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We consider whether disruption of a specific neural circuit related to self-regulation is an underlying biological deficit in borderline personality disorder (BPD). Because patients with BPD exhibit a poor ability to regulate negative affect, we hypothesized that brain mechanisms thought to be involved in such self-regulation would function abnormally even in situations that seem remote from the symptoms exhibited by these patients. To test this idea, we compared the efficiency of attentional networks in BPD patients with controls who were matched to the patients in having very low self-reported effortful control and very high negative emotionality and controls who were average in these two temperamental dimensions. We found that the patients exhibited significantly greater difficulty in their ability to resolve conflict among stimulus dimensions in a purely cognitive task than did average controls but displayed no deficit in overall reaction time, errors, or other attentional networks. The temperamentally matched group did not differ significantly from either group. A significant correlation was found between measures of the ability to control conflict in the reaction-time task and self-reported effortful control.

The current study examined the development of cognitive and neural systems involved in overriding a learned action in favor of a new one using a stimulus-response compatibility task and functional magnetic resonance imaging. Eight right-handed adults (mean age, 22-30 years), and eight children (7-11 years) were scanned while they performed a task. Both children and adults were less accurate for incompatible stimulus-response mappings than compatible ones; the children's performance was significantly worse. The comparison of the incompatible and compatible conditions showed large volumes of activity in the ventral prefrontal cortex, ventral caudate nucleus, thalamus, and hippocampus. Striatal activity correlated with the percentage of errors in overriding the old stimulus-response association. The hippocampal activity correlated with the reaction time to make a response to a new stimulus-response mapping that required the reversal of a prior association between a stimulus and a response location. Developmental differences were observed in the volume of striatal/pallidal and hippocampal/parahippocampal activity in that these regions were larger and extended more ventrally in children relative to adults. These results suggest that with maturation and learning, projections to and from these regions may become more refined and focal. Moreover, these findings are consistent with the role of ventral frontostriatal circuitry in overriding habitual and well learned actions and hippocampal systems in learning and reversing associations between a given stimulus and spatial location.

In this study we combined event-related fMRI with a parametric manipulation of the go nogo paradigm to examine the effect of preceding context on inhibitory processes. Nogo trials were preceded by either 1, 3, or 5 go trials and then compared to one another. Two distinct patterns of activation were associated with behavioral inhibition: First, the ventral prefrontal cortex, cingulate gyrus, and superior parietal regions showed a context effect with an increase in MR signal to nogo trials with increasing number of preceding go trials. Second, anterior regions in the supplementary and premotor cortex showed an increase in MR signal on the nogo condition after 5 preceding go trials, but not after only 1 or 3. A model using the BOLD response in our data was used to verify that the effect of context was not an artifact of the randomization scheme used in the design., (2002 Elsevier Science (USA))

Background: Alterations in amygdala function have been implicated in the pathophysiological characteristics of adult anxiety and depressive disorders. Studies with healthy adults and children, as well as with adults who have amygdala lesions, have found facial expressions of emotion to be useful probes of amygdala activity. Our study examined the amygdala response to fearful and neutral facial expressions in healthy, anxious, and depressed children. We hypothesized that children with anxiety and depression may show atypical amygdala responses to emotional stimuli., Methods: Twelve children (8-16 years of age) with generalized anxiety or panic disorder and 12 healthy comparison children underwent noninvasive functional magnetic resonance imaging while viewing photographs of fearful and neutral facial expressions. In a second comparison, 5 girls with major depressive disorder were compared with 5 anxious and 5 healthy girls from the previous sample., Results: Children with anxiety disorders showed an exaggerated amygdala response to fearful faces compared with healthy children, whereas depressed children showed a blunted amygdala response to these faces. In addition, the magnitude of the amygdala's signal change between fearful and neutral faces was positively correlated with the severity of everyday anxiety symptoms., Conclusions: Our results suggest that amygdala function is affected in both anxiety and depression during childhood and adolescence. Moreover, this disruption appears to be specific to the child's own rating of everyday anxiety.

This study combines measurements of the thermodynamics and kinetics of guest sorption with powder X-ray diffraction measurements of the nanoporous metal organic framework adsorbent (host) at different adsorptive (guest) loadings. The adsorption characteristics of nitrogen, argon, carbon dioxide, nitrous oxide and ethanol and methanol vapors on Ni2(4,4'-bipyridine)3(NO3)4 were studied over a range of temperatures as a function of pressure. Isotherm steps were observed for both carbon dioxide and nitrous oxide adsorption at approximately 10-20% of the total pore volume and at approximately 70% of total pore volume for methanol adsorption. The adsorption kinetics obey a linear driving force (LDF) mass transfer model for adsorption at low surface coverage. At high surface coverage, both methanol and ethanol adsorption follow a combined barrier resistance/diffusion model. The rates of adsorption in the region of both the carbon dioxide and methanol isotherm steps were significantly slower than those observed either before or after the step. X-ray diffraction studies at various methanol loadings showed that the host structure disordered initially but underwent a structural change in the region of the isotherm step. These isotherm steps are ascribed to discrete structural changes in the host adsorbent that are induced by adsorption on different sites. Isotherm steps were not observed for ethanol adsorption, which followed a Langmuir isotherm. Previous X-ray crystallography studies have shown that all the sites are equivalent for ethanol adsorption on Ni2(4,4'-bipyridine)3(NO3)4, with the host structure undergoing a scissoring motion and the space group remaining unchanged during adsorption. The activation energies and preexponential factors for methanol and ethanol adsorption were calculated for each pressure increment at which the linear driving force model was obeyed. There was a good correlation between activation energy and ln(preexponential factor), indicating a compensation effect. The results are discussed in terms of reversible adsorbate/adsorbent (guest/host) structural changes and interactions and the adsorption mechanism. The paper contains the first evidence of specific interactions between guests and functional groups leading to structural change in flexible porous coordination polymer frameworks.

Visuomotor sequence learning was assessed in 4- to 10-year-old children using a serial reaction time (SRT) task with both random and sequenced trials. One-half of the children received exposure to the sequence prior to performing the reaction time (RT) task. In Experiment 1, 7- and 10-year-old children demonstrated sequence-specific decreases in RT. As in the adult SRT literature, participants with explicit awareness of the sequence at the end of the session showed larger sequence-specific reaction time decrements than those without explicit awareness. Contrary to expectation, preexposure to the sequence did not reliably predict the level of awareness attained. Results from Experiment 2 indicate that 4-year-olds also demonstrate significant sequence learning on a variant of the SRT task. This article provides preliminary data regarding developmental changes in sequential learning and the development and use of implicit and explicit knowledge. Age-related differences emerged primarily in explicit rather than implicit knowledge., (Copyright 2001 Academic Press.)

Electrophysiological studies suggest sensitivity of the prefrontal cortex to changes in the probability of an event. The purpose of this study was to determine if subregions of the prefrontal cortex respond differentially to changes in target probabilities using functional magnetic resonance imaging (fMRI). Ten right-handed adults were scanned using a gradient-echo, echo planar imaging sequence during performance of an oddball paradigm. Subjects were instructed to respond to any letter but "X". The frequency of targets (i.e., any letter but X) varied across trials. The results showed that dorsal prefrontal regions were active during infrequent events and ventral prefrontal regions were active during frequent events. Further, we observed an inverse relation between the dorsal and ventral prefrontal regions such that when activity in dorsal prefrontal regions increased, activity in ventral prefrontal regions decreased, and vice versa. This finding may index competing cognitive processes or capacity limitations. Most importantly, these findings taken as a whole suggest that any simple theory of prefrontal cortex function must take into account the sensitivity of this region to changes in target probability., (Copyright 2001 Wiley-Liss, Inc.)

Given recent court decisions, there is a need to investigate less common forms of sexual harassment, including women harassing men and same-gender harassment. The present study was a 2 (harasser gender) x 2 (target gender) x 2 (participant gender) factorial design in which 408 mock jurors made decisions in a hostile work environment case. Women harassing men were more likely to be found guilty than were men harassing women, and harassers in same-gender cases were more likely to be found guilty and were perceived more negatively than harassers in cross-gender cases. Participant gender differences were found in cross-gender, but not same-gender, conditions. Results suggest that the gender composition of the harasser and target may be an extralegal factor influencing managerial and juror decision making.

Background: The amygdala plays a central role in the human response to affective or emotionally charged stimuli, particularly fear-producing stimuli. We examined the specificity of the amygdala response to facial expressions in adults and children., Methods: Six adults and 12 children were scanned in a 1.5-T scanner during passive viewing of fearful and neutral faces using an EPI BOLD sequence. All scans were registered to a reference brain, and analyses of variance were conducted on the pooled data to examine interactions with age and gender., Results: Overall, we observed predominantly left amygdala and substantia innominata activity during the presentation of nonmasked fearful faces relative to fixation, and a decrease in activation in these regions with repeated exposure to the faces. Adults showed increased left amygdala activity for fearful faces relative to neutral faces. This pattern was not observed in the children who showed greater amygdala activity with neutral faces than with fearful faces. For the children, there was an interaction of gender and condition whereby boys but not girls showed less activity with repeated exposure to the fearful faces., Conclusions: This is the first study to examine developmental differences in the amygdala response to facial expressions using functional magnetic resonance imaging.

Objectives: We evaluated the integrity of neural pathways for auditory recognition memory in normal newborn infants (n = 32) and infants of diabetic mothers (IDMs, n = 25). IDMs are at risk for fetal metabolic abnormalities that potentially damage recognition memory pathways. We hypothesized that newborn IDMs would have recognition memory deficits that would be correlated with later cognitive development., Study Design: Recognition memory was assessed with event-related potentials (ERPs). Neonatal ERPs elicited by the maternal voice were compared with those elicited by a stranger's voice. The Bayley Scales of Infant Development were administered at 1 year of age., Results: Infants in both the control and IDM groups demonstrated recognition of the maternal voice, but their ERP patterns differed. Both groups demonstrated increased amplitude and latency for the "P2" peak elicited by the maternal voice compared with the stranger's voice. In the control group the stranger's voice also elicited a negative slow wave, which was attenuated in the IDMs. The negative slow wave correlated significantly with the 1-year Mental Developmental Index., Conclusions: The presence of a specific neonatal ERP pattern indicated better 1-year cognitive development in infants in the control and IDM groups. ERPs from IDMs demonstrated subtle evidence of recognition memory impairments.

Despite significant gains in the fields of pediatric neuroimaging and developmental neurobiology, surprisingly little is known about the developing human brain or the neural bases of cognitive development. This paper addresses MRI studies of structural and functional changes in the developing human brain and their relation to changes in cognitive processes over the first few decades of human life. Based on post-mortem and pediatric neuroimaging studies published to date, the prefrontal cortex appears to be one of the last brain regions to mature. Given the prolonged physiological development and organization of the prefrontal cortex during childhood, tasks believed to involve this region are ideal for investigating the neural bases of cognitive development. A number of normative pediatric fMRI studies examining prefrontal cortical activity in children during memory and attention tasks are reported. These studies, while largely limited to the domain of prefrontal functioning and its development, lend support for continued development of attention and memory both behaviorally and physiologically throughout childhood and adolescence. Specifically, the magnitude of activity observed in these studies was greater and more diffuse in children relative to adults. These findings are consistent with the view that increasing cognitive capacity during childhood may coincide with a gradual loss rather than formation of new synapses and presumably a strengthening of remaining synaptic connections. It is clear that innovative methods like fMRI together with MRI-based morphometry and nonhuman primate studies will transform our current understanding of human brain development and its relation to behavioral development.

On the basis of animal models, it was hypothesized that infants of diabetic mothers (IDMs) would be at risk for suffering damage to the hippocampus primarily because of fetal iron deficiency, chronic hypoxia, and hypoglycemia. This, in turn, may result in impairments in recognition memory at a young age. To test this model, the memory of 6-month-old IDMs and control infants was evaluated with electrophysiological (event-related potential [ERP]) and behavioral (looking time) measures. At 12 months, the Bayley Scales of Infant Development was administered. Our ERP measures showed robust evidence consistent with memory deficits in the IDMs. In contrast, the looking time measures and the Bayley exam failed to distinguish between the groups. From these results it was concluded that the ERP, but not the behavioral, measures are able to detect, in an at-risk population, deficits in recognition memory that are thought to be mediated by damage to the hippocampus.

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.

Background: The neurodevelopment of childhood anxiety disorders is not well understood. Basic research has implicated the amygdala and circuits related to these nuclei as being central to several aspects of fear and fear-related behaviors in animals., Methods: Magnetic resonance imaging was used to measure amygdala volumes and comparison brain regions in 12 child and adolescent subjects with generalized anxiety disorder and 24 comparison subjects. Groups were matched on age, sex, height, and handedness and were also similar on measures of weight, socioeconomic status, and full scale IQ., Results: Right and total amygdala volumes were significantly larger in generalized anxiety disorder subjects. Intracranial, cerebral, cerebral gray and white matter, temporal lobe, hippocampal, and basal ganglia volumes and measures of the midsagittal area of the corpus callosum did not differ between groups., Conclusions: Although these data are preliminary and from a small sample, the results are consistent with a line of thinking that alterations in the structure and function of the amygdala may be associated with pediatric generalized anxiety disorder.

Functional magnetic resonance imaging (fMRI) was used to examine spatial working memory in 8- to 11-year-old children tested under three conditions. In the visual condition, children were asked to examine the location of a dot on a screen. In the motor condition, children were instructed to push a button that corresponded to the location of a dot presented on a screen. In the memory condition, children were asked to remember the location of a dot presented 1 or 2 trials previously. Subtracting the activation of the motor condition from the memory condition revealed activity in the dorsal aspects of the prefrontal cortex and in the posterior parietal and anterior cingulate cortex. These findings were also obtained in the analysis of the memory minus visual conditions except that motor cortex activation was also observed. These findings parallel those reported in comparable studies of adults and suggest that fMRI may be a useful means of examining function-structure relations in developmental populations.

Lysyl oxidase (LO) is a key participant in the accumulation of insoluble fibers of elastin and collagen by virtue of its role in the initiation of the covalent cross-linkages between and within individual molecules comprising these fibers. In view of the essential role played by LO in the accumulation of the fibrotic components of occlusive arterial lesions in atherosclerosis, identification of the signaling molecules which can affect the expression of the LO gene in vascular smooth muscle is of considerable interest. In the present report, we describe evidence for the role of the second messenger, cAMP, in the modulation of the levels of LO in vascular smooth muscle cells. Elevated intracellular cAMP induces the transcription of the LO gene, as revealed by Northern blot analysis and nuclear run on assays. Transient transfection experiments performed with the wild-type LO promoter and with this promoter mutated at a consensus CREB site, located within the region -100 to -93 base pairs relative to the start of transcription, indicate that cAMP-induced transcriptional activation is partially due to the presence of this CREB site within the promoter. Activation of stimulatory adenosine receptors in vascular smooth muscle cells with 5'-N-ethylcarboxamido adenosine (NECA) increases cAMP, LO mRNA, and enzyme activity. These findings point to the importance of cAMP signaling, potentially initiated by a variety of physiological agents, in the upregulation of LO expression in vascular smooth muscle cells., (Copyright 1999 Wiley-Liss, Inc.)

Functional magnetic resonance imaging (fMRI) was used to examine patterns of cortical activity in children during performance of a spatial working memory task. Six children (8-10 years) and six adults (19-26 years) searched a linear array of four boxes for the appearance of a dot. In the visual blocks, participants made no response. In the motor blocks, participants were instructed to indicate the location of the dot on each trial using a button-press response. In the working memory blocks, participants were instructed to indicate at which location the dot had appeared 1 or 2 trials previously. Both children and adults showed activity in the left precentral and postcentral gyri, as well as the right cerebellum for the motor condition as compared to the visual condition. Comparison of the memory and motor conditions revealed reliable activity in the right superior frontal gyrus (BA 8), right dorsolateral prefrontal cortex (BA 10/46), right superior parietal cortex, and bilateral inferior parietal cortex for both adults and children. These results suggest that spatial working memory tasks activate very similar cortical regions for school-age children and adults. The findings differ from previous imaging studies of nonspatial working memory tasks in that the prefrontal activations observed in the current work tend to be more dorsal. Results are discussed in light of the significant behavioral performance differences observed between child and adult participants., (Copyright 1999 Academic Press.)

The present study sought to compare adult and infant ERP responses in analogous tests of visual recognition memory. Adults were tested under two test conditions, in which they were given either explicit instructions to respond to a previously-seen stimulus, or no instructions. Testing in both conditions took place after either a 1- or 5-min delay between familiarization and test. Eight-month-old infants were tested under the same 'passive' (no instruction) conditions as adults. ERP morphology differed across age groups. Adults evinced a P300 that was larger under 'active' as compared to 'passive' test conditions, and a late positive slow wave response that was greater to novel than familiar stimuli. There was no evidence in infants of a P300, although infants did demonstrate a late positive slow wave that was greater to familiar than to novel stimuli. Parallels between the infant and adult findings are discussed and in particular, whether the infant slow wave response is the precursor to the adult P300.

Background: Evidence from in vitro and in vivo studies indicates that damaged elastic fibers can be repaired., Methods: Lipid interstitial pulmonary fibroblasts were cultured for 6 weeks. Cultures were then exposed to 25 microg of porcine pancreatic elastase and fixed in pairs (control, elastase-treated) immediately after exposure and at 1, 2, 3, 4, 7, 10, 14, and 22 days for ultrastructural examination. Elastin was also analyzed biochemically for resistance to hot alkali, an indicator of repair. Steady-state levels of tropoelastin and lysyl oxidase mRNA at 2, 4, and 7 days after elastase treatment were determined by Northern blot analysis., Results: Immediately after exposure to elastase, damaged elastic fibers exhibited a frayed, porous appearance and a granular texture. Through day 4, fibers showed no evidence of repair. By day 7, the granular texture of damaged fibers was no longer evident and a gradual filling-in of porous areas appeared to be taking place. By 22 days, elastic fibers were indistinguishable from elastic fibers in control cultures. The ultrastructural changes were paralleled by changes in hot alkali resistance. Through day 4, there was no change in the level of hot alkali resistant elastin. Between day 4 and day 7, resistance to hot alkali increased sharply and continued to increase at a slower rate, reaching 84% of the control level by day 22. Steady-state levels of tropoelastin and lysyl oxidase mRNA showed no increase over control levels at 2, 4, and 7 days after elastase treatment., Conclusions: Elastic fibers synthesized by lipid interstitial pulmonary fibroblasts in culture were repaired after damage by elastase. This type of repair may have relevance to the prevention of pathological conditions, such as emphysema.

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Disruption of elastic fibers is a major factor in the pathogenesis of pulmonary emphysema. Elastic fibers in culture, injured by exposure to elastase, undergo repair in the presence of elastogenic cells that restores the fibers toward normal as determined by biochemical and ultrastructural methods. The repair appears to be the result of both salvage and de novo repair mechanisms. The evidence for salvage repair is that hot-alkali resistance, lost as a result of elastase treatment, is restored to previously radiolabeled elastic fibers. This repair mechanism has been shown in aortic smooth muscle cell cultures. In order to determine the potential relevance of this mechanism for elastic fiber repair in the lungs, experiments were carried out using neonatal rat lung lipid interstitial fibroblasts (LIF). Treatment of the LIF cultures with elastase, in the absence of serum, caused solubilization of 12% of elastin; however, 81% of the elastin protein and 80% of the elastin-associated radioactivity (EAR) were solubilized by subsequent hot-alkali treatment, indicating that most of the elastin was retained in the matrix but was damaged. Ultrastructurally, the elastic fibers were frayed. After 6 additional wk in culture, hot-alkali resistant elastin protein and EAR were restored to 88 and 62% of control values, respectively, and the ultrastructural appearance of elastic fibers was restored to normal. We calculate that about 42% of the restored elastin represented salvage repair; the remainder was new elastin. No repair occurred in matrices rendered acellular by azide treatment; however, when acellular matrices were replated with LIF, repair was complete at 6 wk. No repair was seen when acellular matrices were replated with a transformed mouse macrophage cell line. We conclude that lung LIF are capable of mounting a robust repair process after elastolytic injury of elastin and that the repair is the result of both salvage and de novo repair mechanisms.

Transforming growth factor-beta 1 (TGF-beta 1) markedly reduced cell proliferation and elevated steady state lysyl oxidase (LO) mRNA 3-fold in neonatal rat aorta smooth muscle cells cultured in medium containing 10% fetal bovine serum. The increase in LO mRNA was prevented by the presence of cycloheximide, indicative of controlling events at the level of protein synthesis. The basal level of mRNA in cells proliferating in 10% fetal bovine serum in the absence of TGF-beta 1 was enhanced 7-fold upon decreasing growth by shifting to medium containing 0.5% serum. Changes in LO activity paralleled those in LO mRNA. Nuclear run-on assays revealed that the stimulation of expression in 0.5% serum involved increased gene transcription whereas that caused by TGF-beta 1 was mostly post-transcriptional in origin. LO mRNA was quite labile (t1/2 approximately 3 h) in 10% serum but was markedly stabilized (t1/2 > 12 h) by the presence of TGF-beta 1 in the 10% serum medium. LO mRNA was also considerably more stable under retarded growth conditions (0.5% serum) in the absence of TGF-beta 1. LO promoter activity in luciferase reporter constructs transfected into these cells was low and not significantly affected by the addition of TGF-beta 1 to the 10% serum medium but was markedly elevated by shifting from 10 to 0.5% serum in the absence of TGF-beta 1. Thus, LO expression is inversely correlated with cell proliferation, and is subject to control at transcriptional and post-transcriptional levels. TGF-beta 1 enhances LO expression in these cells by dramatically stabilizing LO mRNA.

The relationship of task relevance and stimulus probability to P300 morphology, latency and distribution was assessed. Eight year olds and adults completed visual oddball tasks of recognition memory with frequent non-target (60%), infrequent target (20%), and infrequent novel (20%) stimuli. Stimuli consisted of 2 female faces posing neutral expressions, and 40 trial unique novel photographs depicting scenes, animals, objects or abstract patterns. Event-related potentials were recorded from 17 electrodes over frontal, central and parietal scalp, including lateral temporal sites. All stimuli elicited P300 responses at parietal electrodes, with the largest responses to the target stimuli (relevant and infrequent). The P300 responses of adults and children were morphologically dissimilar, with children showing broader peaks and latency shifts across electrodes. In addition, the eight year olds displayed a frontal negativity to novel stimuli which was absent in the responses of adult participants. Results suggest that different anatomical or functional circuitry may be involved in the processing of novelty for adults as compared to eight year olds.

We report the previously undocumented development of an orbital metastasis and skin metastases in a patient with polymorphous low grade adenocarcinoma (PLGA) 15 years after initial presentation. The clinical course, treatment and changing histological features are discussed.

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32-2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including beta-aminopropionitrile, phenylhydrazine, ethylenediamine, alpha, alpha'-dipyridyl, and diethyldithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase.

Interleukin-8 (IL-8) mediates the transendothelial migration and activation of neutrophils to the site of inflammation. Two human IL-8 receptor isotype (A and B) and one rabbit IL-8 receptor isotype (A) cDNAs have been previously cloned and characterized on the basis of their pharmacological profile. Human and rabbit IL-8 receptor subtype A binds IL-8 and structurally related peptide melanoma growth-stimulating activity (MGSA) and neutrophil-activating peptide-2 (NAP-2) according to the following affinity binding profile: IL-8 >>> MGSA > NAP-2, whereas the human IL-8 receptor subtype B profile is IL-8 = MGSA > NAP-2 (LaRosa, G., Thomas, K. M., Kaufmann, M., Mark, R., White, M., Taylor, L., Gray, G., Witt, D., and Navarro, J. (1992) J. Biol. Chem. 267, 25402-25406). In this study, we isolated a cDNA clone (5B1a) from a rabbit neutrophil library encoding a G-protein-coupled receptor of the interleukin-8 receptor family. The 5B1a clone encodes a 358-amino acid protein exhibiting 80% amino acid identity to the human IL-8 receptor B, 74% to the rabbit IL-8 receptor A, and 73% to the human IL-8 receptor A. Tissue distribution by Northern blot analysis reveals that the 5B1a mRNA is expressed preferentially in neutrophils. In contrast to previously described IL-8 receptors, the 5B1a receptor exhibited specific 125I-IL-8 binding with a novel affinity binding profile of IL-8 >> NAP-2 > MGSA. The corresponding apparent Ki values for IL-8, NAP-2, and MGSA were 4, 120, and 320 nM, respectively. IL-8 induced intracellular calcium mobilization and desensitization in Chinese hamster ovary cells stably transfected with 5B1a, indicating that this cDNA encodes a functional IL-8 receptor. Sequence analysis of the 5B1a protein with other IL-8 receptor subtypes within the framework of their pharmacological profile reveals putative structural motifs that may correspond to the ligand binding site of the IL-8 receptor.

IL-8 mediates migration and activation of neutrophils. This study describes the functional and ligand binding specificity of the human intercrine peptides IL-8, neutrophil-activating peptide 2 (NAP-2), melanoma growth stimulatory activity (GRO), and platelet factor 4 (PF4) to rabbit neutrophils and mammalian cell lines transfected with rabbit IL-8 receptor cDNA (F3R). Rabbit neutrophil membranes bound 125I-labeled IL-8 and 125I-labeled NAP-2 but did not bind 125I-labeled GRO or 125I-labeled PF4. Rabbit neutrophils mobilized intracellular Ca2+ in response to IL-8 and NAP-2 but not to GRO or PF4. Monkey kidney cells (COS-7) and hamster lung fibroblasts (CCL-39) were transiently and stably transfected with the rabbit neutrophil IL-8 receptor F3R cDNA. COS-7 cells transfected with F3R cDNA bound 125I-labeled IL-8 but did not bind other IL-8-related peptides such as 125I-labeled NAP-2, 125I-labeled GRO, or 125I-labeled PF4. Furthermore, bound 125I-labeled IL-8 was only displaced by unlabeled IL-8 but not by unlabeled NAP-2, GRO alpha, or PF4. Consistent with this observation, stably transfected CCL 39 cells expressing F3R cDNA mobilized Ca2+ only in response to IL-8. We conclude that F3R cDNA encodes a functional IL-8 receptor isotype with strict ligand binding specificity for IL-8, that rabbit neutrophils do not bind human GRO alpha, and it is suggested that rabbit neutrophils contain in addition to the F3R protein another IL-8 receptor isotype with broad ligand specificity or a distinct NAP-2 receptor.

The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.

Interleukin-8 (IL-8) is a key mediator in the migration of neutrophils from the circulation to the site of inflammation in the tissue. IL-8 is secreted by many cell types in response to proinflammatory stimuli such as interleukin 1, tumor necrosis factor, and lipopolysaccharide and is a potent chemoattractant and activator of neutrophils. Neutrophil activating peptide-2 (NAP-2) and melanoma growth-stimulatory activity (MGSA/GRO) are structurally and functionally related to IL-8 and, like IL-8, bind to specific G protein-coupled receptors on neutrophils. In the present study two closely related cloned IL-8 receptor subtypes are characterized by expression of the cDNA clones in monkey kidney cells (COS-7) or chinese hamster ovary cells and analysis of their ligand binding profiles. Both receptor subtypes bind 125I-labeled IL-8 with similar high affinity, however, the F3R receptor binds IL-8 exclusively, while the 4Ab receptor binds both IL-8 and MGSA/GRO with high affinity and NAP-2 with lesser affinity. Furthermore, we demonstrate with the use of intersubtype chimeric receptors that the specificity of ligand binding to both IL-8 receptor subtypes is dictated by the heterogeneous NH2-terminal domain. The F3R receptor is representative of a restricted IL-8 receptor subtype, and 4Ab represents a nonrestricted receptor subtype. It is proposed that these subtypes be named IL-8 receptors alpha and beta, respectively.

Interleukin-8 (IL-8) is one of the most potent chemotactic agents for neutrophils and has been implicated as a major mediator of inflammation. The IL-8 receptor is expressed exclusively in neutrophils and belongs to the family of G-protein-coupled receptors. In a recent paper we reported the characterization of a cDNA clone, F3R, isolated from a neutrophil cDNA library and showed that it encodes a G-protein-coupled receptor which is exclusively expressed in neutrophils. We also suggested, based on expression studies in Xenopus oocytes, that the F3R protein product is an isoform of the (fMLP) receptor (Thomas, K. M., Pyun, H. Y., and Navarro, J. (1990) J. Biol. Chem. 265, 20061-20064). In this work, the F3R receptor cDNA is expressed in monkey kidney cells (COS-7) and is shown to encode the IL-8 receptor. F3R cDNA does not encode for a fMLP receptor isoform. We show conclusively that the F3R-transfected COS-7 cells express the IL-8 receptor at a density equivalent to that observed in neutrophils. The pharmacological profile of the F3R-transfected cells is the same as that of neutrophils. The apparent Kd values for binding of 125I-IL-8 to neutrophils and F3R-transfected COS-7 cell membranes were 1.2 and 1.4 nM, respectively. Antipeptide antibodies against a partial sequence of the F3R protein product specifically immunoprecipitate the IL-8 receptor from transfected cells as well as neutrophils. The molecular characterization of the IL-8 receptor should provide the basis for further studies on the identification of the binding domain of this inflammatory receptor.

The bacterial chemotactic peptide fMet-Leu-Phe (fMLP) activates neutrophils upon binding to surface receptors. In a previous communication we reported the functional reconstitution of the fMLP receptor in Xenopus laevis oocytes (Coats, W. D., and Navarro, J. (1990) J. Biol. Chem. 265, 5964-5966). In this work we report the isolation of the cDNA encoding the fMLP receptor from neutrophils. A rabbit neutrophil cDNA library was screened with an oligonucleotide probe deduced from the nucleotide sequence of G-protein-coupled receptors, and a cDNA encoding the fMLP receptor was isolated. This cDNA was characterized according to the following criteria: 1) Analysis of the deduced amino acid sequence revealed that the clone belongs to a G-protein-coupled receptor. 2) Tissue distribution analysis of the mRNA indicated that the message is only found in neutrophils. 3) In vitro translation of the message revealed a protein corresponding in size to the deglycosylated fMLP receptor. 4) X. laevis oocytes injected with the fMLP receptor message exhibited fMLP-dependent calcium mobilization and specific binding to the fMLP analog 125I-labeled fNle-Leu-Phe-Nle-Tyr-Lys (where Nle is norleucine and fNle is formylnorleucine). The molecular cloning of the fMLP receptor should provide the framework to analyze the relationship between structure, function, and regulation of this receptor.

We observed a human urine-derived protein complex (IL-2-IN) which competitively inhibits interleukin-2 (IL-2) dependent murine lymphocyte proliferation. Measurements of urinary IL-2-IN have been used to stratify the immune response of patients to bacteria in the bladder. Partial characterization of IL-2-IN indicates that it is a heat-stable, 75 kDa complex comprised of interleukin-2 bound to another protein(s). Although the IL-2-IN complex is stable in physiologic buffers, the complex can be disrupted using acidic or low-ionic strength buffers, thereby liberating IL-2. IL-2-IN activity is susceptible to bacterial and endogenous urinary proteolysis. The IL-2 bound in the IL-2-IN complex cannot be detected using a double monoclonal antibody radioimmunoassay for IL-2. Unlike other IL-2 binding proteins, the IL-2 binding protein of the IL-2-IN complex is not a soluble interleukin-2 receptor. A modification of the bioassay for interleukin-2 activity is the method of choice for the detection and quantification of urinary IL-2-IN.

Crystals of 1,2 dilauroyl-DL-phosphatidyl-ethanolamine:acetic acid are monoclinic with a = 46.2, b = 7.77, c = 9.95 A, beta = 92.0 degrees ; space group P2(1)/c. The structural analysis, based on the visual estimates of 1467 reflection intensities, was achieved by direct methods, and least squares analysis convergence was to R(1) = 0.28. There are marked differences between the observed molecular conformation and those that have been predicted theoretically. The mean planes containing the lipid chains are essentially parallel to one another; the phosphodiester moiety has a double gauche conformation, while intermolecular hydrogen bonding modifies the conformation that could be anticipated for an isolated phosphatidylethanolamine molecule. The intermolecular packing produces the classical lipid bilayer structure, adjacent lipid bilayers being separated by acetic acid molecules of crystallization. The hydrocarbon chain packing can be considered either as a quasi-hexagonal type or as a complex orthorhombic subcell arrangement. One-dimensional electron density profiles across the lipid bilayer at increasing resolution clearly demonstrate the origin of features present on the low resolution profiles of both model and natural membranes.

The effects of the anabolic steroid trenbolone acetate (TBA) on adrenal function and hepatic enzyme activity have been studied in growing female rats. Treatment with TBA resulted in a decrease in the peak of plasma corticosterone concentration which occurred during the afternoon. The enzymes tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase were measured in the livers of treated and control rats. Activities of both enzymes were maximal at 22.00 h, but that of TAT was reduced in TBA-treated rats. After injection of ACTH, TBA-treated rats showed a smaller increase in plasma corticosterone than did controls. Treatment with TBA did not affect the induction of TAT activity after corticosterone treatment. All TBA-treated rats grew significantly faster than controls and the possible relevance of this reduced adrenal function to the increased growth rate is discussed.

A study of 190 tumors of major and minor salivary glands seen in Malawi over a nine-year period showed an overall incidence rate similar to that in Western countries. However, submandibular and palatal tumors are relatively more common, parotid tumors more likely to be malignant, and submandibular tumors less likely to be malignant than in Western countries. No adenolymphomas were seen. Despite the long history given by many patients with pleomorphic adenomas, the rate of malignant change in these tumors was not high; the majority of carcinomas (16% of all tumors) appeared to rise de novo. Histologic features were similar to those of tumors from other countries but the occurrence of tyrosine crystals in 20% of the pleomorphic adenomas was unusual. Melanocyte colonization of a mucoepidermoid tumor and a carcinoma, both in the primary site and in metastases of a salivary gland tumor, has not been previously reported.

A study was made of platelet monoamine oxidase (MAO) activity in non-medicated, newly-admitted schizophrenics and institutionalized chronic schizophrenics both on and off medication. These patients were compared to two control groups: normal subjects and brain-damaged institutionalized patients. No relationship was found between platelet MAO activity and the severity or duration of illness, duration of psychotropic medication, presence of auditory hallucinations or institutionalization. Mean platelet MAO activity did not differ significantly between the schizophrenic subgroups and control groups. Thirty-one patients studied before and after treatment with phenothiazines showed no significant change in platelet MAO activity. The findings did not indicate a relationship between schizophrenia, its treatment or outcome and platelet MAO activity.

The accurate measurement of serum interleukin-2 activity is crucial for assessing the efficacy and toxicity of systemic immunotherapy with recombinant interleukin-2. Incubation of serum at 56 degrees C for 30 minutes facilitates the bioassay for interleukin-2 activity by destroying the interleukin-2 inhibitory activity native to human serum. As this report will demonstrate, however, 30% to 50% of interleukin-2 activity in serum taken from patients or normal volunteers was destroyed by heating at 56 degrees C. No loss of recombinant interleukin-2 activity occurred during heating in serum-free media. The percentage of interleukin-2 activity lost at 56 degrees C varied from patient to patient and also varied with the time of exposure. Native serum interleukin-2 inhibitory activity can be removed, and interleukin-2 activity can be assessed accurately in serial dilutions of the serum beyond 1:64.

Chloroplast DNA from pearl millet (Pennisetum americanum) was used to construct recombinant plasmids. These plasmids contained 97 kilobase pairs of unique DNA sequences. The chloroplast DNA fragments in these plasmids were mapped with the restriction endonucleases SalI, SphI, XhoI, BglI and HpaI. The technique of overlapping hybridization or chromosome walking was used to orient these DNA fragments on a restriction endonuclease map of the chloroplast genome. The size of the chloroplast DNA from pearl millet was estimated in this fashion to be 127-138 kilobase pairs. Twenty one kilobase pairs of the cloned DNA fragments were represented twice on the genome as inverted repeats. Thus, the recombinant plasmids which were isolated contained approximately 86-93% of the nucleotide sequences in the chloroplast genome of pearl millet. Previously characterized cloned chloroplast DNA sequences from other plants were used as hybridization probes to locate the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase, the β-coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.

An inhibitor of interleukin-2 activity (IL-2-IN) is present in the urine of most patients during the acute phase of untreated bacterial cystitis (UTI). We measured urinary IL-2-IN activity in 30 adults with uncomplicated UTIs and followed the patients for an additional 6 months. Urinary IL-2-IN activity ranged between 0 and 1.97 units/mg urine creatinine (U/mg u.c.). Relatively low levels of IL-2-IN (less than 0.5 U/mg u.c.) correlated with a prior history of recurrent UTIs (p less than 0.01), and also were predictive of a subsequent UTI during the 6 month follow-up, regardless of the prior medical history (p less than 0.01). Measurement of urinary IL-2-IN during the untreated phase of a UTI may prove helpful for directing antibiotic prophylaxis against subsequent UTIs.