[The epaulette flap: replantation of osteomyocutaneous forearm segments in interscapulo-thoracic amputation].

Despite sophisticated artificial limb devices available today, forequarter amputation following trauma or tumor resection will lead to severe impairment of function. To close the posttraumatic or oncologically required extended defects and to improve prosthetic supplementation coverage of the thoracic wall should be performed by sparing the whole forearm as an osteomyocutaneous flap. In cases of concomitant serial rib resection radius and ulna will serve as stabilizators of the thoracic wall, thus avoiding a flail chest. Indications, technical details and clinical outcome of three salvage replantations after interscapulo-thoracic ablation will be discussed.


A short latency cortical component of the foveal VEP is revealed by hemifield stimulation.

Transient evoked potentials were recorded simultaneously over 5 electrodes placed in a horizontal row across the occiput. A range of spatial frequencies were presented as either full-field or hemifield stimuli. Subjects were 11 normal observers and 5 patients with lesions causing a homonymous hemianopic field defect. The shortest latency peak response was at approximately 70 msec, a negative potential (N70). For all spatial frequencies, full-field stimuli evoked a lower amplitude N70 at the midline than the sum of N70 amplitudes to two hemifield stimuli, suggesting partial cancellation. The latency and amplitude of N70 increased as spatial frequency increased. N70 and P100 differed in respect to their response to spatial frequency and field size, further suggesting that they may not be subsets of a unitary response. For hemifield stimulation, N70 had an ipsilateral maximum and attenuated or completely reversed in polarity across the midline. Consistent with the data of normals using hemifield stimuli, in 5 patients a full-field stimulus elicited an N70 lateralized contralaterally to the homonymous hemianopia, i.e., the ipsilateral N70 was absent. The absolute amplitude difference between the left and right electrodes was significant for hemifield stimulation in normals and full-field stimulation in the patients, but not for full-field stimulation in normals. Our results imply that the evaluation of N70 hemispheric distribution is useful for the evaluation of paramacular visual field defects.


A VEP study of the visual pathway function in compressive lesions of the optic chiasm. Full-field versus half-field stimulation.

Changes of the pattern reversal visual evoked potentials (VEPs) to half- and full-field stimulation in 50 patients with compressive lesions of the optic chiasm are presented. Temporal half-field stimulation yielded abnormal responses in 85% of the eyes, showing non-recordable P100 in 50% of eyes, while in 35% the P100 was significantly attenuated or delayed. With nasal half-field the percentage of all detectable abnormalities was lower (36% of the eyes). Full-field stimulation revealed VEP abnormalities in 74% of eyes and therefore proved less sensitive than the half-field stimulation. This study adds new evidence that half-field stimulation can be an important adjunct for assessing the function of the optic chiasm.


Subcortical contributions to the surface-recorded flash-VEP in the awake macaque.

Epidural mapping of flash-VEP in awake monkeys revealed a reliable, short latency negativity, N25 (onset: 18-22 msec; peak: 23-27 msec; duration: 15-20 msec), with a broad frontal surface distribution (frontolateral maximum). N25 was dissociable from the electroretinogram (ERG), from cortical VEP and from the high frequency oscillations (wavelets) coextensive with the ERG and with cortical VEP. Depth recordings traced N25 from its surface maximum down to the lateral geniculate nucleus (LGN). Concomitant VEP, current source density (CSD) and multiunit activity (MUA) profiles obtained with multicontact electrodes showed that the peak and later portion of N25 arise primarily from current sinks (associated with MUA increases) that reflect transmembrane current flow attending depolarization of cells in lamina 6, the uppermost lamina, but may also receive contributions from the more ventral LGN laminae. The initial portion of N25 arises from similar processes near the lamina 3/2 border. Wavelets, in contrast, are prominent in VEP, CSD and MUA within LGN, but attenuate rapidly above LGN. LGN laminar and cellular morphology predict volume conduction of N25 over a wide arc lateral and anterior to LGN and roughly horizontal isopotential planes medial and posterior to LGN. Recordings on the brain surface, within LGN, and in the regions surrounding LGN are consistent with these predictions. Possible contributions from other structures and how these results fit with data obtained in humans are considered.


Amplitude abnormalities in the scalp far-field N18 of SSEPs to median nerve stimulation in patients with midbrain-pontine lesion.

Various amplitude ratios were measured in 20 normal controls and 36 patients with midbrain-pontine, thalamic or putaminal lesions in order to evaluate the amplitude abnormalities in scalp far-field N18 following median nerve stimulation. A study of normal controls showed that the distributions of P9/N18, P14/N18 and N18/P14 + N18 resembled a gaussian distribution and could be used as criteria for determining the decrease in N18 amplitude in each patient. There was a decrease in N18 amplitude, or the absence of N18, in patients with midbrain-pontine lesions, but not in those with thalamic or putaminal lesions. Nine amplitude ratios (P11/P9, P14/P9, N18/P9, P9/P11, P9/P14, P9/N18, N18/P14, P14/N18 and N18/P14 + N18) were compared statistically for normal controls and 3 groups of patients based on non-parametric, Wilcoxon's non-pairs and signed-rank tests. A decrease in N18 amplitude in midbrain-pontine lesion was shown by significant changes in N18/P9, P9/N18, N18/P14, P14/N18 and N18/P14 + N18, no amplitude decreases in P11 and P14 being found from the amplitude ratios of P11/P9, P9/P11, P14/P9 and P9/P14. No significant changes were seen in any of the 9 amplitude ratios when the normal controls and patients with thalamic and putaminal lesions were compared. The amplitude ratios of N18 can be used to detect a decrease in N18 amplitude in patients with midbrain-pontine lesions. The data obtained support the hypothesis that N18 originates in the midbrain-pontine region and that neither the thalamus nor thalamocortical radiation make major contributions to the formation of the N18 peak.


The N30 component of somatosensory evoked potentials in patients with dystonia.

We recorded short-latency median nerve somatosensory evoked potentials (SEPs) in 10 patients with dystonia (6 with focal dystonia, 3 with generalized dystonia, and 1 with segmental dystonia) and compared them with those of 10 normal controls. The EEG was recorded from 29 sites on the scalp with linked earlobe electrodes for reference. Latencies and amplitudes of P15, postcentral N20 and P45, and frontal N30 were evaluated. The latencies of all potentials were the same in patients and controls. The amplitudes of P15, N20 and P45 were also the same in both groups, but the N30 amplitude of the patients was larger than of the controls. The amplitude of N30 did not vary from the affected side to the unaffected side. Previous work has shown decreased N30 amplitude in patients with Parkinson's disease. Changes in N30 amplitude may be indicative of abnormal excitatory effects on cortex resulting from disorders of the basal ganglia.


Current source density mapping of somatosensory evoked responses following median and tibial nerve stimulation.

Potential recording of brain activities always encounters the problem resulting from the activation of reference electrodes. Current source density (CSD) computation does not take reference sites into account and consequently may better localize the generator sources. In the past, several attempts have been made to record CSDs of the somatosensory evoked responses (SERs) following median nerve stimulation. In order to compare the generating mechanisms of SERs following median nerve and tibial nerve stimulation, the scalp CSD distributions of the median nerve SER and the tibial nerve SER were compared in 5 normal subjects. In the median nerve SER, far-field potentials such as P14 and N16 were abolished in the CSD records. N20, P25 and N35 showed almost identical CSD distributions, albeit P25 had a reversed polarity. By contrast, the tibial nerve SER showed similar distributions for P40 and P60 CSDs, but N50 had a different distribution from the others. In the potential records, P40 and P60 were distributed predominantly ipsilateral to the stimulus (paradoxical lateralization), whereas the P40 and P60 CSDs formed a dipole localized over the contralateral foot somatosensory area. N50 disclosed the same tendency, although it had a slightly different CSD pattern from that of P40 and P60. These findings suggest that the median nerve and tibial nerve SER components are not necessarily comparable and that under certain circumstances CSDs are better indicators of local electrical events than the corresponding potentials.


Steady-state vibration evoked potentials: descriptions of technique and characterization of responses.

Steady-state scalp evoked potentials were recorded in response to amplitude modulated vibration applied to the fingers and palmar surface of one hand. Evoked response dependence on the frequency of amplitude modulation in the 2-40 Hz range was studied in a group of normal young adult volunteers. Response amplitude was greatest at low amplitude modulation frequencies. The greatest signal to EEG noise ratios were found at modulation frequencies near 26 Hz. At modulation frequencies near 26 Hz the steady-state response latency was found to be 58 +/- 14 msec. Inverse dipole modeling localized the steady-state evoked response generators in or near somatosensory cortex with the dipole moment orientation being predominantly in the anterior-posterior direction.


Potentials evoked in human and monkey medial temporal lobe during auditory and visual oddball paradigms.

Event-related potentials (ERPs) were recorded from epileptic patients with electrodes chronically implanted in the medial temporal lobe (MTL) and other intracranial locations, and from monkeys with epidural, transcortical, and MTL electrodes. For both humans and monkeys, the eliciting events consisted of trains of auditory or visual stimuli in which a random 10-20% deviated in pitch or pattern from the remaining stimuli. The distribution of ERPs elicited by the rare (oddball) stimuli in both species was similar, consisting of a P3 recorded from the scalp or cortical surface and a slightly later, but temporally overlapping, focal negativity in the hippocampus and nearby MTL structures. The similarity between the patterns of ERPs in humans and monkeys establishes the feasibility of studying the electrogenesis of P3-like activity with detailed intracranial recordings in an animal model. The data also establish that the MTL ERPs in human patients represent a normal neurophysiological process unrelated to epilepsy.


The possible role of gliotoxin in health and disease.

Demonstration of the unique therapeutic properties of penicillin initiated an extensive investigation of other mould metabolites in the hope of finding other useful chemotherapeutic agents. While many hundreds of active substances were studied very few indeed satisfied the criteria necessary for clinical use. Among those whose toxicity ruled them out was gliotoxin. Recently, however a chance observation, in quite a different context, suggests that gliotoxin may prove valuable in human tissue transplantation.


Sensory mechanisms on the molecular level.

As sensory perception is the channel by which we learn about the nature of the world around us it has been the subject of philosophical inquiry since classical antiquity. With the emergence of science in its modern sense in the 17th century, experimental investigation began to be possible but it was only with the relatively recent advent of molecular biology that sensory perception can be studied at its basic level.


The effect of different high-pass filter settings on peak latencies in the event-related potentials of schizophrenics, patients with Parkinson's disease and controls.

Eighteen schizophrenic patients, 16 patients with idiopathic Parkinson's disease, and the same numbers of age, sex and education matched controls were examined with oddball experiments for the generation of P3. Individual averages were high-pass filtered at different cut-off frequencies with single-pole digital filters with equivalent analogue Butterworth filter profiles. The purpose of this procedure was to simulate analogue high-pass filters used in clinical studies from different centres and to examine their potential effect on group differences. Increasing high-pass filters resulted in a phase lead for all peaks examined (N1, P2, N2, P3). The only group differences were found for P3, which showed a greater phase lead in controls than in the patient groups, usually resulting in a more pronounced group difference. Similar wave forms and filter properties could be modelled by synthetic wave forms consisting of sine waves of different frequencies.


Creation of an autocrine model of insulin-like growth factor-I action in transfected FRTL-5 cells.

Although there is much evidence that insulin-like growth factor-I (IGF-I) is delivered to its target tissues via the circulation from distal sites of synthesis, many other observations suggest that it is synthesized in or near its target tissues and acts by autocrine and/or paracrine modalities. Studies of the mechanisms of such local actions, however, have been problematic, because in vivo studies of a single tissue are technically difficult and confounded by many variables, whereas in vitro studies of autocrine/paracrine actions have been limited by low levels of IGF-I expression and/or lack of dramatic or clearly defined responses to IGF-I. We, therefore, set about to create IGF-I expression in FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells that have been extensively studied as a model of TSH action. The modest increase in thymidine incorporation stimulated by TSH in wild type FRTL-5 cells is markedly increased in the presence of exogenous IGF-I. By transfecting these cells with a chimeric IGF-IA gene, driven either by the mouse metallothionein-1 or IGF-II 5' genomic regulatory regions, we were able to generate stable cell lines that synthesize and secrete mature IGF-I. This was demonstrated by RIA, by Northern analysis, and by polyacrylamide gel electrophoresis characterization of the radiolabeled intracellular and extracellular products that reacted with an IGF-I antibody. The mitogenic responses to TSH in IGF-I-expressing transfected FRTL-5 cells were indistinguishable from those stimulated by TSH and IGF-I in wild type or control-transfected cells (FRTL-5 cells stably transfected with a similar transgene that does not encode IGF-I). Basal DNA synthesis was higher and the peak of thymidine incorporation was earlier in IGF-I-expressing transfected FRTL-5 cells than in wild type or control cells (18-24 h vs. 30-36 h). The concentrations of TSH that maximally stimulate the incorporation of thymidine were not altered by IGF-I expression, and transfected cells did not appear to be transformed, as judged by their inability to form colonies in soft agar. TSH-stimulated DNA synthesis was blocked in IGF-I-expressing FRTL-5 cell by a monoclonal antibody to IGF (Sm 1.2). Thus, secretion of IGF-I appears to be required for the autocrine effects observed. These IGF-I-expressing FRTL-5 cell lines provide a model in vitro system to study the intracellular processing of IGF-I and the mechanisms by which IGF-I acts in an autocrine manner.


Mental movement simulation affects the N30 frontal component of the somatosensory evoked potential.

It is known that somatosensory evoked potentials can be influenced by several centripetal and centrifugal factors which modify their amplitude. The present study shows for the first time that the frontal waves N30 and to a lesser extent N23 are specifically attenuated during mental movement simulation (MMS) activity. This gating phenomenon, tested on 16 normal subjects, occurred when repeated fast finger movements on the stimulated side are mentally simulated. In contrast, no significant modification appeared when the subject performed the MMS activity with the hand contralateral to that receiving electrical stimuli or when the subject performed a mental operation unrelated to the MMS. The MMS was shown by Roland et al. (1980) to increase the regional blood flow exclusively in the supplementary motor area (SMA). Our experimental data therefore indicate that the SMA could play an important role in the generation of the frontal N30.


Brain-stem auditory evoked potentials in adults with Down's syndrome.

Brain-stem auditory evoked potentials (BAEPs) were examined in 37 adult patients with Down's syndrome and in 37 age-matched normal subjects. All absolute and interpeak latencies except for the interpeak latency IV-V were shorter in patients than in normal subjects. The amplitude of wave V and the amplitude ratio V/I were smaller in patients than in normal subjects. Short latencies in patients were considered to be due to the smaller size of the brain-stem or to faster conduction velocity. The prolonged interpeak latency IV-V and the smaller wave V may indicate physiological dysfunctions between the upper pons and the lower midbrain.


Middle latency auditory evoked potentials improve the detection of abnormalities along auditory pathways in multiple sclerosis patients.

Brain-stem and middle latency auditory evoked potentials (BAEPs and MLAEPs) have been studied in 34 multiple sclerosis (MS) patients. We were able to detect a central nervous system auditory pathway involvement in 17 (50%) of the patients: 38% by BAEPs alone (I-V inter-peak latency) and 47% by MLAEPs alone (Na and Pa peak latency). Five patients had abnormal MLAEPs with normal BAEPs whereas the opposite was detectable in only 1 patient. In addition, most MLAEP parameters in the MS group statistically differed from those obtained in the control group. Therefore, our results demonstrated that the auditory pathway impairment could frequently be located at a rostral level along the auditory radiation. In conclusion, even if only Na and Pa components were considered, MLAEPs succeeded in improving the sensitivity of the auditory evoked potential examination without increasing the false positive rate.


Effects of rat alpha-fetoprotein administration on estradiol free fraction, the onset of puberty, and neural and uterine nuclear estrogen receptors.

The cause of the onset of puberty in the rat or any other mammalian species is unknown. According to one theory, puberty is initiated through switching of the brain "gonadostat." It is hypothesized here that puberty in the rat is the consequence of the appearance of free, and therefore physiologically active, estrogen in the circulation. To test this, the unbound fraction of estradiol in serum of immature female rats was measured in relation to the nuclear receptor occupancy of estradiol in the hypothalamus, preoptic area, and uterus at various times after birth. In addition, an attempt was made to alter the free fraction of estradiol by injection of the estradiol-binding protein alpha-fetoprotein (AFP) into immature female rats. The free fraction of estradiol was low (less than 1%), but began rising at about 20 days of age, and a significant increase in nuclear bound estradiol was observed in 23-day-old rats (P less than 0.001). By day 30, unbound levels attained adult values (3.99 +/- 0.15%). At this time, nuclear bound estradiol in all tissues examined fell (P less than 0.01), but by day 40, these were greatly increased in rats in estrus (P less than 0.001), being trebled in the preoptic areas and doubled in the hypothalamus. Injection of AFP into immature female rats extended the period of low free estradiol (1.22 +/- 0.08%), while in albumin-injected rats, the free fraction was 4.44 +/- 0.1%. Injection of AFP resulted in levels of nuclear-bound estradiol that were less than half those measured in nuclei from AFP-injected animals (P less than 0.001), and AFP delayed puberty. The affinity of the reaction between estradiol and nuclear receptors in brains of immature and mature rats was not significantly different; the Kd fell within the range of 0.05-0.08 nM. It is suggested that in the rat, puberty is the result of the appearance in the circulation of physiologically active estradiol after day 20.


Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism.

In this study we investigated the mechanism(s) by which insulin-like growth factor-binding protein-3 (IGFBP-3) potentiates IGF-I action in cultured bovine fibroblasts. Preincubation of cells with glycosylated or nonglycosylated recombinant human IGFBP-3 enhanced responsiveness to IGF-I in a time-dependent manner. A preincubation period of at least 24 h with IGFBP-3 was required to see a significant effect. Pretreatment with IGFBP-3 for 72 h resulted in a 2- to 4-fold augmentation of IGF-I-stimulated [3H]aminoisobutyric acid uptake; IGFBP-3 had no effect on basal [3H]aminoisobutyric acid uptake. During the preincubation period, exogenous IGFBP-3 associated with the fibroblast surface and exhibited time-dependent processing to lower mol wt forms that retained the ability to bind radiolabeled IGF-I. Initial surface adherence (preincubation time of 24 h or less) was readily reversible. However, IGFBP-3, once processed, appeared to be closely associated with the cell. After 72 h of exposure to bovine fibroblasts, cell-associated IGFBP-3 had a 10-fold lower affinity for IGF-I compared to intact IGFBP-3 in solution. In addition, incubation of bovine fibroblasts with IGFBP-3 had modifying effects on type I IGF receptor-mediated signalling because 1) the bioeffectiveness of [Gln3,Ala4,Tyr15,Leu16]IGF-I and insulin, IGF-I receptor activators with little or no affinity for IGFBP-3, was potentiated by preincubation with IGFBP-3; and 2) fibroblast responsiveness to IGF-I analogs with different affinities for the type I IGF receptor was enhanced in direct relation to the ability of the peptide to bind to the receptor. There was no evidence for an increase in receptor number or affinity as a result of IGFBP-3 treatment. These data suggest that IGFBP-3 potentiation of IGF-I action in bovine fibroblasts may involve changes in IGFBP-3 and type I IGF receptor responsiveness. Thus, cell-associated IGFBP-3 may provide a mechanism for optimal presentation of IGF-I to its receptor as well as a means to heighten receptor reactivity to IGF-I and related peptides.


Progesterone inhibits the estrogen-induced expression of c-fos messenger ribonucleic acid in the uterus.

Estradiol produces a large increase in the uterine level of c-fos mRNA, which is maximum in 3 h. The administration of progesterone antagonizes this estrogen-induced increase in protooncogene transcript levels in both the rat and mouse. The inhibitory effect of progesterone is observed within 1 h after hormone treatment and persists for 9-18 h. In the rat, this effect can be observed at a dose of 0.25 mg progesterone and is maximum at a dose of 2.5 mg. A similar inhibition of fos mRNA levels after estrogen administration is produced by the glucocorticoid dexamethasone, but not by androgens or mineralocorticoids. Progesterone does not block the induction of c-jun or c-myc mRNA by estradiol. Uterine levels of c-fos mRNA observed after treatment with the phorbol ester phorbol 12-myristate 13-acetate are not decreased by a 3-h pretreatment with progesterone. Under the conditions of our experiments, progesterone does not decrease occupied levels of nuclear estrogen receptors in the uterus after estradiol administration. These findings are consistent with a mechanism in which progesterone inhibits transcriptional activation by the estrogen receptor at the level of the c-fos gene.


Anatomical relationships in the patterns of insulin-like growth factor (IGF)-I, IGF binding protein-1, and IGF-I receptor gene expression in the rat kidney.

The rat kidney is both a target of circulating insulin-like growth factor-I (IGF-I) and a site of local IGF-I production. In order to identify which renal structures produce IGF-I and the functionally related IGF binding protein 1 (IGFBP-1), and which structures are potential sites of circulating or endogenous renal IGF action, we have employed in situ hybridization to localize IGF-I, IGFBP-1, and IGF-I receptor messenger RNAs (mRNAs) in the rat kidney. The effects of hypophysectomy (Hx) and GH replacement on renal IGF-I, IGFBP-1, and IGF-I receptor gene expression have also been evaluated. IGF-I and IGFBP-1 mRNAs are both localized in the epithelial cells of medullary thick ascending limbs (TALs) of Henle's loops in the normal rat kidney. IGF-I receptor mRNA is also abundant in TALs, but, in addition, is distributed throughout the distal nephron and collecting duct, and in the glomerulus, with lowest levels found in proximal tubules. Hx and GH treatment had complex effects on patterns of renal IGF-I and IGFBP-1 gene expression. In general, Hx resulted in decreased IGF-I and increased IGFBP-1 mRNA levels, and GH treatment produced the opposite effects, while IGF-I receptor mRNA levels were not significantly effected by either treatment. However, the most dramatic effect produced by the interruption of the pituitary-renal axis was the demonstration of reciprocal changes in IGF-I vs. IGFBP-1 gene expression in individual kidneys and even in individual nephrons, suggesting a local interaction between IGF-I and IGFBP-1 in the regulation of their respective mRNA levels. Functional implications issuing from these anatomical relationships in renal patterns of IGF-I, IGFBP-1, and IGF-I receptor gene expression are that IGF-I, if secreted into the tubular lumen, possibly carried or modulated by IGFBP-1, may act on luminal TAL and downstream receptor sites. The specific physiological role of IGF-I produced in TALs is open to speculation. Glomerular IGF-I receptor sites, based on their localization upstream and distant from local sources of IGF-I production, are predicted to be targets for circulating IGFs.


Differential regulation by growth hormone (GH) of insulin-like growth factor I and GH receptor/binding protein gene expression in rat liver.

We have reported that female hypophysectomized (hypox) rats replaced with T4 and cortisone and treated for 7 days with GH injections (4 x 12.5 micrograms/day) had significantly greater growth and increase in serum insulin-like growth factor-I (IGF-I) than did hypox rats continuously infused with GH (50 and 250 micrograms/day), whereas GH binding to liver membranes was increased only by infusion. We now report the effects of hypophysectomy, T4 and cortisone replacement, and the aforementioned continuous vs. intermittent GH treatment on liver IGF-I and GH receptor (GHR)/binding protein (GHBP) gene expression in female rats. Concentrations of IGF-I peptide were measured in acid-extracted sera and liver tissues. Total GH binding to liver membranes was determined in MgCl2-treated homogenates and serum GH binding activity was assessed by gel filtration of serum incubated with 125I-bovine GH. The abundance of messenger RNA (mRNA) transcripts encoding IGF-I, the GHR, and the GHBP was quantified by Northern hybridization analysis of liver poly(A)+ RNA. Hypophysectomy in female animals decreased serum IGF-I and liver IGF-I mRNA concentrations by 95% and 87%, respectively, serum GHBP activity, and total liver GH binding by 50% (P less than 0.001 compared with intact controls), and liver GHR and GHBP mRNA abundance by 30-35% (P less than 0.05 vs. controls). These changes were not reversed by T4 and cortisone treatment. Repeated injections of GH produced a 13-fold increase in liver IGF-I peptide and a 5-fold increase in liver IGF-I mRNA concentrations (vs. saline-treated hypox rats), whereas continuous GH infusions induced only 7-fold and 2-fold increases in IGF-I peptide and mRNA, respectively. Serum GHBP activity was not changed in the GH-injected animals, but rose 2- to 3-fold in the GH-infused rats, an increase similar to that reported for their liver GH binding sites. No major change in liver concentrations of GHR and GHBP mRNAs was seen after repeated GH injections. Differential regulation of the two GHR/GHBP gene products was observed after continuous infusion of GH, with a net 60-70% increase in liver GHBP mRNA abundance contrasting with no apparent change in the GHR mRNA transcript. These results indicate that pulsatile GH administration is more effective than continuous GH infusion in stimulating liver IGF-I gene expression, and this effect is not mediated by an increase in GHR mRNA or protein.(ABSTRACT TRUNCATED AT 400 WORDS)


Steroids and tissue-specific modulation of galanin gene expression in the male rat reproductive system.

Galanin is a neuropeptide widely distributed throughout the vertebrate neural and endocrine system. Galanin can influence pituitary hormone secretion, intestinal motility, and other biological activities. The precise physiological role of galanin is unknown. We studied the control of galanin gene expression in peripheral organs in the male rat using Northern blot and in situ hybridization techniques. In the adrenals and prostate, galanin mRNA was undetectable in the controls and did not change after the administration of dexamethasone (0.0001-10.0 mg/kg, ip) and diethylstilbestrol (0.1 mg/kg, ip). In the testis, thymus, seminal vesicles, medial basal hypothalamus, and colon, galanin message was detectable, but was not influenced by steroids. On the other hand, dexamethasone (0.5-10.0 mg/kg) was very effective in enhancing galanin expression in the vas deferens and epididymis (4- to 7-fold in the vas deferens), with a peak 6-9 h after the treatment. Diethylstilbestrol (0.1 mg/kg) stimulated galanin mRNA transcription only in the vas deferens (2- to 3-fold), with a peak 1-3 h after the treatment. Dihydrotestosterone treatment (0.2-0.4 mg/kg) was ineffective in all tissues examined. In the vas deferens and seminal vesicles, galanin mRNA has been localized at a cellular level by in situ hybridization. In these tissues only fibroblast-like cells contained the message. These data demonstrate that galanin is expressed in the male rat reproductive system and that steroid hormones participate in the control of galanin gene expression in a tissue- and hormone-specific fashion.


Steroidal regulation of hypothalamic neuropeptide Y release and gene expression.

Neuropeptide Y (NPY) readily stimulates the release of hypothalamic LHRH and pituitary LH release in intact and gonadal steroid-primed gonadectomized rats. We have now tested the hypothesis that the release and synthesis of hypothalamic NPY may be regulated by gonadal steroids. To measure the effects of gonadal hormones on NPY release, a permanent push-pull cannula was implanted in the anterior pituitary (AP) of sham castrated (controls) or castrated (CAST) male rats, and 1 week later, the AP was perfused with artificial cerebrospinal fluid over a 3-4 h period. NPY concentrations in the perfusates collected at 10-min intervals were measured by RIAs. The NPY release pattern in the AP was episodic in both intact and CAST rats, and the frequency of NPY episodes was similar in two groups. However, the amount of NPY detected in the AP of CAST rats was significantly less than that of intact rats because the mean rate of release and the amplitude of NPY episodes in the perfusates of CAST rats were significantly reduced. This observation of attenuated hypothalamic NPY output in vivo and previous evidence of decreased hypothalamic NPY contents after CAST implied that the synthesis of hypothalamic NPY may be regulated by testicular secretions. Therefore, the effects of testosterone (T)-replacement on preproNPY messenger RNA (mRNA) in the medial basal hypothalamus (MBH) was evaluated. Rats were CAST and received either empty or T-filled Silastic capsules sc. Two weeks later, the level of perproNPY mRNA in the MBH was determined by solution hybridization/ribonuclease protection assay using a complementary RNA probe complementary to the rat NPY precursor mRNA. We observed that the levels of preproNPY mRNA were 2-fold higher in the MBH of T-replaced CAST as compared to control CAST rats. These findings are consistent with the hypothesis that gonadal steroids enhance the neurosecretory activity of hypothalamic NPYergic neurons, and for the first time reveal a coupling between the level of gene expression and the secretion of a neuropeptide involved in the regulation of hypothalamic LHRH and pituitary LH release.


Protein phosphatase inhibitors and bone resorption: inhibition by okadaic acid and biphasic actions of calyculin-A.

PTH receptors on osteoblasts and calcitonin receptors on osteoclasts are coupled to adenylate cyclase. Despite similar transduction mechanisms, these hormones have opposing physiological actions. We investigated the consequences of persistent protein phosphorylation on bone resorption in neonatal mouse calvariae using okadaic acid (OA) and calyculin-A, two inhibitors of protein phosphatase-1 and -2A. These two inhibitors caused different responses in bone at picomolar and low nanomolar concentrations. OA inhibited, in a dose-dependent manner, bone resorption stimulated by PTH, 1,25-Dihydroxyvitamin D3, phorbol ester, and prostaglandin E2 (PGE2). OA did not inhibit the generation of the second messengers cAMP or PGs and did not have nonspecific toxic effects, as measured by protein and RNA synthesis. Thus, OA appeared to mimic the global inhibitory action of calcitonin on bone resorption. Unlike OA, calyculin-A elicited a biphasic dose response. At concentrations of 3.3 nM and greater, calyculin-A inhibited, in a dose-dependent manner, stimulated bone resorption. However, calyculin-A alone, at 0.625 and 2.5 nM, stimulated bone resorption via a PG-independent pathway. In calvariae, OA and calyculin-A increased phosphorylation of a 58- to 60-kilodalton protein. A protein of similar molecular mass was hyperphosphorylated in OA-treated ROS 17/2.8 osteoblast-like cells. We conclude that in addition to hormonal regulation of protein kinase activity, protein dephosphorylation plays a functionally important role in the modulation of bone resorption.


Induction of avidin messenger ribonucleic acid in the chick oviduct by progesterone and other steroids.

Avidin gene expression was analyzed using an avidin immunoassay and RNA hybridization analysis. To ascertain whether the induction of the avidin gene by progesterone remains specific also during secondary restimulation with diethylstilbestrol, chicks were given different steroid hormones or hormone combinations. Progesterone-specific induction of avidin protein and messenger RNA (mRNA) was 15- to 30-fold over the control even after secondary restimulation with diethylstilbestrol. A functional difference between the progesterone response element and glucocorticoid response element was suggested, since dexamethasone alone did not induce avidin in vivo. In spite of progesterone specificity, a combination of progesterone with other steroids nevertheless generated a synergistic increase in the amount of avidin mRNA. This may indicate that binding of progesterone receptor to the progesterone response element may be important to alter the functional activity of other hormone response elements present on the avidin gene. The time response curve of the avidin mRNA induction by progesterone was also determined. Avidin mRNA was detectable 8 h after progesterone induction, and its amount was maximal after 16-24 h. This would indicate that the avidin gene belongs in the so-called late responder genes, which also include chicken ovalbumin, ovomucoid, and lysozyme genes.


Expression of transforming growth factor-beta isoforms (beta 2 and beta 3) in the mouse uterus: analysis of the periimplantation period and effects of ovarian steroids.

Expression of beta-type transforming growth factor genes (TGF beta 2 and TGF beta 3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17 beta-estradiol (E2) and progesterone (P4) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGF beta 2 or TGF beta 3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGF beta 2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGF beta 2 immunostaining was also detected in decidual cells. In contrast, TGF beta 3 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (D1-D8). Antisense TGF beta 2 and TGF beta 3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF beta 2 transcripts (approximately 6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)+ RNA on D1-D6 and in poly(A)+ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF beta 2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGF beta 3 transcript in total uterine poly(A)+ RNA on D1-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGF beta 2 and TGF beta 3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF beta 2 or TGF beta 3 mRNA persisted in ovariectomized mice. However, an injection of E2 induced a rapid (6 h), but transient, induction (approximately 3- to 4-fold) of TGF beta 2 mRNA. An injection of P4 had no effect on TGF beta 2 mRNA levels, and coinjection of P4 with E2 did not antagonize the E2-stimulated transient accumulation of TGF beta 2 mRNA. In comparison, neither an injection of E2 nor one of P4 exerted significant effects on TGF beta 3 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)


Regulation of avian calbindin-D28K gene expression in primary chick kidney cells: importance of posttranscriptional mechanisms and calcium ion concentration.

Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.


Production of mitogenic factor(s) by ovine corpora lutea throughout the estrous cycle.

To evaluate secretion of mitogenic factors by ovine corpora lutea (CL) at several stages of development, luteal explants from days 5 (n = 12 ewes), 10 (n = 6 ewes), and 15 (n = 6 ewes) of the estrous cycle were incubated in serum-free medium for 24 h. Luteal-conditioned media (LCM) were evaluated for their ability to stimulate proliferation of endothelial, BALB/3T3, and ovarian granulosa cells. After mitogenic activity of LCM from individual animals was evaluated, pools of LCM from each day of the estrous cycle were subjected to anion-exchange, cation-exchange, and heparin-affinity chromatography to characterize mitogenic activity. Pools of LCM also were utilized for ultrafiltration, heat-treatment, trypsin-treatment, and immunoneutralization studies. Results demonstrated that ovine CL secrete mitogenic activity that stimulates (P less than 0.01) proliferation of endothelial (135.7 +/- 5.3% of control) and granulosa cells (188.9 +/- 2.9%) but not 3T3 (103.2 +/- 2.5%) cells. Differences between stages of luteal development were not observed. The mitogenic activity bound to diethylaminoethyl-Sephacel and heparin-agarose, but not to carboxymethyl-Sepharose, indicating that ovine luteal mitogenic factor(s) is anionic and may belong to the heparin-binding growth factor (HBGF) family. In addition, the mitogenic activity was heat-labile, trypsin-sensitive, and appeared to have a M(r) greater than 100,000. Mitogenic activity for endothelial cells was partially neutralized with a specific antibody against HBGF-1 and was completely abolished with a specific antibody against HBGF-2. Moreover, HBGF-1 and HBGF-2 were immunolocalized in histological sections of CL from days 5 (n = 5 ewes), 10 (n = 5 ewes), and 15 (n = 5 ewes) after estrus. These findings are the first report of a major mitogenic factor(s) produced by cyclic ovine CL and indicate this factor is an HBGF-2-like molecule.


Retinoic acid regulates insulin-like growth factor II expression in a neuroblastoma cell line.

Insulin-like growth factors (IGF-I and IGF-II) are mitogenic polypeptides that play an important role in normal growth and development. IGF-II has been shown to stimulate the growth of neuroblastoma tumors in an autocrine and paracrine fashion. Critical in determining the role of IGF-II in tumorigenesis is the necessity to delineate factors affecting the transcription of IGF-II in normal and tumor tissues. To date such factors are poorly characterized. In this study we find that retinoic acid (RA), a naturally occurring morphogen, that has been shown to be indispensable in the development of the chick limb bud, stimulates an increase in IGF-II messenger RNA (mRNA) in the Lan-1-15N neuroblastoma cell line. This increase in IGF-II is coincident with RA mediated inhibition of DNA synthesis. An increase in the steady state levels of IGF-II mRNA is detectable within 2 h of RA treatment and maximal by 24 h. In RA-treated Lan-1-15N cells, IGF-II mRNA levels are regulated at the level of new gene transcription and result in an increase in IGF-II protein in the culture supernatant. These studies suggest one mechanism affecting the production of IGF-II in vivo may be mediated by RA and detail a model system by which transcriptional regulation of IGF-II mRNA can be analyzed.


Follistatin steady state messenger ribonucleic acid levels in confluent cultures of a rat renal mesangial cell line are regulated by multiple factors.

The regulation of steady state levels of follistatin (FS) messenger RNA (mRNA) was examined in a rat renal mesangial cell line in tissue culture. A specific 32P-radiolabeled antisense probe was used which corresponds to the 3' end of exon 5 together with the 5' end of exon 6 of the rat FS gene, and which distinguishes between the two different forms of FS mRNA. In addition, a specific 35S-radiolabeled probe for the ubiquitous protein cyclophilin was developed and used as an internal standard. Total RNA was harvested from confluent cell cultures to yield four independent samples per treatment/time point, and equal amounts of RNA from every sample in a given experiment were subjected to S1-nuclease analysis for the estimation of specific mRNA levels. Treatment of the cultured cells with epidermal growth factor (10 nM) caused an 8- to 9-fold increase in the FS mRNA level after 4 h, but no consistent change was observed after treatment with basic fibroblast growth factor (0.28 or 0.56 nM), somatostatin (3.7-73 nM), angiotensin II (0.1-2500 nM), or FS itself (0.29 nM) for between 4 and 48 h. Neither activin (0.5 or 1.2 nM) nor inhibin (0.64 nM) changed the FS mRNA level in the mesangial cell line during a 24-h treatment. FS mRNA levels in the cells also were not affected by a 48-h treatment with the steroids dihydrotestosterone (1-1000 nM), estradiol (1 and 100 nM), and the antiprogesterone RU 486 (1000 nM), whereas 100 nM RU 28362 (a synthetic glucocorticoid) caused a 5- to 6-fold increase and 1000 nM progesterone increased the FS mRNA level up to 3.5-fold above control. Retinoic acid, a vitamin A derivative, significantly increased the FS steady state mRNA level at 3 nM, and at 1000 nM stimulated FS mRNA up to 5-fold within 4 h, whereas incubation of the cells with 30 microM prostaglandin E2 for 4 h caused a 10-fold increase. The FS mRNA level increased 3- and 4-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, 13-acetate, and 25 microM forskolin, respectively, whereas the calcium ionophore A23187 (1-100 microM) caused no change within this timespan. None of the tested hormones had an obvious effect on the ratio of the two different forms of FS mRNA (FS 344:FS 317).(ABSTRACT TRUNCATED AT 400 WORDS)


Purified human alpha fetoprotein inhibits growth factor-stimulated estradiol production by porcine granulosa cells in monolayer culture.

Purified alpha fetoprotein (AFP) synergizes with transforming growth factor alpha (TGF alpha) and insulin-like growth factor I (IGF-I) to enhance proliferation of porcine granulosa cells (pGC) in primary culture, suggesting a role for AFP in the modulation of growth factor-mediated cell growth. TGF alpha stimulates basal estrogen production by pGC and is in fact more potent than FSH in these cells. In this study, we investigated the effects of AFP on growth factor-stimulated estradiol (E2) production by pGC. Basal production of E2 was not altered by the addition of AFP. AFP dose-dependently inhibited TGF alpha-stimulated E2 production with statistically significant inhibition observed with 2.5 micrograms/ml. We have previously shown that the mitogenic effects of AFP are maximized with TGF alpha+IGF-I. E2 production was even more sensitive to AFP inhibition when the two growth factors were combined. Human serum albumin (HSA; 10 micrograms/ml) was without effect. AFP did not interfere with the E2 RIA, affect the uptake of or display specific in vitro binding of the androgen substrate. Furthermore, human AFP and HSA did not exhibit specific in vitro binding of E2, in contrast to purified rat AFP (positive control). These data indicate that physiological concentrations of purified AFP significantly and dose-dependently inhibit growth factor-stimulated E2 production by pGC in culture. Since AFP is known to increase TGF alpha+IGF-I mediated cell growth, these data suggest that AFP may be inhibiting the differentiated function (steroidogenesis) of pGC while enhancing the proliferation of these cells.


Enhanced histamine release from lung mast cells of guinea pigs exposed to sulfuric acid aerosols.

To clarify the relationship between air pollution and mast cell response, the effects of sulfuric acid aerosols on histamine release from lung mast cells of guinea pigs were investigated. Guinea pigs were exposed to 0.3, 1.0 and 3.2 mg/m3 sulfuric acid (H2SO4) aerosols or 4 ppm nitrogen dioxide (NO2) for 2 and 4 weeks. After the exposure, lung mast cell suspensions were isolated by collagenase treatment and antigen- or A23187-induced histamine release was measured. Antigen-induced histamine release from mast cells was significantly enhanced by the exposure to 1.0 and 3.2 mg/m3 H2SO4 for 2 weeks, but exposure to H2SO4 for 4 weeks did not show the enhancement of antigen-induced histamine release. A23187-induced histamine release was significantly enhanced by the exposure to 1.0 mg/m3 H2SO4 or 4 ppm NO2 for 2 weeks, but suppression of histamine release from lung mast cells stimulated with A23187 was observed by the exposure to 3.2 mg/m3 H2SO4 for 4 weeks. The exposure to 0.3 mg/m3 H2SO4 showed no changes in antigen- and A23187-induced histamine release. The combination of 1.0 mg/m3 H2SO4 with 4 ppm NO2 for 2 weeks resulted in no changes in antigen- and A23187-induced histamine release. These results suggested that functional properties of lung mast cells may be altered by a low concentration of H2SO4 aerosol exposure.


Small RNA helices as substrates for aminoacylation and their relationship to charging of transfer RNAs.

RNA microhelices that reconstruct the acceptor stems of transfer RNAs can be aminoacylated. The anticodon-independent aminoacylation is sequence-specific and suggests a relationship between amino acids and nucleotide sequences which is different from that of the classical genetic code. The specific aminoacylation of RNA microhelices also suggests a highly differentiated adaptation of the structures of aminoacyl-tRNA synthetases to sequences in the acceptor stems of transfer RNAs.


Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.


Attenuation in the rph-pyrE operon of Escherichia coli and processing of the dicistronic mRNA.

We have substituted on a plasmid the native promoter of the Escherichia coli rph-pyrE operon with an inducible transcription-initiation signal. The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following induction of transcription. The results showed that dicistronic rph-pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNa was the major transcription product in high-UTP pools, thus supporting an UTP-controlled attenuation mechanism for regulation of pyrE gene expression. However, the dicistronic rph-pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high-UTP pools. Furthermore, the major 3' end of the pyrE mRNA was mapped near a palindromic structure of similarity to the family of repetitive extragenic palindromic sequences, 35 nucleotide residues after stop codon of the pryE gene.


Determination of the nucleotide sequences in mouse U14 small nuclear RNA and 18S ribosomal RNA responsible for in vitro intermolecular base-pairing.

U14 small nuclear RNA (snRNA) is an evolutionarily conserved RNA species that plays a role in rRNA processing. The conserved ability of fungal, amphibian and mammalian U14 snRNAs to hybridize with both homologous and heterologous eukaryotic 18S rRNAs indicates a potential role for this intermolecular RNA/RNA interaction in U14 snRNA function. To understand better the possible role of this intermolecular base-pairing in rRNA processing, we have defined those nucleotide sequences in mouse U14 snRNA and 18S rRNA responsible for the observed in vitro hybridization. We have constructed, using synthetic DNA oligonucleotides, a U14 snRNA gene which has been positioned behind a T7 RNA polymerase promoter site and then inserted into a plasmid. The presence of natural or engineered restriction endonuclease sites within this construct has permitted the in vitro transcription of full-length mouse U14 snRNA transcripts (an 87-nucleotide mouse U14 snRNA minus 5' or 3' leader sequences) or 3' terminally truncated U14 snRNA fragments. Hybridization of full-length or truncated fragments of U14 snRNA to mouse 18S rRNA demonstrated the utilization of a previously proposed 18S rRNA complementary sequence located near the 3' end of mouse U14 snRNA (nucleotides 65-78) for intermolecular hybridization. Conversely, RNase-T1-generated fragments of 18S rRNA capable of hybrid-selection by U14 snRNA have been isolated and sequenced. A nested set of hybrid-selected 18S rRNA fragments define a mouse 18S rRNA sequence (nucleotides 459-472) which exhibits perfect complementarity to the defined U14 snRNA sequence 65-78. Primer-extension/chain-termination mapping of mouse U14-snRNA.18S-rRNA hybrids has confirmed the formation of the proposed hybrid structure. A second set of observed complementary sequences in mouse U14 snRNA (nucleotides 25-38) and mouse 18S rRNA (nucleotides 82-95) are not used for the in vitro hybridization of these two RNAs. Presumably the involvement of this second 18S-rRNA-complementary sequence in the secondary/tertiary folding of mouse U14 snRNA prevents its base-pairing with 18S rRNA. However, the strong evolutionary conservation of both U14-snRNA.18S-rRNA hybrid structures and their juxtapositioning within the folded secondary structure of 18S rRNAs argues for a biological role for each in U14 snRNA function.


Murine endothelial leukocyte-adhesion molecule 1 is a close structural and functional homologue of the human protein.

Human endothelial leukocyte-adhesion molecule 1 (ELAM-1), a cell-surface glycoprotein expressed solely on cytokine-activated endothelial cells, mediates the adhesion of blood neutrophils, memory T-cells and some monocytes. ELAM-1, also known as E-selectin or leukocyte endothelial-cell-adhesion molecule 2, is a member of the lectin/epidermal-growth-factor/complement-regulatory-protein-like cell-adhesion molecule family, which includes structurally related molecules referred to as selectins. They are all involved in cell/cell adhesion, playing roles in leukocyte trafficking which are currently only partially defined. We report here the isolation and characterization of the murine equivalent of human ELAM-1. Murine ELAM-1 is encoded by a single-copy gene, spanning about 13 kb, which is structurally organized into 14 exons and 13 introns; very similar to that of its human counterpart. The exon/intron architecture exactly parallels the domain structure of the encoded protein. A murine ELAM-1-specific cDNA was cloned from heart tissue of an interleukin-1-(IL-1)-treated mouse. Its nucleotide sequence shows an overall similarity of 70% to human ELAM-1 cDNA. Transiently expressed in Cos cells, the encoded protein promotes the adhesion between recombinant cells and both human polymorphic nuclear cells, as well as HL60 cells expressing S-Lewis-x sugar moiety. Northern blot studies revealed by far the highest expression of the murine ELAM-1 gene in heart tissue and only low expression in lung tissue of IL-1-treated mice. Within the promoter, most of the recently identified regulatory elements are conserved. An exception is the nuclear factor (NF) kappa B box sequence, which, in the murine ELAM-1 promoter, does not correspond to the consensus NF kappa B sequence (Lenardo and Baltimore, 1989). Band-shift analyses show no binding to NF kappa B-like proteins. However, fusion of the murine ELAM-1 promoter to a chloramphenicol acetyltransferase reporter confers cytokine-inducible transcription, although at a lower level, when compared to the human ELAM-1 promoter. Our results demonstrate the existence of a murine homologue of the human gene and demonstrate for adhesion functional equivalence between the homologous proteins from the two species. In addition, we provide the first evidence of the utility of the murine model in addressing biological questions about the role which ELAM-1 plays in inflammation.


The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively.

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence. In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.


Formation of heterodimers of human-immunodeficiency-virus-type-1 reverse transcriptase by recombination of separately purified subunits.

Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66,000 subunit and its C-terminally truncated form of M(r) 51,000, but, when expressed as a recombinant M(r) 66,000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66,000 and 51,000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66,000 protein. Heterodimers having greater asymmetry have also been prepared by recombining Mr 66,000 subunits containing single-point or deletion mutations, with wild-type M(r) 51,000 subunits, and the resulting heterodimers analysed.


Monoclonal antibodies against Pseudomonas aeruginosa elastase: a neutralizing antibody which recognizes a conformational epitope related to an active site of elastase.

We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme.


Fusion-inhibiting monoclonal antibodies and their relevant antigens in relation to sexual process of Dictyostelium discoideum.

Sexual cell fusion occurs between NC4 and HM1, the heterothallic strains in Dictyostelium discoideum. Cells of these strains are fusion incompetent when cultured on agar plates in the light and become fusion competent upon cultivation in a liquid medium in darkness. Two cell-surface components, gp70 and gp138, have been identified and characterized as being relevant to sexual cell fusion. Both are glycoproteins, and the former is detected only in fusion-competent HM1 cells, while the latter is detected both in fusion-competent HM1 and fusion-competent NC4 cells. We therefore suspect gp 70 to be responsible for cell recognition and gp138, for membrane fusion. Therefore, NC4 cells are expected to possess specific surface molecule(s) that can be recognized by HM1 cells. In the present study, we raised monoclonal antibodies (mAbs) against membrane fractions of NC4 cells and selected fusion-inhibiting mAbs to identify novel molecules related to sexual cell fusion in D. discoideum. Out of the five mAbs we obtained three, DE1, GG6, and HH9, were characterized. DE1 recognized antigens that specifically existed in fusion-competent NC4 cells but not in fusion-incompetent NC4 or HM1 cells. GG6 recognized cell-surface proteins with approximate molecular weights of 125 and 32 kDa in both fusion-competent NC4 and fusion-competent HM1 cells. In addition GG6 also recognised other proteins commonly present in fusion-incompetent cells. The 125 kDa protein appeared to be the same as gp138. The epitope recognized by HH9 was sodium dodecyl sulfate (SDS)-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)


Hormonal regulation of adult type keratin gene expression in larval epidermal cells of the frog Xenopus laevis.

Triiodothyronin (T3) is known to induce amphibian metamorphosis but other hormones such as glucocorticoids accelerate T3 action. The increase in plasma concentration of both T3 and glucocorticoids during metamorphic climax is correlated with the transformation of the epidermis from larval type (uncornified) to adult type (cornified). Previously we have shown that T3 induced adult-type 63 Kd keratin gene expression and cornification of the larval epidermis. In this study, we have examined the effects of T3 and hydrocortisone (HC) on the conversion of larval to adult epidermal cells in vitro. When larval epidermal cells were treated with both T3 and HC, they had a synergistic effect on adult-type keratin synthesis (both 63 Kd and 49 Kd keratins) and epidermal cornification. The synergistic effect between T3 and HC required a pretreatment with T3 for 3 days. During this time, addition of HC to cultures containing T3 did not change the amount of 63 Kd keratin mRNA. Thus, HC did not reduce the lag time for epidermal cells to respond to T3. After 4 days of hormone treatment, T3 increased the amount of 63 Kd keratin mRNA 9-fold while T3 and HC induced it 18-fold. When cultures were pretreated with T3 for 3 days, a 1 day treatment with HC was sufficient to obtain the synergistic effect. Thus the induction of 63 Kd keratin gene expression by T3 required a much longer lag (3 days) than the lag required for the synergistic action of T3 and HC (less than 1 day).(ABSTRACT TRUNCATED AT 250 WORDS)


Methylene chloride--an inhalation study to investigate pathological and biochemical events occurring in the lungs of mice over an exposure period of 90 days.

Male B6C3F1 mice were exposed to 4000 ppm methylene chloride (MC) for 6 hr/day, 5 days/week for up to 13 weeks. Groups of mice were killed at intervals from Day 2 to Week 13. Whole lungs were examined morphologically, immunocytochemically, and biochemically. Biochemical and morphological examination was also performed on isolated Clara cells. The major initial morphological effect seen in lungs was acute Clara cell damage after one exposure to MC. However, this damage appeared to resolve after five consecutive daily exposures to MC. After a 2-day interval the Clara cell lesion reappeared on subsequent reexposure to MC. However, the severity of the lesion decreased over the duration of the study. The appearance and disappearance of the lesion in the Clara cell correlated well with the activity of cytochrome P450 monooxygenase in the Clara cell as assessed immunocytochemically (cytochromes P450IIB 1 and 2) in the whole lung and biochemically in the freshly isolated Clara cell (determined by ethoxycoumarin O-dealkylation and aldrin epoxidation). When there was a marked decrease in cytochrome P450 monooxygenase activity the lesion was not present. This suggested that with time the lung (Clara cell) has developed tolerance to MC possibly due to the inactivation of a cytochrome P450 isozyme. The glutathione S-transferase metabolism of MC by the lung cytosol remained virtually unaltered throughout the study. Events accompanying the discussed changes include (1) a significant increase in nonprotein sulfhydryl in the lungs of all exposed animals, (2) altered plating characteristics of the isolated Clara cells from exposed lungs after 24 hr in culture, and (3) an increase in the number of bronchiolar cells in the S-phase after the first exposure to MC. The study also demonstrates the advantages of target cell isolation and study over whole lung biochemical investigation alone.


Application of molecular encapsulation for toxicology studies: comparative toxicity of p-Chloro-alpha, alpha, alpha-trifluorotoluene in alpha-cyclodextrin vehicle versus corn oil vehicle in male and female Fischer 344 rats and B6C3F1 mice.

The application of alpha-cyclodextrin (alpha-CD) as an alternative vehicle for water insoluble and volatile chemicals was investigated in toxicity studies of p-chloro-alpha, alpha, alpha-trifluorotoluene (CTFT). Groups of F344 rats and B6C3F1 mice of each sex were administered CTFT (97% pure) by gavage in either corn oil or alpha-CD aqueous formulations daily for 14 consecutive days. The dose levels used were 10 (mice only), 50, 400, and 1000 mg/kg for corn oil vehicle and 10, 50, and 400 mg/kg (maximum achievable dose at gavage volume of 5 ml/kg) for alpha-CD vehicle. With both vehicles CTFT and alpha 2u-globulin were found to accumulate in the male rat kidney after 14 days of exposure and a dose-related toxic nephropathy was observed at dose of 50 mg/kg or higher. The hepatocellular hypertrophy and cytoplasmic vacuolation of the adrenal cortex which appeared in dosed male and female rats were also found to be independent of vehicle. Clinical pathology findings suggested a mild anemia and cholestasis in rats. With both vehicles no tissue bioaccumulation of CTFT was found in male or female mice. Vehicle-independent hepatocellular hypertrophy and cholestasis were also observed in mice at doses of 400 and 1000 mg/kg. In conclusion, the alpha-CD vehicle does not affect the toxic responses of CTFT in both sexes of both species. The results of the studies suggest that alpha-CD may be an appropriate alternative vehicle for toxicity studies.


Localization of the cytoadhesin integrins in the human cornea. An immunohistochemical study using monoclonal antibodies.

Cell-matrix interactions play a fundamental role in normal and pathological conditions. They can be mediated by the cytoadhesin subgroup of the integrin superfamily of adhesion molecules. Its members include the vitronectin receptor (VNR) and the platelet glycoprotein IIb/IIIa (GP IIb/IIIa). Both receptors are composed of an alpha-chain (alpha v and alpha IIb, respectively) coupled to a beta 3-chain. Using in situ immunohistochemistry and monoclonal antibodies, the authors studied the distribution of GP IIIa (common beta 3-chain), GP IIb/IIIa (alpha IIb-chain) and VNR (alpha v-chain) in normal and pathological corneal tissues. In the normal cornea, the limbal vascular endothelium was weakly alpha v-positive. Occasionally, faint and granular staining was seen in the epithelium. In the pathological samples, an upregulated expression of the alpha v-chain was noticed on the corneal epithelium as well as on fibroblasts and corneal endothelium. The alpha IIb and beta 3-chains were consistently absent. These data suggest that expression of the VNR-alpha v-chain in the human cornea is modulated by soluble factors released during inflammation and wound healing. Dissociation of expression of the alpha v and beta 3-chains suggests usage of an alternative beta-chain by the VNR-alpha v-chain.


Pulmonary function status of shopkeepers of Ahmedabad exposed to autoexhaust pollutants.

The study deals with evaluation of pulmonary function status (VC, FEV1% and FEF25-75%) in Ahmedabad shopkeepers stationed near different traffic junctions and relating them with the levels of oxides of nitrogen (NOx) near these junctions categorised as Heavy, Medium and Low polluted area junctions. The pulmonary function test (PFT) values of heavy polluted and medium polluted area shopkeepers is compared with low polluted area shopkeepers. The influence of smoking habits and duration of exposure over PFT values was seen. The prevalence of airway obstruction in shopkeepers was compared with USA population. The results indicated significant impairment in FEV1% and FEF25-75% value in high polluted area shopkeepers where NOx level is much higher than TLV value. In medium polluted area, where NOx level is slightly higher than TLV value, shopkeepers demonstrated significant impairment in FEF25-75%. Smoking is found to have an additive effect. A linear increase in the prevalence of pulmonary impairment with increasing duration of exposure was evidenced. Shopkeepers exhibited higher prevalence of impairment in both smokers and non-smokers than USA population attributing it to the effect of autoexhaust pollutants. This study also denoted that FEF25-75% is an early indicator of obstruction in smaller airways which is the primary site of deposition of inhaled pollutants.


Effects of the new 5-lipoxygenase inhibitor E6080 on leukotriene release in vitro.

Fundamental studies were conducted to examine the release of histamine and leukotriene (LT) C4 from lung fragments of guinea pigs and the effects of E6080 on the release of LTB4 and LTC4 from lung fragments or inflammatory cells. The release of histamine and LTs showed large interindividual variations and a marked dependence on experimental conditions. Addition of 10 mM L-cysteine significantly increased LTC4 release compared with that in its absence (about 1.7 times, in terms of mean value). E6080 inhibited antigen-stimulated LTB4 and LTC4 release from passively sensitized human (IC50: LTB4 0.08 microM, LTC4 0.2 microM) and guinea-pig lung fragments (IC50: LTC4 1.1 microM). The LTB4 and LTC4 releases from healthy human polymorphonuclear leukocytes (calcium ionophore A23187) and from allergic patients' leukocytes (basophils, antigen) were inhibited by E6080 with IC50 values of below 1.0 microM. Furthermore, the LTC4 release from rat alveolar macrophages (silica particles) was inhibited by E6080 with an IC50 of 0.2 microM. The potent inhibition by E6080 might be a result of the inhibition of 5-lipoxygenase, since 5-lipoxygenase in rat basophilic leukemia cell was inhibited by E6080 with an IC50 of 0.2 microM. The results confirm the potent inhibitory effects of E6080 on the release of LTs.


Hemopoietic growth factors regulate the survival of human basophils in vitro.

Human basophils were purified from normal peripheral blood, using density gradient followed by negative panning selection. We tested the effects of hemopoietic growth factors on the survival of these basophils in vitro. In the absence of exogenous factors, basophils (purity greater than 90%) decreased in number rapidly. At day 7 only 11% of the cells remained alive in cultures; less than 1% of cells survived at day 14. Interleukin (IL)-3 maintained numbers of viable cells; cell viability was 67% at day 7 and 45% at day 14. Granulocyte-macrophage (GM)-colony-stimulating factor (CSF) exhibited slight effect on the survival; 33% of cells remained at day 7. Other growth factors including granulocyte (G)-CSF, macrophage (M)-CSF, and IL-4 had no significant effect on the survival of basophils at all. Morphological and functional characterization of cells maintained by IL-3 revealed that they belonged to the basophil lineage. These observations indicate that normal basophils possess functional receptors for IL-3 and GM-CSF and that both factors modulate immediate- and delayed-type hypersensitivity reactions by prolonging the life span of basophils.


Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines.

Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.


Biochemical and antigenic properties of the Campylobacter flagellar hook protein.

The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.


Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions.

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.


Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25.

Two FK506 binding proteins of molecular mass 12 kDa (FKBP12) and 13 kDa (FKBP13) have been identified as common cellular receptors of the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and overexpression of a 25-kDa rapamycin and FK506 binding protein (termed FKBP25) with peptidylprolyl cis-trans-isomerase (PPIase) activity. The amino acid sequence, predicted from the FKBP25 cDNA, shares identity with FKBP12 (44%) and FKBP13 (47%) in the C-terminal 97 amino acids. Unlike either FKBP12 or FKBP13, the nucleotide sequence of FKBP25 contains a number of putative nuclear localization sequences. The PPIase activity of recombinant FKBP25 was comparable with that of FKBP12. The PPIase activity of FKBP25 was far more sensitive to inhibition by rapamycin (IC50 = 50 nM) than FK506 (IC50 = 400 nM). PPIase activity of 100 nM FKBP25 was almost completely inhibited by 150 nM rapamycin while only 90% inhibition was achieved by 4 microM FK506. These data demonstrate that FKBP25 has a higher affinity for rapamycin than for FK506 and suggest that this cellular receptor may be an important target molecule for immunosuppression by rapamycin.


Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.


EPR detection of heme and nonheme iron-containing protein nitrosylation by nitric oxide during rejection of rat heart allograft.

The paramagnetic molecule nitric oxide (NO), produced from L-arginine by a specific enzyme (NO synthase), has been shown to be involved in a surprising variety of mammalian cellular responses, including the regulation of T cell immunity to alloantigens in vitro. In cytotoxic activated macrophages, NO production results in a characteristic pattern of alteration of iron-containing enzyme function that is mimicked by exposure to NO. Electron paramagnetic resonance (EPR) studies have shown the formation of iron-nitrosyl species during macrophage activation and also during sepsis, indicating that alteration of iron-containing protein function may be the result of the well-documented tendency of NO to bind to metal ions. We have recently shown that the NO synthesis induced during alloantigenic activation of rat splenocytes inhibits lymphocyte proliferation and cytotoxic T-lymphocyte generation. This report demonstrates that iron-nitrosyl EPR signals similar to those observed in macrophages and during sepsis are present in the blood and in the grafted tissue of rats during the rejection of allogeneic (but not syngeneic) heart grafts. These signals are found in the blood and at the site of allograft rejection, but are not found in other tissues (such as spleen and lung), and are obliterated by administration of the immunosuppressant FK506. These results directly demonstrate the formation of iron-nitrosyl complexes during vascularized allograft rejection and suggest that consequent destruction of iron-containing protein function plays an important role in the rejection response.


The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.


P-selectin and E-selectin. Distinct but overlapping leukocyte ligand specificities.

P-selectin on platelets and endothelial cells and E-selectin on endothelial cells are leukocyte receptors that recognize lineage-specific carbohydrates on neutrophils and monocytes. The proposed ligands for these receptors contain the Le(x) core and sialic acid. Since other investigators have shown that both E-selectin and P-selectin bind to sialylated Le(x), we evaluated whether E-selectin and P-selectin recognize the same counter-receptor on leukocytes. The interaction of HL60 cells with Chinese hamster ovary (CHO) cells expressing P-selectin or E-selectin was studied. To determine whether a protein component is required in addition to sialyl Le(x) for either P-selectin or E-selectin recognition, HL60 cells or neutrophils were digested with proteases, including chymotrypsin, elastase, proteinase Glu-C, ficin, papain, or thermolysin. Cells treated with these proteases bound E-selectin but not P-selectin. Fucosidase or neuraminidase treatment of HL60 cells markedly decreased binding to both E-selectin- and P-selectin-expressing CHO cells. Growth of HL60 cells in tunicamycin inhibited the ability of these cells to support P-selectin-mediated binding and, to a lesser extent, E-selectin-mediated binding. Purified P-selectin inhibited CHO:P-selectin binding to HL60 cells, but incompletely inhibited CHO:E-selectin binding to HL60 cells. However, purified soluble E-selectin inhibited CHO:P-selectin and CHO:E-selectin binding to HL60 cells equivalently and completely. COS cells, unable to bind to E-selectin or P-selectin, bound E-selectin but not P-selectin upon transfection with alpha-1,3-fucosyltransferase or alpha-1,3/1,4-fucosyltransferase. Similarly, LEC 11 cells expressing sialyl Le(x) bound E-selectin- but not P-selectin-expressing CHO cells. Sambucus nigra lectin, specific for the sialyl-2,6 beta Gal/GalNAc linkage, inhibited P-selectin but not E-selectin binding to HL60 cells. Although sialic acid and Le(x) are components of the P-selectin ligand and the E-selectin ligand, these results indicate that the ligands are related, having overlapping specificities, but are structurally distinct. A protein component containing sialyl Le(x) in proximity to sialyl-2,6 beta Gal structures on the P-selectin ligand may contribute to its specificity for P-selectin.


CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.


The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s).

The autophosphorylation, from [gamma-32P]ATP, of insulin and epidermal growth factor receptors in rat liver endosomes peaked at 2-5 min and declined thereafter. When autophosphorylation from either [gamma-32P]ATP or unlabeled ATP was stopped after 5 min by adding excess EDTA +/- ATP, the phosphotyrosine (PY) content of each receptor decreased at 37 degrees C with a t 1/2 of 1.6 min. This was equally so whether the PY content of 32P-labeled receptors was analyzed by autoradiography of KOH-treated gels or by Western blotting with PY antibodies of immunoprecipitated receptors. The dephosphorylation reaction was strictly dependent on the presence of sulfhydryl, was unaffected by the addition of rat liver cytosol, and was temperature-dependent. The phosphotyrosine phosphatase(s) (PTPase(s)) appeared to be tightly anchored to the endosomal membrane, since the dephosphorylation reaction was unaffected by sodium carbonate and 0.6 M KCl treatments. However, treatment with Triton X-100 abolished dephosphorylation, implying an intimate association between the PTPase(s) and its substrate in an intact membrane environment. The powerful insulinomimetic agent pervanadate was the most potent inhibitor (50% inhibition at 1 microM). Increasing the dose of injected ligand augmented the rate of insulin and decreased that of EGF receptor dephosphorylation, respectively. Immunoblotting with specific antibodies failed to identify PTPase 1B or T-cell PTPase in ENs, whereas positive signals were seen in plasma membrane. These studies indicate that the phosphorylation state of receptor tyrosine kinases is dynamically regulated, with dephosphorylation, by closely associated PTPase(s), playing an important role.


Inactivation of human fibroblast growth factor-1 (FGF-1) activity by interaction with copper ions involves FGF-1 dimer formation induced by copper-catalyzed oxidation.

Although the angiogenic proteins acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) both interact with the transition metal copper, itself a putative modulator of angiogenesis, a role for copper in FGF function has not been established. Using nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we detect the complete conversion of recombinant forms of human FGF-1 monomer protein to FGF-1 homodimers after exposure to copper ions. In contrast, not all forms of bovine FGF-1 isolated from bovine brain or a recombinant preparation of human FGF-2 completely formed homodimers after exposure to copper ions under similar conditions. Since the copper-induced FGF-1 homodimers reverted to the monomer form in the presence of dithiothreitol, specific alkylation of cysteine residues by pyridylethylation prevented FGF-1 homodimer formation, and preformed FGF-1 homodimers could not be dissociated by the metal chelator EDTA, FGF-1 dimer formation appeared to result from the formation of intermolecular disulfide bonds by copper-induced oxidation of sulfhydryl residues. FGF-1 homodimers bound with similar apparent affinity as FGF-1 monomers to immobilized copper ions, both eluting at 60 mM imidazole. Both human FGF-1 monomer and dimer forms had a 6-fold higher apparent affinity for immobilized copper ions, as compared with human FGF-2, which eluted in the monomer form at 10 mM imidazole. Further, in contrast to FGF-1 monomers, which dissociate from immobilized heparin in 1.0 M NaCl, preformed FGF-1 homodimers had reduced apparent affinity for immobilized heparin and eluted at 0.4 M NaCl. In contrast, the apparent affinity of human FGF-2 for immobilized heparin was unaffected after exposure to copper ions. Heparin appeared to modulate the formation of copper-induced intermolecular disulfide bonds for FGF-1 but not FGF-2, since co-incubation of heparin and copper with FGF-1 monomers resulted in dimers and other oligomeric complexes. FGF-1 copper-induced homodimers failed to induce mitogenesis in [3H]thymidine incorporation assays, an effect which could be reversed by treatment with dithiothreitol, whereas FGF-2-induced mitogenic activity was relatively unaffected by pretreatment with copper. The differences between human FGF-1 and FGF-2 in protein-copper interactions may be due to differing free thiol content and arrangement between the two proteins. A recombinant human FGF-1 mutant containing the two cysteines conserved throughout the FGF family of proteins but lacking a cysteine residue (Cys 131) present in wild-type human FGF-1 but not human FGF-2 readily formed copper-induced dimers.(ABSTRACT TRUNCATED AT 400 WORDS)


Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.


Isolation and characterization of alpha 2-macroglobulin-protease complexes from purified mouse mammary tumor virus and culture supernatants from virus-infected cell lines.

Cleavage of oligopeptide substrates mimicking the maturation sites in the Gag polyproteins of the mouse mammary tumor virus was assayed using lysed virus. Cleavage at the expected P1-P1' positions was detected in four of seven synthetic peptides. However, studies with specific inhibitors of retroviral proteases showed that only two of them could be unequivocally attributed to the viral enzyme. In an attempt to characterize other proteolytic activities that copurify with the virus, we isolated a multicatalytic high molecular mass protease (700 kDa) that copurifies with the virus. This protein has been identified as an alpha 2-macroglobulin-protein complex according to its biochemical properties and ultrastructure. The proteases forming these complexes are mainly serine proteases and can be inhibited by phenylmethylsulfonyl fluoride. However, other compounds such as chymostatin and elastatinal are more effective inhibitors. The relative efficacy of each compound depends on the substrate, since the complexes described herein appear to be multicatalytic. Elastatinal is a very good inhibitor of the cleavages found at Ala-Ala bonds in peptides representing the capsid/nucleocapsid site, while chymostatin inhibits certain cleavages at the carboxyl terminus of bonds involving leucine and valine in three of the substrates used. Therefore, the alpha 2-macroglobulin present in the cell culture medium is able to bind proteases, forming high molecular weight complexes, which are active against peptide substrates, copurify with the virus and are responsible for the nonviral proteolytic activities found in the purified virus. Elastase appears to be the main proteolytic activity which can be detected in the alpha 2-macroglobulin-protease complexes associated with the virus.


The sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase.

The cDNA sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase is reported. This is the first complete eukaryotic sequence of the flavoprotein subunit to be characterized, and it encodes a 665-amino acid protein that consists of a presequence and a 621-residue mature protein. The deduced bovine sequence shows homology to the corresponding peptides of prokaryotic succinate dehydrogenase and the related fumarate reductases; in particular, there is good overall homology (48%) to the flavoprotein subunit of Escherichia coli succinate dehydrogenase. The conserved sequences comprising the active site and those involved in FAD binding are also found in the bovine protein. The active site of the bovine polypeptide contains a cysteine that confers sensitivity of the enzyme to sulfhydryl reagents; this cysteine is only present in some sequences and thus provides a discriminatory biochemical marker. A putative flavoprotein subunit of human placental succinate dehydrogenase (partial sequence) that lacks this critical cysteine (Malcovati, M., Marchetti, T., Zanelli, T., and Tenchini, M. L. (1991) in Flavins and Flavoproteins 1990 (Curti, B., Ronchi, S., and Zanetti, G., eds) pp. 727-730, Walter de Gruyter & Co., Berlin) has only 16% homology to the bovine heart flavoprotein subunit. However, we show that the enzyme from human placenta is as sensitive to N-ethylmaleimide as that from bovine tissues. In addition, a transcript in human placenta and muscle hybridizes to the bovine heart flavoprotein cDNA and is the same size as that in bovine tissues.