led to a transient decrease in secretoneurin tissue levels in the prefrontal cortex after 4h followed by an increase in secretoneurin tissue levels after 12h. Repeated phencyclidine treatment (10mg/kg/day) for five days resulted in elevated secretoneurin levels in cortical areas whereas chromogranin A and chromogranin B tissue levels were unchanged. After the same treatment, a significant increase in the number of secretoneurin containing neurons was found in cortical layers II-III, and V-VI as revealed by immunocytochemistry. The increases in secretoneurin levels were paralleled by an increased number of secretogranin II messenger RNA containing neurons as well as by an increased expression of secretogranin II by individual neurons. The present study shows that secretoneurin II tissue concentration and secretogranin II messenger RNA expression is distinctly altered after acute and subchronic phencyclidine application. From these results we suggest that phencyclidine may induce synaptic alterations in specific brain areas and may contribute to a better understanding of synaptic dysfunction which may also occur in schizophrenia.

Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas.

To investigate the constituents of the matrix of endocrine secretory granules, we analyzed endocrinoilogically silent ("non-functioning") human pituitary adenomas for the occurrence of the chromogranins/secretogranins (granins), a protein family normally stored together with many different hormones. When five non-functioning pituitary adenomas were analyzed by immunoblotting using polyclonal and monoclonal antibodies specific for individual members of the granin family, chromogranin A was detected in four cases and chromogranin B and secretogranin II were detected in all cases. The cellular distribution of the granins and of various hormones known to be expressed in the anterior pituitary was studied by immunocytochemistry in fixed, frozen tissue sections from five additional adenomas. Of the eight hormones investigated, only thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone were detected, occurring in only two of the five adenomas. In contrast, granins were found in all five tumors. Chromogranin B and secretogranin II were detected in each of the adenomas in virtually every cell studied, whereas chromogranin A exhibited such a widespread cell distribution in only three adenomas, being focally present in one and absent from the other tumor. The subcellular localization of the granins and the three glycoprotein hormones was investigated by double immunoelectron microscopy. Chromogranin A and chromogranin B were mainly co-localized in secretory granules, whereas secretogranin II was either co-localized with the other two granins or segregated to different secretory granules. When present, glycoprotein hormones were immunodetected in both the secretory granules containing all three granins and those containing mainly secretogranin II. Our data indicate that in non-functioning pituitary adenomas chromogranin A is differentially expressed from chromogranin B and secretogranin II. Moreover, the granins appear to be the most widespread constituents of endocrine secretory granules known, forming the dense-core matrix irrespective of the presence or absence of hormones.

Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.

The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent. According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.

Endothelin A and B receptors change their expression levels during development of human placental villi.

Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33-35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development.

Differential amygdala activation in schizophrenia during sadness.

Several studies have reported impaired emotion processing in schizophrenic patients. However, the corresponding functional cerebral correlates of such impairment have not been fully understood, leaving the neurobiological basis of their affective symptoms unknown. Functional magnetic resonance imaging (fMRI) was utilized to examine brain activity in subcortical and cortical regions of 13 medicated male schizophrenic patients and 13 matched healthy controls during happy and sad mood induction. Results show brain activity in the amygdala of normal controls during negative affect, which is in line with previous neuroimaging findings. Unlike controls, schizophrenic patients have not demonstrated amygdala activation during sadness despite matched ratings to normal controls indicating a similar negative affect. Recognizing that structural abnormalities exist in the amygdala of schizophrenic patients, our results provide new evidence of functional abnormalities in the limbic system.

fMRI reveals amygdala activation to human faces in social phobics.

Functional magnetic resonance imaging was used to determine the activation of the amygdala while seven social phobics and five healthy controls were exposed to slides of neutral faces as well as aversive odor stimuli. The amygdala was selectively activated in the social phobics during presentation of the face stimuli. The data show for the first time that the amygdala is active in human phobics when they are exposed to potentially fear-relevant stimuli. Further research is needed to determine the extent to which overactivation of the amygdala precedes or is a consequence of phobia.

Computer-controlled flushing for long-term cannulation in freely moving rats.

An in vivo long-term perfusion system is presented, which is based on automated, computer-controlled high-frequency heparin (10 U/mL) flushing of the cannula inserted into the tail artery of freely moving rats. The short flushing intervals as achieved with this system guarantee stable conditions throughout an experiment of several days, and reduce blood clotting and the infiltration of fibroblasts. In addition, a specially developed software enables the periodical application of test substances into the blood stream under time-controlled conditions over long periods. The system can be used continuously to follow up blood parameters in single animals without significant elicitation of stress-sensitive plasma catecholamines for up to 12 days.

The impact of diets with different magnesium contents on magnesium and calcium in serum and tissues of the rat.

The impact of three different magnesium diets (70, 1,000 and 9,000 ppm) on total, ionized and bound magnesium as well as ionized calcium in serum and total calcium and magnesium in femoral bone, skeletal muscle, heart and liver of male Sprague-Dawley rats was investigated. The percentage of ionized serum magnesium was unproportionally high in rats fed a low magnesium (70 ppm) diet. Femoral magnesium was correlated with ionized and total serum magnesium. In contrast, there was generally no correlation between total serum magnesium and the magnesium fractions in skeletal muscle, heart and liver. In rats fed the magnesium deficient diet, total cardiac concentration of magnesium was even significantly increased along with total calcium content, while there were no effects on total muscle and liver magnesium. Within the single groups, ionized serum calcium was never proportional to dietary magnesium, but in all three magnesium diet groups together, it was inversely correlated with dietary magnesium. Moreover, ionized serum calcium was inversely correlated with both ionized and total serum magnesium. In all 3 groups together, the concentrations of total calcium and magnesium in heart and skeletal muscle were correlated, within the single groups correlation existed only in the 1000 ppm group. Magnesium influx via calcium channels during low magnesium intake has been seen in non cardiac tissues [35,36], but nothing similar is known about non selective channels for divalent cations in the heart [33]. Thus, magnesium uptake by cardiac cells along with calcium seems to be possible, especially at low intracellular magnesium concentrations, but is still poorly investigated. We suggest that the calcium-antagonistic effect of magnesium is related to the turnover rate of magnesium rather than to its tissue concentrations.

Preparation and physiological properties of alkyl esters of 2,3-dihydroxypropionic acid.

In order to investigate the physiological behavior of alkyl esters of 2,3-dihydroxypropionic acid two such compounds have been synthesized. One of them, the 1-dodecylester of 2,3-ditetradecyloxypropionic acid was subjected to digestion by pancreatic lipase. The substance remained unaffected. For an in vivo experiment a doubly labelled homolog, the [1'-14C]decyl ester of 2,3-di[1'-3H]hexadecyloxypropionic acid was synthesized. This compound was fed by stomach tube to three groups of male albino rats. The experimental animals were killed after 2,4 and 6 h, those of the control groups after 6 h. Blood, urine, small intestines and livers were examined for radioactivity. From the recovery rates it could be derived that the molecule had been metabolized and absorbed. Obviously, the alkyl chain labelled with 14C was split off first and the alkyl chains labelled with 3H were split off thereafter. As the substance is metabolized in vivo it cannot be utilized as a 'non-fattening fat'.

Correspondence of emotional self-rating with facial expression.

Emotional processing abilities are difficult to measure psychometrically. Ultimately their quantification has to rely on 'subjective' judgment thereby leaving open the problem of response biases. Assessments of autonomic arousal similarly provide a mere unspecified measurement of a specific emotion. A standardized mood induction procedure capable of obtaining reliable happy and sad mood changes in healthy subjects was used to demonstrate the effectiveness of this procedure. We performed a two-part experiment using a rater-based analysis of facial expressions. This entailed analyzing the emotion portrayed in the faces. The faces of 24 healthy subjects were videotaped during the mood induction procedure of happiness and sadness, respectively. A group of 20 raters naive to the experimental task and conditions rated the facial expressions on six basic emotions. Results showed that ratings corresponded with the facial expressions, which were reflecting the mood of the task condition. Subjects' facial expressions together with self-ratings demonstrate the successful applicability of this standardized mood induction procedure for eliciting happy and sad mood.

Generality and specificity of emotion-recognition deficit in schizophrenic patients with positive and negative symptoms.

Schizophrenic patients with positive and negative symptoms, as well as non-patient control subjects, were asked to recognize emotional stimuli of happy, sad, and neutral facial expressions. Dependent measures were the percentage of correct responses, and the incorrect use of an emotion category owing to false recognition. Schizophrenic patients with negative symptoms exhibited a generalized emotion-recognition deficit, and their use of emotion categories during false recognition was random. Schizophrenic patients with positive symptoms showed a deficit in their recognition of 'sad' emotion and were 'positively biased' to the category 'happy' as reflected by its most frequent usage during false recognition.

Functional MRI reveals left amygdala activation during emotion.

The potential of functional magnetic resonance imaging (fMRI) for experimental studies of the brain and behavior considerable given its superior time and spatial resolution, but few studies have attempted to validate them against established methods for measuring cerebral activation. In a previous study absolute regional cerebral blood flow was measured in 16 healthy individuals using quantitative H215O-PET during standardized happy and sad mood induction and during two non-emotional control conditions. During sad mood, blood flow increased in the left amygdala and these changes correlated with shifts towards a negative affect. In the present study blood oxygenation level dependent (BOLD) changes were measured with fMRI during the same experimentally controlled mood states and control tasks. Twelve right-handed normal subjects were examined with a T2*-weighted FLASH sequence. A significant increase in signal intensity was found during sad as well as happy mood induction in the left amygdala. This converging evidence supports the potential of fMRI for advancing the understanding of neural substrates for emotional experience in humans.

Somatotosensory evoked potentials during baroreceptor stimulation in chronic low back pain patients and normal controls.

Nineteen chronic low back pain patients (aged 19-63) and 17 controls (aged 20-41) received electrical pain stimuli during manipulation of their carotid baroreceptors. The non-invasive mechanical manipulation of baroreceptors, using the PRES technique (Phase Related External Suction), simulates the end-effects of phasic blood pressure changes. This technique was developed to assess pain responses induced by changes in blood pressure without the typical shortcomings of pharmacological manipulation or lack of a control condition. During maximum baroreceptor activity, there was an unexpected increase in the amplitude of the somatosensory evoked potentials (SEPs) elicited by the electrical pain stimuli condition (N150-P260 peak-to-peak). In most other studies the opposite effect was found, with decreased pain responses during maximum baroreceptor activity. The chronic pain group reported greater pain during highest baroreceptor activation than did the controls. In addition, the chronic pain group showed lower diastolic blood pressure. To determine whether pain and baroreceptor responses observed in the chronic pain group depended on lower blood pressure levels, a second experiment with a non-clinical sample was performed. Results showed that lower tonic blood pressures are associated with greater baroreceptor activity amplifying pain, while higher blood pressure is associated with pain dampening during high baroreceptor activity. Data suggested that the differences in pain responses found in low back pain patients were associated with their lower tonic blood pressure levels. It is proposed that in general, lower blood pressures may be associated with greater pain during baroreceptor activation.