YMXM motifs of IRS-1 define substrate specificity of the insulin receptor kinase.

Of 34 tyrosine residues in insulin receptor substrate 1 (IRS-1), 14 are adjacent to acidic residues, suggesting that they might be phosphorylation sites. Synthetic peptides corresponding to sequences surrounding these tyrosines were used as substrates of the insulin receptor kinase. Surprisingly six of these, each within YMXM motifs, were phosphorylated with greatest efficiency (Km, 24-92 microM; kcat/Km, 0.6-2.1 x 10(4) M-1.sec-1). Substituted YMXM peptides revealed a strong preference of the insulin receptor kinase for methionine at Y + 1 and Y + 3 positions. When phosphorylated, related YMXM sequences are recognition motifs for binding to proteins with src-homology (SH2) domains. The combined hydrophobic and flexible nature of methionine side chains adjacent to the targeted tyrosines provides a versatile contact for recognition by diverse proteins involved in signal transduction.


Differentiation of multiple domains in the herpes simplex virus 1 protease encoded by the UL26 gene.

Previous studies have shown that the herpes simplex virus 1 gene UL26 encodes a 635-amino acid protease that cleaves approximately 20 amino acids from the carboxyl terminus of itself and of a 329-amino acid product of the UL26.5 gene. The results of studies with a variety of protease inhibitors showed that the UL26 protease was inhibited by serine protease inhibitors but not by inhibitors of cysteine protease, aspartic acid protease, or metalloprotease. Mutations resulting in amino acid substitutions, deletions, or insertion of stop codons in the gene or of 20-amino acid stretches into the protease have delineated the dispensable domains I and IV at the amino and carboxyl domains of the gene product. The essential carboxyl-proximal domain (III) can be separated from the essential amino-proximal domain (II) by at least 20 amino acids. The amino-proximal domain is the most conserved region among varicella-zoster virus and human cytomegalovirus homologues of UL26. Of the conserved aspartic acid, histidine, or serine amino acids in this domain, only histidine-61 and -148 could not be replaced without impairment of the proteolytic activity.


Homodimerization and constitutive activation of the erythropoietin receptor.

The erythropoietin receptor (EPO-R) is a member of the recently described cytokine receptor superfamily. A constitutively active (hormone independent) form of the EPO-R was isolated that has a single amino acid change in the exoplasmic domain, converting arginine-129 to cysteine (R129C). Since EPO-Rs containing R129S, R129E, and R129P mutations are functionally wild type, the presence of cysteine at residue 129, and not the loss of arginine, is required for constitutive activity. Several mutant forms of the EPO-R were analyzed; all constitutively active mutants form disulfide-linked homodimers, whereas EPO-responsive or inactive forms of the receptor do not. Monomers and disulfide-linked dimers of the constitutive receptor are present on the plasma membrane and bind EPO with a single affinity. Homodimerization of the EPO-R is likely to play a role in ligand-induced signal transduction, and disulfide-linked dimerization of the constitutive receptor may mimic this step.


Thrombomodulin gene regulation by cAMP and retinoic acid in F9 embryonal carcinoma cells.

Thrombomodulin (TM) expression was investigated during differentiation of F9 embryonal carcinoma cells into primitive or parietal endoderm. Exposure of F9 cells to retinoic acid (RA) triggers differentiation into primitive endoderm and induces the appearance of barely detectable amounts of TM mRNA, whereas treatment with dibutyryl cAMP plus theophylline (CT) augments the levels of TM mRNA to a 4-fold greater extent than RA. Exposure of F9 cells to RA plus CT initiates differentiation into parietal endoderm and synergistically increases the levels of TM mRNA by 10- to 12-fold compared with CT. The time-dependent establishment of cooperativity between RA and CT appears to be secondary to RA-induced differentiation to primitive endoderm. The above alterations in TM mRNA levels occur by a transcriptional mechanism as judged by nuclear run-on experiments. Transient gene expression experiments show that the human TM promoter is transactivated by coexpression of the human RA receptor beta. Thus, the mechanism of induction of TM expression in F9 cells undergoing differentiation to parietal endoderm appears to be similar, but not identical, to that noted for other late response genes.


Detection of intrahepatic replication of hepatitis C virus RNA by in situ hybridization and comparison with histopathology.

A nonisotopic in situ hybridization (NISH) assay was used to detect hepatitis C virus (HCV) RNA. A synthetic oligonucleotide complementary to bases 252-301 of the highly conserved 5' noncoding region of the HCV genome was end-labeled by terminal deoxynucleotidyltransferase using digoxigenin-conjugated dUTP. The hybridized oligomer was revealed by an immunohistochemical reaction after incubation with an alkaline phosphatase-conjugated anti-digoxigenin antibody and subsequent amplification with a complex of alkaline phosphatase and anti-alkaline phosphatase antibodies. The intracellular distribution of HCV RNA was monitored in the livers of two chimpanzees experimentally infected with the H strain of HCV and compared with the serum alanine aminotransferase activity, serum HCV RNA, and liver histopathology. Most cells were stained in the cytoplasm as early as 2 days after inoculation, 1 and 2 days, respectively, before the appearance of viral RNA in the serum. The time course of HCV RNA replication was correlated with increases in serum alanine aminotransferase. However, neither one paralleled the appearance of liver cell necrosis nor showed any correlation with the inflammatory response. The NISH signal was not found in liver biopsy specimens taken from these two animals before inoculation with HCV, from chimpanzees with acute hepatitis type A, B, or delta, or from two animals never experimentally infected with any hepatitis agent; moreover, it disappeared when the positive specimens were predigested with RNase and it was not observed after hybridization of positive controls with a labeled oligomer unrelated to HCV RNA. Thus, detection of liver HCV RNA by NISH is a sensitive and specific method for studying HCV replication at the cellular level. Intracellular replication of HCV did not appear to be associated with histopathologic changes in the liver, although the correlation with increases of liver enzyme activity in the serum suggested possible damage to the liver cell membrane.


Multiple retinoid-responsive receptors in a single cell: families of retinoid "X" receptors and retinoic acid receptors in the Xenopus egg.

In a search for nuclear hormone receptors expressed in early development we found that Xenopus laevis eggs contain mRNAs from two retinoic acid receptor genes (xRAR alpha and xRAR gamma) and two retinoid "X" receptor genes (xRXR alpha and xRXR gamma). We also show that RXRs are members of a family of at least three genes, thus expanding the number of genes encoding retinoic acid-responsive transcription factors to six. With the exception of xRXR gamma, these maternal mRNAs are degraded before gastrulation. The RXRs isolated are differentially activated by retinoic acid and by 3,4-didehydroretinoic acid. Considered together, these four receptors provide a molecular basis for the pleiotropic effects of retinoic acid on early development, and their pattern of expression suggests a role for retinoic acid at the earliest stages of embryonic determination.


Direct effects of luteinizing hormone-releasing hormone agonists and antagonists on MCF-7 mammary cancer cells.

The binding of luteinizing hormone-releasing hormone (LH-RH) analogues to the human mammary tumor cell line MCF-7 and their effect on the cell proliferation was studied to elucidate their direct action on estrogen-dependent mammary tumors. The growth rate of these cells was doubled by the addition of 1 nM estradiol to cells maintained in an estrogen-deficient medium. Although the basal growth rate was only slightly inhibited by the LH-RH antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), the estrogen-stimulated growth was completely abolished by the antagonist. In contrast, the LH-RH agonist buserelin stimulated cell growth in estrogen-deficient medium, whereas it had no effect in the presence of estrogen. 125I-labeled buserelin was used for the measurement of LH-RH receptors on MCF-7 cells. A Scatchard plot analysis of buserelin-specific binding revealed a nonlinear plot, which suggested the presence of one high-affinity binding site with a Kd of 1.4 +/- 1.0 nM and the remaining sites with low affinity (Kd = 1.3 +/- 1.0 microM). The binding of 125I-labeled buserelin was displaced equally well by unlabeled buserelin and by the LH-RH antagonist SB-75, suggesting that both analogues are bound to the same receptor. When parallel experiments were performed with 125I-labeled SB-75, the binding was displaced by unlabeled SB-75 and other antagonists, but only partially displaced by unlabeled buserelin. The results suggest that in these mammary tumor cells there is a LH-RH antagonist binding site that is not recognizable by LH-RH agonists. This hypothesis was tested by measuring cell growth in the presence of both agonists and antagonists. It was found that SB-75 inhibited the stimulation of growth by buserelin, but buserelin did not prevent the inhibition by the antagonist of the estrogen-dependent growth. These results suggest that antagonists directly inhibit mammary tumor growth, not only by competing with LH-RH high-affinity receptors, but also by other mechanisms mediated by low-affinity antagonist binding sites.


The rat trk protooncogene product exhibits properties characteristic of the slow nerve growth factor receptor.

Two distinct nerve growth factor receptor (NGFR) complexes are present on NGF-responsive cell types; these correspond to 100 kDa and 158 kDa for the fast (fNGFR) and the slow (sNGFR) NGFRs, respectively. Previous studies indicate that each complex is derived from a separate gene product and that the sNGFR contains tyrosine kinase activity. The cDNA encoding the fNGFR has previously been cloned. In this report, a rat trk protooncogene cDNA has been isolated from PC12 cells and Trk has been shown to bind NGF, generating a complex of 158 kDa. Characterization of NGF-Trk interactions indicates that Trk and NGF dissociate more slowly than do NGF and the fNGFR. Moreover, NGF-bound Trk is not destroyed by trypsin digestion whereas the NGF-fNGFR complex is sensitive to trypsin digestion. These observations suggest that the trk protooncogene product, expressed in the absence of the fNGFR, binds NGF with properties characteristic of the sNGFR, which was identified as the high-affinity NGFR on primary neurons and PC12 cells.


Relaxin binding in the rat heart atrium.

Relaxin is a member of the insulin family of polypeptides that is best known as a reproductive hormone. In an effort to elucidate the mechanism of action of relaxin we previously localized the specific binding sites of a 32P-labeled relaxin in the rat uterus and brain. These studies suggested that, in addition to its classical role in pregnancy, relaxin might have other physiological functions. In the present paper we describe the specific and high-affinity binding of relaxin to the cardiac atrium of both male and female rats. The relaxin binding could not be displaced by peptides belonging to the same family [insulin, insulin-like growth factor I (IGF-I)] or by peptides that were identified in the atrium or were known to have cardiovascular functions (atrial natriuretic peptide, angiotensin II). The dissociation constant for relaxin in the atrium was estimated to be 1.4 nM, which was similar to that found in the uterus (1.3 nM) and the brain (1.4 nM). In view of the close association of relaxin with reproduction, an experiment was also performed to compare the relaxin binding in the uterus and heart after gonadectomy and sex steroid treatment. It was found that the relaxin binding in the rat uterus was diminished by 53% overall following ovariectomy but was restored to 90% of normal levels when treated with estrogen (but not with testosterone). In contrast, the relaxin binding in the rat heart was not affected by castration or sex steroid treatment. We conclude that specific and high-affinity relaxin receptors exist in the atrium of both the male and female rat heart and that these are regulated differently than the relaxin receptors in the uterus.


Crystal structure of the neutral form of fructose 1,6-bisphosphatase complexed with regulatory inhibitor fructose 2,6-bisphosphate at 2.6-A resolution.

The three-dimensional structure of the complex between fructose 1,6-bisphosphatase (EC 3.1.3.11) and the physiological inhibitor beta-D-fructose 2,6-bisphosphate (Fru-2,6-P2), an analogue of the substrate (fructose 1,6-bisphosphate), has been refined at 2.6-A resolution to a residual error (R) factor of 0.171. The rms deviations are 0.012 A and 2.88 degrees from ideal geometries of bond lengths and angles, respectively. The Fru-2,6-P2 occupies the active sites of both polypeptides C1 and C2 in the crystallographic asymmetric unit in the space group P3(2)21. The furanose and 6-phosphate of Fru-2,6-P2 are located at the fructose 6-phosphate site established earlier, and the 2-phosphate binds to the OH of Ser-124, the NH3+ of Lys-274, and the backbone NH of Gly-122 and Ser-123. Backbone displacements of 1 A occur for residues from Asp-121 to Asn-125. Model building of substrate alpha-D-Fru-1,6-P2 based on the binding structure of Fru-2,6-P2 in the active site shows interactions of the 1-phosphate with the backbone NH of Ser-123 and Ser-124. In the AMP sites, density peaks attributed to Fru-2,6-P2 are seen in C1 (and C4) but not in C2 (and C3). This minor binding of Fru-2,6-P2 to AMP sites partially explains the synergistic interaction between AMP and Fru-2,6-P2 and the protection of the AMP site from acetylation in the presence of Fru-2,6-P2. In the synergistic interaction between AMP and Fru-2,6-P2, inhibition of catalytic metal binding by the presence of Fru-2,6-P2 at the active site, and propagation of structural changes over some 28 A along beta-strand B3 from residues 121 to 125 in the active site to Lys-112 and Tyr-113 in the AMP site, as well as movement of helices across the interdimeric interfaces, may affect AMP binding and the subsequent R-to-T transition. In addition, occupancy of Fru-2,6-P2 at the AMP sites of C1 and C4 may favor binding of AMP to the remaining unoccupied AMP sites and thus promote the accompanying quaternary conformational changes.


Three-dimensional structure of Theiler murine encephalomyelitis virus (BeAn strain).

Depending on the strain, Theiler murine encephalomyelitis virus (TMEV) may cause acute encephalitis or chronic demyelinating disease, which is associated with viral persistence in mice. Persistent central nervous system infection and demyelination by the less-virulent TMEV has provided a useful animal model for the human demyelinating disease multiple sclerosis. The less-virulent BeAn strain of TMEV was crystallized and its atomic structure was determined by x-ray crystallography. The alpha-carbon coordinates of the closely related Mengo virus were used to calculate the initial phases to 3.5 A resolution and the interpretable electron density map was produced by 10 cycles of 30-fold noncrystallographic molecular replacement averaging. The structure revealed a high degree of overall structural similarity to Mengo virus as well as substantial differences in the surface loops. These structural changes might be correlated with TMEV host-specific recognition, pH-related stability, and neurovirulence.


Differential low density lipoprotein receptor-dependent formation of eicosanoids in human blood-derived monocytes.

We studied the ability of low density lipoproteins (LDLs) to provide arachidonic acid (AA) for eicosanoid biosynthesis in human blood-derived monocytes. When incubated in the presence of reconstituted LDL that contained cholesteryl [1-14C]arachidonate (recLDL-[14C]AA-CE), resting monocytes formed three labeled products of the prostaglandin (PG) H synthase pathway: 6-keto-PGF1 alpha, thromboxane B2, and PGE2. The amounts of these eicosanoids in response to recLDL-[14C]AA-CE were comparable to or exceeded those that were produced in response to the addition of 10 microM unesterified [1-14C]AA. By contrast, resting monocytes formed only small amounts of products of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4 from either recLDL-[14C]AA-CE or [14C]AA, indicating preferential utilization of AA in the PGH synthase reaction. However, they converted LDL-derived [14C]AA efficiently into LTB4 and LTC4, when they were first incubated with recLDL-[14C]AA-CE and subsequently stimulated with the chemotactic peptide N-formylmethionylleucylphenylalanine or the Ca2+ ionophore A23187. The classical LDL receptor pathway mediated the synthesis of all of the above eicosanoids from LDL but not from unesterified AA. These results demonstrate that the LDL receptor pathway preferentially promotes the synthesis of PGH synthase products in resting human blood-derived monocytes and that an additional mechanism is required to promote effective synthesis of 5-lipoxygenase pathway products from AA that originates in LDL cholesteryl esters.


Regulatory genes linked to the albino locus in the mouse confer competence for inducible expression on the structural gene encoding serine dehydratase.

A cluster of unlinked genes encoding gluconeogenic enzymes in the mouse is characterized by the failure of normal hormone-inducible expression in animals homozygous for one of several overlapping deletions mapping on chromosome 7 near the albino locus. Previous investigations have shown hormones and their receptors to be normal in the mutants and therefore not responsible for the abnormalities of inducibility. Instead, these studies have implicated a possible failure of the affected structural enzyme genes themselves to attain during prenatal development the competence for inducible gene expression. The results reported here add serine dehydratase (EC 4.2.1.13) and its structural gene to the affected group of gluconeogenic enzymes and their genes. Even though, in deletion homozygotes, serine dehydratase is expressed normally on the constitutive level, hormone-inducible expression fails to develop. The abnormality appears to reside in a defect of prenatal differentiation of cis-acting regulatory elements of the structural gene essential in the pathway of inducible gene expression.


Absolute requirement for GTP in activation of human neutrophil NADPH oxidase in a cell-free system: role of ATP in regenerating GTP.

Guanine and/or adenine nucleotides appear to be involved in the activation of the superoxide-generating NADPH oxidase of phagocytic cells. Their precise roles, however, are unclear, as much of the evidence for their involvement comes from experiments in which nucleotides have been added to complex systems already rich in both endogenous nucleotides and enzymes capable of interconverting them. To circumvent this problem we have examined the role of nucleotides in neutrophil NADPH oxidase activation by using a cell-free system in which adenine and guanine nucleotide concentrations were carefully controlled and monitored by (i) depletion of endogenous nucleotides by extensive dialysis and charcoal treatment; (ii) reconstitution of the depleted system with reagents analyzed for purity; and (iii) measurement of nucleotide levels in cytosol preparations and in oxidase reaction mixtures by HPLC analysis. In contrast to previous reports that have demonstrated only a several-fold enhancement of oxidase activity by GTP or its analogs, we have shown that oxidase activation was absolutely dependent upon GTP in reactions containing dialyzed cytosol in which the total endogenous nucleotide levels were reduced by greater than 99.5%. Kinetic studies revealed that GTP is required at or before the rate-limiting step in oxidase activation. Two nonhydrolyzable analogs of GTP, guanosine 5'-(gamma-thio)triphosphate and guanylyl imidodiphosphate, were even more active than GTP, suggesting the involvement of one or more GTP-binding proteins. In contrast, ATP was neither necessary nor sufficient for oxidase activation. If reaction mixtures were contaminated with GDP and/or GMP, however, ATP (but not its nonhydrolyzable analog adenylyl imidodiphosphate) could indirectly support oxidase activation by means of endogenous enzymes that catalyze the ATP-dependent conversion of GMP and GDP to GTP.


Production of high efficiency of plating hepatitis A virus in primary African green monkey kidney cells.

High-titered hepatitis A virus, strain HM-175, was produced in primary African green monkey kidney cells (5.5 x 10(10) tissue culture ID50/850 cm2 roller bottle). The virus preparation had an efficiency of plating of 15 particles per infectious unit. Single-cycle growth kinetics of the adapted virus indicated that after an eclipse period of 2 days, maximal yields were attained 6 days after infection.


Stimulation of vascular Na-K pump with subpressor angiotensin II in rats.

The effect of subpressor doses of angiotensin II (ANG II) on vascular Na-K pump activity and Na-H exchange, two transmembrane signals of trophic stimulation of vascular muscle, was investigated. Male Sprague-Dawley rats (350-400 g) were given subpressor doses of ANG II by osmotic minipump intraperitoneally for 24 hr or 7-10 days. Control rats received sham procedure/vehicle infusion. Na-K pump activity (86Rb uptake), total and intracellular (Li exchange at 4 degrees C) Na content, and amiloride-sensitive and -insensitive Na uptake of aortas were measured ex vivo. Ouabain-sensitive 86Rb uptake of aortas of rats receiving 80-100, 160-180, and 240-260 ng/kg.min-1 of ANG II for 24 hr was 26.6 +/- 3.5, 28.8 +/- 3.4, and 29.1 +/- 2.6 nmol/mg dry wt.15 min-1 (mean +/- SD, n = 7-12), respectively, compared with 25.2 +/- 3.8 in controls (n = 23, P less than 0.01). These increases were maintained at 7-10 days. After 24 hr and 7-10 days of ANG II treatment, the total Na content of aortas was increased by 9.2% (P less than 0.01) and 7.6% (P less than 0.02), respectively, without a change in intracellular Na content, indicating accumulation of excess extracellular Na. Total and amiloride-sensitive Na uptake of the aorta was unchanged after 24 hr or 7-10 days of ANG II administration. The dry weight of anatomically defined segments of the aorta was 40 +/- 3.8 mg/kg body wt (n = 25) after 24 hr and 42 +/- 4.4 (n = 20) after 7-10 days of ANG II administration, compared with 37 +/- 4.8 (n = 15, P less than 0.05) and 37 +/- 4.9 (n = 17, P less than 0.01) in appropriate controls. Increased Na-K pump activity may signal the onset of trophic stimulation of vascular muscle by ANG II.


Blockade of conditioned taste aversion by scopolamine and N-methyl scopolamine: associative conditioning, not amnesia.

The anticholinergic, scopolamine, consistently disrupts one-trial passive avoidance conditioning but the effects of such drugs on one-trial conditioned taste aversion (CTA) are variable and contradictory. In the present study, treatment of rats with scopolamine impaired the suppression of sucrose intake by post-ingestion administration of lithium chloride (LiCl) in a two-bottle choice test. A similar effect was obtained by using N-methyl scopolamine which penetrates the brain only to a limited degree on acute administration. The blockade of CTA could be prevented in three ways: (i) by exposing the rats to sucrose only on the training day, (ii) by pre-exposing the rats to both sucrose and scopolamine, and (iii) by using a less palatable sucrose/ascorbate mixture. The results demonstrate that the effect of scopolamine on taste aversion is not mediated by the central nervous system, and can be modified by altering the novelty and relative salience of the taste conditioned stimulus. These experiments suggest that conditioned associations between taste and LiCl, and scopolamine and LiCl may underlie the blockade of CTA by scopolamine.


Spinal cord alpha-2 noradrenergic receptors mediate conditioned analgesia.

The present experiment investigated the effects of direct spinal administration of the monoaminergic receptor blockers yohimbine, phentolamine and methysergide on the expression of conditioned analgesia. Animals in the Paired group received classical conditioning trials in which one context was paired with footshock administration (1 mA shock for 15 s). Animals in the Unpaired control group were administered shock in a second, different, context. On the test day animals within each condition were administered saline (20 microliters), yohimbine (30 micrograms), phentolamine (30 micrograms), or methysergide (30 micrograms) prior to receiving a hot plate test (50 degrees C) in the context previously used to shock the Paired group. These ligands were administered into the spinal fluid through a chronic, indwelling spinal catheter. Animals in the Paired group which received saline displayed longer paw lick latencies than saline-treated animals in the Unpaired group. Yohimbine, but not phentolamine or methysergide, attenuated this conditioned analgesia. These results suggest that spinal cord noradrenergic substrates mediate conditioned analgesia, and that this mediation occurs specifically through the alpha-2 noradrenergic receptor.


Cocaine facilitation of prefrontal cortex self-stimulation: a microstructural and pharmacological analysis.

A novel self-stimulation methodology involving a fixed-interval (FI-5 s) schedule of reinforcement, microanalysis and threshold evaluation was used to investigate the effects of cocaine on rats lever pressing for electrical stimulation of the prefrontal cortex. Cocaine (15 mg/kg) increased medial prefrontal cortex (MPC) self-stimulation rates under FI-5 by a mean of 269% and reduced current thresholds for self-stimulation. A similar facilitation was evident with self-stimulation of the sulcal prefrontal cortex. Microanalysis showed that cocaine decreased inter-response times and post-reinforcement pauses, increased responding in the second and third quartiles of the inter-reinforcement interval (IRI) and decreased responding in the fourth IRI quartile. Schedule control of responding was still evident following cocaine despite the profound facilitation of response rates. Increased response rates were seen up to 48 h following a single dose of cocaine, suggesting sensitization of the PFC reinforcement substrate. The acute effects of cocaine on MPC self-stimulation were completely reversed by the dopamine (DA) D1 antagonist SCH 23390 0.02 mg/kg) and the D2 antagonist raclopride (0.3 mg/kg) but not by naloxone (0.5 mg/kg). These results are consistent with previous studies demonstrating the PFC as part of the neural substrate mediating cocaine reward. Further, these results implicate DA receptors in the reinforcing properties of both cocaine and MPC self-stimulation.


Infantile stimulation and the role of the benzodiazepine receptor system in adult acquisition of two-way avoidance behavior.

The present study shows that postnatal "consistent" handling (CH) of rats had long-lasting improving effects on coping with an stressful task (i.e. two-way active avoidance), and that such effects were partially prevented by acute Ro 15-1788 (antagonist of benzodiazepine receptor-BZR; 5 mg/kg) administration. Long-lasting detrimental effects in the same task were also observed in rats which received postnatal "inconsistent" handling (INCH), effects that were slightly increased by acute Ro 15-1788 treatment. Finally, Ro 15-1788 tended to increase avoidance acquisition in non-handled (NH) animals. The observed effects of Ro 15-1788 could be partially attributed to a differential modulation of the process of avoidance acquisition depending on postnatal treatments producing different levels of emotionality.


Down-regulation of cortical beta-adrenoceptors by chronic treatment with functional NMDA antagonists.

Down-regulation of cortical beta-adrenoceptors is observed in laboratory animals following chronic treatment with many clinically effective antidepressant therapies. [3H]Dihydroalprenolol (DHA) binding to cortical beta-adrenoceptors was examined in mice treated with the functional NMDA antagonists 1-aminocyclopropane-carboxylic acid (ACPC) and MK-801. ACPC and MK-801 reduced [3H]DHA binding by 19 (P less than 0.05) and 21% (P less than 0.05), respectively, while imipramine produced a 23% (P less than 0.05) reduction. No corresponding changes in the KD of [3H]DHA were observed. These findings are consistent with the observation that functional NMDA antagonists are active in animal models commonly used to evaluate antidepressants and may represent a novel approach to the treatment of depression.


Breast cancer recurrence after lumpectomy and irradiation: role of mammography in detection.

Between 1977 and 1989, 102 of 1,145 patients treated with lumpectomy and radiation as an alternative to mastectomy required subsequent biopsy for suspected ipsilateral recurrence. The authors reviewed the mammograms of 58 of those patients for whom at least two sets of mammograms were available, including one set obtained within 3 months of the subsequent repeated biopsy. Of 38 biopsy-proved recurrences, 13 (34%) were detected with mammography alone, 17 (45%) with palpation alone, and eight (21%) with both mammography and palpation. While standard mammographic criteria for biopsy were followed, distortions and calcifications inherent to posttreatment appearances necessitated judicious modifications. As anticipated, sensitivities and positive predictive values showed more impairment within the lumpectomy quadrant. In addition, all cases of purely in situ cancer were detected solely with mammography. Mammography plays an important complementary role to physical examination in posttreatment follow-up. On the basis of these findings, a follow-up algorithm for the ongoing evaluation of these patients was developed.


Multisection FLASH: method for breath-hold MR imaging of the entire liver.

One hundred ten patients with various focal liver lesions were imaged with a multisection fast low-angle shot (FLASH) gradient-echo sequence with an echo time of 4.6 msec. This sequence enabled the acquisition of 19 T1-weighted magnetic resonance (MR) images of the liver within a single 26-second breath hold. Patients were also examined with standard T1- and T2-weighted spin-echo (SE) sequences. The multisection FLASH sequence provided significantly higher (P less than .01) liver-spleen contrast, liver-spleen signal-difference-to-noise ratio (SD/N), liver-tumor contrast, and liver-tumor SD/N than the T1-weighted SE sequence but lower values than the T2-weighted SE sequence. Motion artifacts were reduced with the multisection FLASH sequence compared with both SE sequences (P less than .01). The overall image quality of the multisection FLASH images was similar to that of the T1-weighted SE images and superior to that of T2-weighted SE images. The most important characteristics of the multisection FLASH technique in MR imaging of the liver are the high T1 contrast, the prevention of motion artifacts, and a dramatic reduction in imaging time.


Mucin-producing pancreatic tumor: CT findings and histopathologic correlation.

Twenty patients with mucin-producing pancreatic tumor and 60 with other pancreatic diseases underwent computed tomography (CT) to establish the CT characteristics of mucin-producing pancreatic tumor. Scans were obtained with thin sections by administering a large volume of contrast material (200 mL). Mucin-producing pancreatic tumors were divided into three subgroups, and the CT characteristics were as follows: Main duct type tumors consisted of a cystic mass in or communicating with the dilated main pancreatic duct (MPD). Excrescent nodules and/or septa were found in the cyst. The MPD was markedly dilated over its entire length. Branch duct type tumors consisted of clustered small cysts that were all approximately the same size in diameter (1-2 cm). Excrescent nodules or septa were not always seen. The MPD near the lesion was often slightly dilated. Peripheral type tumors consisted of a well-defined cystic mass with excrescent nodules and/or septa. Even if the cyst was multilocular, a large main cyst was in it. The MPD usually was not dilated. The CT findings corresponded to macroscopic findings. Mucin-producing pancreatic tumor can be differentiated from other pancreatic diseases with these criteria.


Adenocarcinoma of the pancreatic ducts: comparative evaluation with CT and MR imaging at 1.5 T.

A prospective comparison of computed tomography (CT) and magnetic resonance (MR) imaging at 1.5 T was performed in 50 patients with the suspected diagnosis of pancreatic carcinoma. CT scans were obtained before and after administration of contrast material in 41 of 50 patients (82%); 34 of 41 postcontrast scans (83%) were obtained with dynamic CT. MR images were interpreted without knowledge of the results of CT, ultrasound, cholangiography, or endoscopic retrograde cholangiopancreatography in 48 patients (96%). Surgical correlation of findings at CT and MR imaging was performed in 24 patients (48%) at laparotomy and in two patients (4%) at autopsy. On T1-weighted MR images, relatively diminished signal intensity of tumor compared with that of the adjacent pancreas was a consistent finding. MR imaging proved superior to CT in identification of pancreatic carcinoma (particularly in smaller intrapancreatic tumors), peripancreatic extension, vascular and portal vein invasion, and duodenal invasion. These results suggest that MR imaging of the pancreas is superior in many instances to CT in preoperative evaluation of pancreatic carcinoma.


Immunoglobulin A-induced shift of Epstein-Barr virus tissue tropism.

Increased immunoglobulin A (IgA) antibodies to the Epstein-Barr virus (EBV) appear months to years before the clinical onset of nasopharyngeal carcinoma and define populations at high risk for this EBV-associated epithelial cancer common in south China. In the human HT-29 epithelial cell line, polymeric IgA (pIgA) specific for EBV promoted infection of the otherwise refractory epithelial cells. When bound to pIgA, EBV entered epithelial cells through secretory component-mediated IgA transport but no longer infected B lymphocytes. Such an immune-induced shift in EBV tissue tropism provides a paradigm for endogenous spread of EBV in the immune host that predicts infectious sequelae of epithelium.


Cold-preserved rat liver viability testing by proton nuclear magnetic resonance relaxometry.

The results of a series of experiments in the cold-preserved rat liver, applying a newly developed method of pretransplant viability testing, are described. The livers were stored either under simple hypothermic conditions (KHB) or in EC, HTK, or UW preservation solution. Livers were stored up to 48 hr and reperfused after a period of hypothermic storage of 1, 7, 14, or 21 hr. In a parallel series of experiments, with livers stored under identical conditions, repeated proton relaxometry measurements (0, 1, 7, 14, 24, 32, 48 hr) were undertaken; and ATP, ADP, AMP (Atkinson's energy charge), and water content of livers, as well as pH of storage solution, were estimated. Based on a strong correlation between proton spin-spin relaxation time T2 and tissue water content (edema), this new method may be useful to estimate the amount of cell swelling during hypothermic storage from a surgical biopsy of about 200 mg within a few minutes. There was, however, no significant correlation found between energy charge and/or pH and water content, T2, or bile flow. Our method could be useful as a rapid test method in experimental cold liver storage models and may be of interest in human liver transplantation as a viability indicator in combination with other parameters.


A comparison of triple-therapy with double-therapy immunosuppression in cadaveric renal transplantation.

Triple-therapy (low-dose cyclosporine-azathioprine-prednisone) immunosuppression regimen was compared with double-therapy (cyclosporine-prednisone) in 91 consecutive nonrandomized adult cadaveric renal transplant recipients. Both groups were comparable with respect to ethnic diversity, prior transplants, and diabetes. The majority of patients with delayed function (ATN) were maintained on triple therapy, and the use of antilymphocyte agents was more common in the triple-therapy group (52% vs. 7%; P = 0.0001). Triple-therapy patients experienced increased acute rejection episodes (1.4 vs. 0.8 per patient, P = 0.03), required more courses of additional steroid pulse therapy (4.3 vs. 1.6 per patient; P = 0.001), and developed serious infections more frequently (37% vs. 15%; P = 0.05), especially CMV infections (17% vs. 0; P = 0.03), compared with double-therapy patients. However, the increased overall infection rate and CMV infection rate were observed only in those patients who received antilymphocyte agents compared with those who did not (46% vs. 21%; P = 0.02 for all infections, 26% vs. 4%; P = 0.006 for CMV). Additional steroid pulse therapy was associated with increased CMV infections (24% vs. 0; P = 0.03) but not with overall infections. One-year allograft and patient survival were equivalent in both groups. Exclusion of ATN patients from analysis did not alter the findings. This experience confirms the overall efficacy of triple-therapy immunosuppression in renal transplant recipients but suggests that triple therapy may be associated with more acute rejection episodes, greater immunosuppression requirements, and a resultant increase in infections, especially CMV.


Serum levels and receptor expression of tumor necrosis factor-alpha following human allogeneic and autologous bone marrow transplantation.

We have investigated tumor necrosis factor-alpha levels in serum samples of patients before and after allogenic (16 patients) or autologous (8 patients) bone marrow transplantation. A sensitive immunoradiometric assay for monitoring levels of endogenous tumor necrosis factor-alpha was used. The serum levels of tumor necrosis factor-alpha were found to be relatively low (ranging from less than 15 to 77 pg/ml). Among 13 patients having graft-versus-host disease following allogeneic bone marrow transplantation 8 patients did not have detectable tumor necrosis factor-alpha (less than 15 pg/ml) while 4 out of 8 patients undergoing autologous bone marrow transplantation had detectable tumor necrosis factor-alpha levels (15 pg/ml), indicating a lack of correlation between tumor necrosis factor-alpha serum levels and the occurrence of graft-versus-host disease. Because the tumor necrosis factor-alpha levels detected in patient sera could be regulated by TNF-receptor expression, the presence of TNF-receptor on patients' peripheral blood mononuclear cells was also studied using fluorescent liposome-conjugated tumor necrosis factor-alpha and immunofluorescence analysis. Our data indicate that peripheral blood mononuclear cells of some patients receiving either autologous or allogeneic bone marrow transplantation expressed significant levels of TNF-receptors, suggesting a lack of correlation between TNF-receptor expression and graft-versus-host disease development.


Amyloid enhancing factor activity is associated with ubiquitin.

Crude amyloid enhancing factor (AEF) drastically reduces the pre-amyloid phase on passive transfer and induces amyloid deposition in the recipient mice in 48-120 h. We attempted to purify AEF from murine amyloidotic liver and spleen extracts by using gel filtration, preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ion exchange chromatography and isolated a 5.5 kDa peptide. In the mouse bioassay, this peptide induced accelerated splenic AA deposition in a dose-dependent manner. Based on structural, electrophoretic and immunochemical criteria the peptide was identified as ubiquitin. A polyclonal rabbit anti-bovine ubiquitin IgG antibody (RABU) abolished the in vivo AEF activity of crude murine AEF in a dose-dependent manner. Monomeric ubiquitin and its large molecular weight adducts were isolated from crude AEF using cyanogen bromide-activated sepharose conjugated to RABU and size exclusion chromatography methods. These were assayed and were found to possess AEF activity. Furthermore, increased levels of ubiquitin, a phenomenon similar to that of AEF, were detected by immunocytochemistry in mouse peritoneal leucocytes prior to and during amyloid deposition. Since AEF shares a number of biological and functional properties with ubiquitin, we suggest a possible role of ubiquitin as an AEF, and that serum amyloid protein A and ubiquitin, the two reactants generated during inflammatory stress conditions, may converge to induce AA amyloid deposition.


Translation of potato virus S RNA in vitro: evidence of protein processing.

RNA from potato virus S (PVS), a member of the carlavirus group, was translated in vitro in rabbit reticulocyte lysate. Time-course experiments revealed the largest product of Mr 190 kD, decreasing in intensity after 60-min incubations, correlating with the accumulation of a 150-kD peptide. This apparent processing could be blocked by the addition of the amino-acid analogues p-fluorophenylalanine and L-canavanine for phenylalanine and arginine, respectively. L-canavanine also appeared to specifically reduce the quantity of PVS (34 kD) coat protein, concomitant with the synthesis of a 36-kD peptide. Sucrose gradient-fractionated genomic RNA directed the synthesis of predominantly 190-kD peptides that appeared not to be processed in the absence of small molecular weight (subgenomic) RNA products.


Conservation of amino-acid sequence motifs in lentivirus Vif proteins.

The nonstructural/regulatory genes of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are believed to play an important role in the replication and pathogenesis of these viruses. In HIV-1 and other lentiviruses, the vif (viral infectivity factor) open reading frame (ORF) (also termed sor or Q in some lentivirus genomes) is located in the central region, overlapping the 3' end of the pol ORF, but in a different reading frame. Among the lentiviruses, only equine infectious anemia virus lacks a vif ORF. The predicted Vif protein sequences from 38 lentiviruses were analyzed for the presence of global and local sequence similarity. The Vif proteins of closely related lentiviruses are highly conserved (HIV-1HXB2:HIV-1mn = 91% identity), while those of more distantly related lentirviruses have diverged significantly (HIV-1HXB2:simian immunodeficiency virusmax = 30% identity). A search for local sequence similarity revealed that a unifying feature of predicted lentivirus Vif proteins is the presence of at least one of two short, highly conserved sequence motifs, SL(I/V)X4YX9Y and SLQXLA. SLQXLA was present in 34 of 38 lentiviruses examined, while the remaining four lentiviruses had one (three viruses) or two (one virus) substitutions in this motif (of five total substitutions, three were conservative changes). The SL(I/V)X4YX9Y motif was found only in primate lentiviruses and in bovine immunodeficiency-like virus. Based on these findings, we suggest that the locus designation vif be used to denote all lentivirus ORFs previously called vif, Q, or sor.


A comparison of simultaneous cervical cytology, HPV testing, and colposcopy.

This pilot study explores the screening techniques for premalignant and malignant disease of the cervix. Given current knowledge of the etiology and progression of cancer of the cervix, should family physicians screen patients with Papanicolaou (Pap) smears, Human Papillomavirus (HPV) smears, colposcopy, or some combination of these three? In a retrospective audit of 75 patients comparing simultaneous Pap smears and colposcopy, 5 of 8 patients with biopsy-proven cervical intraepithelial neoplasia (CIN) and one with invasive cancer had normal Pap smears. Because of this high false-negative Pap smear rate, a prospective study comparing a simultaneous Pap smear, HPV smear, and colposcopic examination (with biopsy when indicated) was undertaken. Seventy consecutive patients seen by the author for routine pelvic examinations consented to and were enrolled in the study. Seven patients had biopsy-proven CIN lesions; 2 were found by Pap smear, 3 by colposcopy, and 5 by HPV smear. The Pap smear missed 5 lesions, colposcopy missed 4, and the HPV smear missed 2. Further studies are needed to determine the optimal screening method for CIN.


Colposcopy in family practice: pilot studies of pain prophylaxis and patient volume.

In a family practice office with volume representative of an average one-person practice, the implementation of colposcopy services was studied. During six months, 257 patients received Papanicolaou (PAP) smears. These patients represented 16.2% of the total 1,585 female patient visits for ages greater than 16. Of the 257 cytology specimens, 10.1% (26) revealed dysplasia or evidence of Human Papilloma Virus (HPV), indicating the need for colposcopic examination. Pathology reports of all biopsied cases confirmed the presence of dysplasia or HPV. These results suggest that the average family physician might reasonably anticipate about 100 colposcopy sessions per year, assuming each patient receives a diagnostic and a therapeutic colposcopic evaluation. In the course of colposcopy services in a family practice, 46 patients underwent 67 colposcopic examinations with cervical biopsy and/or cryotherapy in the office. Some patients received nonsteroidal anti-inflammatory medication 30-60 minutes before the procedure, with selection according to physician preference. All patients rated the pain experienced during the procedure on a 10-point visual-analog scale. Pain scores were significantly lower in those who received medication. Overall, patients tolerated the procedure well and there were no reported complications. Because of the possibility of such factors as the placebo effect influencing these results, a randomized, placebo-controlled, double-blind study will be needed to verify the findings.


Glucocorticoids decrease vitamin D receptor number and gene expression in human osteosarcoma cells.

The mechanisms by which glucocorticoids (GC) inhibit some actions of vitamin D [1,25-(OH)2D3] are not well understood, but there is growing evidence that GC alter vitamin D receptor (VDR) number. We studied the effects of dexamethasone (DEX) on VDR number and mRNA in the human osteosarcoma cell line, MG-63. The effects of DEX on 1,25-(OH)2D3 binding were examined by incubating confluent cells overnight in media without or with 10(-6) M DEX. DEX decreased VDR number (B max) by approximately 70% (110 versus 32 fmol/mg cellular protein, p less than 0.001) without significantly changing the apparent affinity (K'D) of 1,25-(OH)2D3 for its receptor (3.8 versus 2.2 x 10(-10) M, p greater than 0.05). Overnight incubation of MG-63 cells with DEX produced a time- and dose-responsive decrease in VDR mRNA compared to untreated controls (p less than 0.01). To determine the mechanism of the DEX-mediated decrease in VDR mRNA, the effect of DEX on VDR mRNA stability was studied. We found that the half-life for the VDR mRNA was approximately 5.7 h and was not significantly changed when the cells were incubated with DEX (approximately 6.3 h). We conclude that DEX decreases both VDR number and mRNA in MG-63 osteosarcoma cells. Since the half-life of VDR mRNA was not significantly modified by dexamethasone, glucocorticoids appear to decrease VDR mRNA by inhibiting VDR gene transcription or by affecting the processing of VDR mRNA.


Assessment of immune modulation of beta-adrenergic pathways in human dilated cardiomyopathy: influence of methodologic factors.

Sera from patients with dilated cardiomyopathy contain autoantibodies modifying cardiac beta-adrenergic pathways. The influence of the methodology used to determine the prevalence of these antibodies was examined by comparing in 51 patients with idiopathic dilated cardiomyopathy the results of three assays: (1) isoproterenol-sensitive adenylate cyclase; (2) ligand binding inhibition; (3) enzyme-linked immunoassay (ELISA) of beta-receptor peptides. In 20% of the patients, all three tests gave positive results, while the concordance of the adenylate cyclase assay with either of the other two tests was 65%. ELISA was positive for a beta 1-receptor peptide corresponding to the second extracellular loop, but negative for a beta 2-receptor peptide. The results suggest that autoantibodies in dilated cardiomyopathy sera interact with several components of the beta-receptor-adenylate cyclase, and therefore a combination of methodologic approaches is needed to evaluate the prevalence and consequences of beta-receptor autoimmunity.


[Chemonucleolysis using chymopapain and collagenase. 3-year results of a prospective randomized study].

100 patients were prospectively and randomized treated by chemonucleolysis either by collagenase (n = 50/400 ABC-U/disc) or by chymopapain (n = 50/4000 I.U.). The success rate after 1 year was 70% for collagenase and 78% after chymopapain, and 72%/80% after 3 years, respectively. Successful results increased significantly during the first year after treatment and remained stable after that point. After chymopapain, one case of successfully treated anaphylaxis (2%) occurred. After collagenase, 3 cases of secondary sequestrations were observed in cases with primarily closed discograms with intact dorsal longitudinal ligament.


Plasma lipid and lipoprotein concentrations of men consuming a low-fat, high-fiber diet.

This study assessed the influence of a low-fat, high-fiber diet on blood lipid concentrations of 42 men with desirable or moderately elevated cholesterol concentrations. A low-fat diet (19% fat, 4% saturated fatty acids, 4.6 g fiber/MJ) was compared with a high-fat diet (41% fat, 15% saturated fatty acids, 2.0 g fiber/MJ) and with subjects' self-selected diets. Substituting the low-fat for the high-fat diet decreased total, low-density-lipoprotein, and high-density-lipoprotein cholesterol by 17-20%. Lipid changes between 6 and 10 wk were minor. A reduction in plasma cholesterol of greater than 0.52 mmol/L was achieved with the low-fat diet in 59% of men changing from their self-selected diets and in 79% changing from the high-fat diet. Percent reduction was independent of subjects' cholesterol classification. Results indicate that significant reductions in plasma cholesterol can be achieved by the majority of men committing to a low-fat, high-fiber diet.


Guar, but not psyllium, increases breath methane and serum acetate concentrations in human subjects.

Guar and psyllium are fermented by human fecal bacteria in vitro. To see if they were fermented in vivo, eight subjects were studied over 3 separate days, in random order, while eating a polysaccharide-free diet. Twenty grams guar or psyllium, taken at breakfast, had no effect on breath hydrogen levels over 14 h. Mean breath methane and serum acetate concentrations after guar, 37 +/- 1 ppm and 93 +/- 6 mumol/L, respectively, were significantly greater than after control, 20 +/- 2 ppm (P less than 0.05) and 62 +/- 4 mumol/L (P less than 0.01), and psyllium, 20 +/- 2 ppm (P less than 0.05) and 78 +/- 6 mumol/L (P less than 0.05). Serum acetate increased after guar (area under curve 193 +/- 56 mumol.h/L; P less than 0.02) but decreased after psyllium and control. We conclude that guar is fermented in the human colon, producing rises in breath methane and serum acetate but not hydrogen. Although psyllium had no effect on hydrogen, methane, or acetate, this does not prove that it is not fermented.


Colonic fermentation and markers of colorectal-cancer risk.

The aim of this study was to determine the effect of soluble fiber on indexes of colon-cancer risk in postpolypectomy and nonpolyp patients. Forty-five postpolypectomy and 49 nonpolyp volunteers completed 2-wk metabolic studies where half of the group received oat-bran supplements and the other half took wheat-brain supplements. Colonic biopsies taken before and after the intervention showed no difference in the index of thymidine colonic-crypt-cell labeling, thymidine-labeling pattern, or nuclear aberrations. Nevertheless, fecal pH was significantly reduced by 0.23 +/- 0.07 pH units (P less than 0.002) as an index of increased colonic fermentation on oat bran. This was not associated with increased basal breath hydrogen concentrations; fecal butyrate concentrations were higher on wheat bran. We conclude that soluble fiber as oat brain appears to have no advantage over wheat bran in modifying putative risk factors for colonic cancer.


Dietary changes in older Americans, 1977-1987.

This study compares dietary practices of persons aged greater than or equal to 65 y surveyed as part of the 1977-78 and 1987-88 Nationwide Food Consumption Surveys. Intakes of high-fat beef and pork, whole milk, and white bread decreased with increases in low-fat beef, pork, poultry and fish, low-fat milk, and whole-grain breads. However, consumption of many important sources of calories and fat (high-fat desserts, butter, and margarine) and fiber (fruits, high-fiber cereals, and vegetables) changed little between 1977 and 1987. The food-consumption trends translated into modest changes in overall nutrient intake. Gender differences were small and contradict the prevailing feeling that women are changing their diets more rapidly than are men. The authors suggest that public health messages have focused too heavily on foods to avoid while not giving adequate guidance for how to plan and prepare meals that will enable older Americans to meet the current diet and health recommendations.


Phosphate transport by plasma membranes of enterocytes during development: role of 1,25-dihydroxycholecalciferol.

The present studies were designed to investigate phosphate transport across the brush border and basolateral membranes of enterocytes and to determine the effect of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on these processes in suckling and adolescent rats. Vitamin D deficiency was induced in suckling rats by feeding pregnant dams a vitamin D-deficient diet 48 h after insemination; they were then kept in the dark. Vitamin D deficiency in the adolescent rats was induced by feeding the vitamin D-deficient diet to weanling rats for 4 wk. V max values for Na(+)-dependent phosphate uptake in the brush border membranes of vitamin D-deficient and 1,25(OH)2D3-injected suckling rats was 0.7 +/- 0.1 and 1.5 +/- 0.2 nmol.mg protein-1.10 s-1 (P less than 0.01), respectively; V max values in adolescent rats were 0.2 +/- 0.05 and 0.36 +/- 0.04 nmol.mg protein-1.10 s-1 (P less than 0.05), respectively. Vmax values for Na(+)-dependent phosphate uptake in basolateral membranes of vitamin D-deficient and 1,25(OH)2D3-treated suckling rats were 0.006 +/- 0.001 and 0.047 +/- 0.006 nmol.mg protein-1.10 s-1 (P less than 0.01).


A study of oral etoposide, infusional cisplatin, and infusional 5-fluorouracil for locally advanced or metastatic non-small-cell lung cancer. A Mid-Atlantic Oncology Program study.

A combination of oral etoposide, infusional cisplatin (24-hr) and infusional 5-fluorouracil (5-day) was used to treat 87 patients with non-small-cell lung cancer in a Phase II trial. Twenty-six patients were Stage IIIB, and 61 patients were Stage IV (new international classification). The regimen was well tolerated, with 49% grade 3 or 4 toxicities of all types. Response rates, partial and complete, were 40%, (95% confidence interval: 30%, 51%) for Stage IV patients and 20% (95% confidence interval: 10%, 32%), in Stage IIIB. An additional 68% of patients in Stage IIIB and 45% of patients in Stage IV achieved stable disease and had a median survival of 8.8 months, similar to that of patients in partial remission. Median survival was 5.6 months (95% confidence interval: 4.4 months, 10.8 months) for Stage IV patients and 11.0 months (95% confidence interval: 8.8 months, 12.4 months), for Stage IIIB. Of interest was the finding of a higher response rate in patients with a shorter duration of symptoms (less than 6 months versus greater than 6 months).


Mitomycin C, vindesine, and cisplatin in advanced non-small-cell lung cancer. A phase II study.

Between August 1985 and June 1986, 49 previously untreated patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) were treated with the combination of cisplatin 80 mg/m2 i.v. on day 1, vindesine 3 mg/m2 i.v. on days 1 and 8, and mitomycin-C 8 mg/m2 i.v. on day 1 (MVP), repeating after an interval of 4 weeks, and thereafter every 6 weeks. The median age for all patients was 62 years, with a range of 21 to 77 years. All patients had a performance status of 0, 1, or 2 (ECOG scale) and measurable disease. Histologic types included squamous cell carcinoma (22 patients), adenocarcinoma (22 patients), and large-cell carcinoma (6 patients). Forty-eight patients were evaluable for response. Out of 48 patients, one (2%) achieved a complete response and 24 patients (50%) achieved a partial response, resulting in an overall response rate of 52% (95% confidence interval, 38-68%). The response rates were 52% for squamous cell carcinoma, 45% for adenocarcinoma, and 80% for large-cell carcinoma, respectively. The median duration of response was 4.2 months and the median duration of survival for all patients was 10.6 months. The major toxicity was myelosuppression. Leukopenia and thrombocytopenia of grade 3 or 4 occurred in 85% and 33%, respectively. One patient died of sepsis associated with leukopenia. Other toxicities were manageable and reversible. In conclusion, the MVP regimen was active and tolerable in patients with advanced NSCLC. Prospective randomized study comparing the MVP regimen with the two-drug combination of vindesine and cisplatin is warranted.


Non-small-cell lung cancer (NSCLC): chemotherapy in advanced disease. Our experience in ten years.

This is a review of the therapeutic schedules used in our service during the past 10 years for the therapy of advanced non-small-cell lung cancer. During the first years, nonrandomized trials were conducted and several combinations were tested: MACC (methotrexate, doxorubicin, cyclophosphamide, and CCNU), cisplatin-etoposide, and cisplatin-vindesine. The results of these trials were invariably discouraging: objective responses hardly reached 30%, while the survival was around 15 months in the best case. On December 1985 a new randomized trial, based on the combination MIP (mytomicin, ifosfamide, cisplatin) was designed; 60.7% of objective responses were achieved, with 9 complete remissions (17.6%) and 22 partial remissions (43.1%). Median survival was 15 months. In order to reduce the toxicity of this combination, carboplatin was substituted for cisplatin. Unfortunately, results were very poor. No complete remission, and only 5 partial responses (20%) were achieved. At the present time, a new randomized trial is being conducted. In it, MIP combination is compared with VIP (vindesine, ifosfamide, cisplatin). Preliminary results have shown no differences between both arms in response, toxicity, or survival. New therapeutic approaches, as neoadjuvant therapy, are being explored.


Intra-arterial and intravenous use of 4' epidoxorubicin combined with 5-fluorouracil in primary hepatocellular carcinoma. A randomized comparison.

Between October, 1986, and March, 1990, 20 consecutive untreated and noncirrhotic patients with measurable and histologically and/or cytologically confirmed unresectable primary liver cancer were randomly assigned to intravenous (10 patients) or intra-arterial (10 patients) therapy. Patients were treated every 4 weeks with a combination chemotherapy regimen containing 4' epidoxorubicin and 5-fluorouracil. A 3-min bolus injection of 4' epidoxorubicin was followed by 5-fluorouracil given in a 90-min infusion. The dose of 4' epidoxorubicin was escalated: the starting dose was 40 mg/m2, the second dose was 50 mg/m2, and thereafter 60 mg/m2 during subsequent cycles. The dose of 5-fluorouracil was always 800 mg/m2. Objective response rates (20%) were similar in both treatments; two patients had partial responses in the intra-arterially treated group and one complete and one partial response were recorded in the intravenously treated group. The median survival time was 15.2 months for the patients treated intra-arterially and 13.8 months for the patients treated intravenously. Toxicity was mainly mild in both groups with less hematopoietic toxicity in the I.A.-treated group. 4' epidoxorubicin combined with 5-fluorouracil given intra-arterially is not superior to the intravenous therapy, but it may diminish systemic toxicity.


A serosurvey of pathogens associated with shellfish: prevalence of antibodies to Vibrio species and Norwalk virus in the Chesapeake Bay region.

Recent concerns regarding the safety of shellfish consumption have focused on the risk posed by naturally occurring marine bacteria such as Vibrio species and by viruses such as Norwalk and related agents. Despite the widespread environmental presence of Vibrio species in the Chesapeake Bay, the rate of reported infections remains low; there have also been no reports of major Norwalk outbreaks associated with shellfish in this area. As infections with these agents may not always be recognized because of difficulties in making the diagnosis and/or their mild or subclinical presentation, a serosurvey was conducted among healthy volunteers living in the Chesapeake Bay region. Serum and questionnaire data were collected during the fall of 1987 from 267 persons with varying levels of exposure to shellfish: shellfish industry workers, persons attending a local seafood festival, and Seventh-day Adventists (who traditionally abstain from eating shellfish). In comparisons among groups, a significant association could not be demonstrated between shellfish consumption or contact and antibody response to Vibrio cholerae O1 or Norwalk virus. Rates of seropositivity were high for both agents (up to 22% seropositive with a V. cholerae O1 Inaba vibriocidal assay, 14% with an enzyme-linked immunosorbent assay for cholera toxin, and up to 70% seropositive with an enzyme-linked immunosorbent assay for antibodies to Norwalk virus); the basis for these responses in population-based studies remains to be determined. Shellfish industry workers did have a significantly elevated antibody response to the unencapsulated phase variant of Vibrio vulnificus as compared with the other groups studied. Infection with V. vulnificus may be relatively common among persons with high levels of exposure to shellfish.


Epidemic poliomyelitis in The Gambia following the control of poliomyelitis as an endemic disease. I. Descriptive findings.

An epidemic of type 1 poliomyelitis involving 305 cases occurred in The Gambia (estimated 1986 population, 768,995) from May through November 1986, following a 6-year period when only five cases were reported. Cases were identified by physician reporting during the epidemic and by a national village-to-village search conducted after the epidemic. The national attack rate was 40 cases per 100,000 people. Cases lived in all parts of the country except the capital, Banjul. The peak month of the epidemic was August (139 cases). The highest attack rate by year of age was in 1-year-old children (394 cases per 100,000 persons), and 75% of cases were 3 years of age or less. A vaccination coverage survey showed that 64% (95% confidence interval 60-68) of 1- to 2-year-old children were vaccinated with at least three doses of trivalent oral polio vaccine at the beginning of the epidemic. Fifty-seven cases became paralyzed more than 2 weeks after a national mass campaign in which 95% of children 1-7 years old were reported to have received a dose of trivalent oral polio vaccine. Experience in The Gambia shows that a several-year period of excellent control of endemic poliomyelitis by a vaccination program can be followed by a major epidemic and that a mass vaccination campaign may be only partially successful in ending the epidemic.


Oral folic acid supplementation for cervical dysplasia: a clinical intervention trial.

We attempted to evaluate the effect of oral folic acid supplementation on the course of cervical dysplasia. A total of 235 subjects with grade 1 or 2 cervical intraepithelial neoplasia were randomly assigned to receive either 10 mg of folic acid or a placebo daily for 6 months. Clinical status, human papillomavirus type 16 infection, and blood folate levels were monitored at 2-month intervals. Outcome data were subjected to chi 2 analysis. The prevalence of human papillomavirus type 16 infection initially was 16% among subjects in the upper tertile of red blood cell folate versus 37% in the lower tertile (trend p = 0.035). After 6 months no significant differences were observed between supplemented and unsupplemented subjects regarding dysplasia status, biopsy results, or prevalence of human papillomavirus type 16 infection. Folate deficiency may be involved as a cocarcinogen during the initiation of cervical dysplasia, but folic acid supplements do not alter the course of established disease.


Immunofluorescent staining and corneal sensitivity in patients suspected of having herpes simplex keratitis.

We examined immunofluorescent staining and corneal sensitivity in 25 control subjects (25 eyes) with normal corneas, six patients (eight eyes) with possible herpes simplex keratitis, and 44 patients (48 eyes) with corneal lesions (recurrent erosion, superficial punctate keratitis, marginal ulcer, and follicular keratoconjunctivitis) in whom herpes simplex keratitis was not suspected. On immunofluorescent staining, all 25 control subjects had negative reactions, all eight eyes suspected of having herpes simplex keratitis had positive reactions, and 11 (23%) of the 48 eyes not suspected of having herpes simplex keratitis had positive reactions; the remaining 37 eyes had negative reactions. Of the 11 eyes not suspected of having herpes simplex keratitis but that had positive reactions on immunofluorescent staining, nine had recurrent erosions and the remaining two eyes had superficial punctate keratitis. Of the eight eyes with possible herpes simplex keratitis, seven (88%) had decreased corneal sensitivity. Of the 11 eyes not suspected of having herpes simplex keratitis but that had positive reactions on immunofluorescent staining, eight (73%) had decreased corneal sensitivity. Of the 37 eyes not suspected of having herpes simplex keratitis that had negative reactions on immunofluorescent staining, 11 (30%) had decreased corneal sensitivity.


Hypoxia-induced increased permeability of endothelial monolayers occurs through lowering of cellular cAMP levels.

Prolonged exposure to hypoxia, as at high altitude, results in increased vascular permeability that may be ameliorated by administration of glucocorticoids. To understand mechanisms underlying these observations, cultured bovine aortic and pulmonary artery endothelial cells (ECs) were subjected to hypoxia, and changes in monolayer permeability and adenosine 3',5'-cyclic monophosphate (cAMP) levels were assessed. Exposure of both types of cultured ECs to hypoxia (PO2 approximately 14 Torr) led to a time- and dose-dependent increase in monolayer permeability, as measured by diffusion of radiolabeled solutes, which was associated with a progressive decrease in EC cAMP levels from 60 to 15 pmol/mg protein, and a decrease in EC adenylate cyclase activity. The change in endothelial barrier function was prevented by addition of cAMP analogues. Pertussis toxin protected EC monolayers from hypoxia-mediated increase in permeability while maintaining cAMP levels and adenylate cyclase activity. Addition of dexamethasone to EC monolayers before or simultaneously with their incubation under hypoxic conditions blocked the hypoxia-mediated increase in monolayer permeability. Dexamethasone pretreatment also prevented the decline in cAMP and adenylate cyclase levels in oxygen-deprived cultures. These data indicate that hypoxia decreases EC barrier function by lowering adenylate cyclase activity and cellular cAMP levels. They suggest that dexamethasone may exert its protective effect, in part, by preventing the hypoxia-induced decline in adenylate cyclase activity, leading to an increase in cellular cAMP and maintenance of EC barrier function.


Juxtaglomerular cells cultured on a reconstituted basement membrane.

Circulating renin levels are regulated by release from juxtaglomerular (JG) cells. Here, for the first time, we describe the primary culture of rat juxtaglomerular cells on a reconstituted basement membrane. In addition, primary cultures were transformed with a temperature-sensitive SV40 large T antigen gene to promote the development of a continuous JG cell line. Both primary cultures and transformed JG cells maintain a highly differentiated state and secrete active renin. These preparations now provide a system in which characterization of the cellular mechanisms of regulation of renin synthesis and release is possible.


Distribution of actin filament lengths measured by fluorescence microscopy.

We analyzed the distribution of actin filament lengths by optical microscopy (OM). OM avoids possible alterations in the size or structure of actin filaments occurring during sample preparation for electron microscopy (EM). Images of F-actin labeled with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin were analyzed for both size distribution and flexibility. In the standard buffer [25 mM potassium acetate, 4 mM MgSO4, 25 mM tris(hydroxymethyl)aminomethane acetate, pH 7.5, 20 mM beta-mercaptoethanol] filaments did not aggregate into bundles and remained stable at nanomolar concentrations for at least 1 h. At the same concentration, actin labeled directly with rhodamine (no phalloidin) formed unstable filaments whose average length decreased with time. The number average length of TRITC-phalloidin labeled filaments (Ln) was 4.90 microns, the ratio (rho) of the weight average length to the number average length was 2.06, and the correlation length (1/lambda) was 8.33 microns. These parameters were in good agreement with the values determined by EM for filaments shorter than 8 microns. Passing G-actin through a Sephadex G-150 column before polymerization did not have a significant effect on the distribution of lengths but made filaments more stiff (1/lambda = 12.5 microns). Millimolar concentration of ATP increased the correlation length, and gelsolin had the expected fragmenting effect on filaments. These results show that OM can be used as a fast and reliable method to analyze the distribution and flexibility of actin filaments and suggest that, in spite of extensive manipulation of actin filaments during sample preparation, EM is a valid tool for determination of size parameters of actin filaments.


Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosa cells.

Rat and bovine adrenal zona glomerulosa (ZG) cells possess a low-threshold, voltage-dependent Ca2+ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membrane potentials above -80 mV with maximal peak Ca2+ current elicited near -30 mV. Inactivation of the Ca2+ current was half-maximal between -74 and -58 mV, depending on the external Ca2+ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Cai) levels. Under basal conditions, Cai is stable in single rat ZG cells, whereas more than half of the bovine ZG cells produce repeated Cai transients. These Cai transients, which are blocked by removal of external Ca2+ or addition of Ni2+, are likely due to repetitive electrical activity in bovine ZG cells. Cai responses can be elicited by small increases in external K+ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+ channels. However, these Cai changes remain robust at high external K+ concentrations (20-40 mM). In experiments combining Cai measurements and whole cell voltage clamp, a steep dependence of Cai on membrane potential was revealed beginning at depolarizing voltages near a holding membrane potential of -80 mV. A maximal increase in Cai occurred near -30 mV (equivalent to an external K+ concentration of 40 mM), a membrane voltage at which sustained current through low-threshold Ca2+ channels should be negligible. These data raise the possibility of additional voltage-dependent pathways for Ca2+ influx.


P2 purinergic receptors and cellular calcium metabolism in A 431 human epidermoid carcinoma cells.

Stimulation of P2 purinergic receptors on A 431 human epidermoid cells with ATP rapidly mobilized intracellular calcium and increased cytosolic free Ca2+ ([Ca2+]i). Incorporation of 45Ca2+ was also stimulated by ATP at a rate less than that of [Ca2+]i elevation. Among a number of nucleosides, nucleotides, and their analogues examined, ATP, GTP, UTP, ADP, UDP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and 5'-adenylylimidodiphosphate (AMP-PNP) increased both [Ca2+]i and 45Ca2+ influx, whereas others did not; these latter two analogues (ATP gamma S and AMP-PNP) blocked the ATP-stimulated 45Ca2+ influx only very slightly, suggesting that they are not prominent antagonists but rather agonists. A high correlation between [Ca2+]i increase and 45Ca2+ influx, in terms of nucleotide specificity, suggests the involvement of [Ca2+]i in influx of 45Ca2+. It appeared that [Ca2+]i elevated by several nucleotides or nucleotide analogues opened a calcium gate, thus allowing the influx of 45Ca2+. P2 purinergic receptors on these cells had such a characteristic that they were rapidly desensitized. These nucleotides or analogues also affected epidermal growth factor (EGF) receptors by inhibiting the EGF binding. The differences of ligand or substrate specificities between P2 purinergic receptors and ecto-nucleotidases indicates that the two components are different molecules involved in different systems.


Sarcolemmal Na(+)-K(+)-ATPase activity in congestive heart failure due to myocardial infarction.

Because the Na+ pump is considered to modulate the contractile force development by the cardiac muscle and depressed cardiac pump function is the hallmark of congestive heart failure, we characterized the sarcolemmal Na(+)-K(+)-ATPase activity in failing rat hearts after myocardial infarction. For this purpose, the left ventricular coronary artery was ligated, and hearts were examined 4, 8, and 16 wk later; sham-operated animals served as controls. Hemodynamic assessment revealed the presence of abnormal cardiac function at 4, 8, and 16 wk. Although accumulation of ascites in the abdominal cavity was present in experimental animals at 4 wk, other clinical signs of congestive heart failure in experimental rats including lung congestion and cardiac dilatation were evident 8 and 16 wk after induction of myocardial infarction. The depression in Na(+)-K(+)-ATPase activity in purified sarcolemmal membrane from the uninfarcted experimental left ventricle at 8 wk was associated with depressed Vmax without any changes in the affinities for Mg-ATP, Na+, and K+ or the pH optimum for the enzyme. The Kd values of both the high- and low-affinity binding sites for [3H]ouabain, which is believed to interact with Na(+)-K(+)-ATPase, were increased; however, no change in the density of either class of ouabain binding site was evident. The depression of Na(+)-K(+)-ATPase activity in failing hearts at 16 wk of myocardial infarction was not different from that observed at 8 wk but the enzyme activity was not altered at 4 wk of coronary occlusion. These data support the view that depression of Na(+)-K(+)-ATPase activity may serve as an adaptive mechanism during the development of congestive heart failure.


Regulation of glucose transport and GLUT1 glucose transporter expression by O2 in muscle cells in culture.

The effect of varying cellular oxygenation on L6 muscle cell 2-deoxy-D-glucose transport, glucose utilization, lactate production, and expression of GLUT1 and GLUT4 transport proteins was investigated. Incubation of L6 myotubes in 3% O2 (mimicking a state of hypoxia) elevated glucose uptake by 6.5-fold over 48 h relative to cells incubated in 21% O2 (normoxia). Incubation of L6 cells in hyperoxic conditions (50% O2) significantly depressed glucose uptake by 0.4-fold. These effects were fully reversible. Incubation in 3% O2 also caused lactate accumulation and enhanced glucose consumption from the medium. Hypoxia elevated 2-deoxy-D-glucose transport even when the concentration of glucose in the medium was kept constant, suggesting that glucose deprivation alone was not responsible for increased cellular glucose uptake. Incubation in 3% O2 also elevated 3-O-methylglucose uptake but not amino acid uptake. Cycloheximide prevented the hypoxia-induced increase in glucose uptake, indicating that de novo synthesis of glucose transport-related proteins was the major means by which cells increased glucose uptake. The content of GLUT1 glucose transporter was significantly elevated in total membranes of cells incubated in 3% O2 and depressed in membranes from cells incubated in hyperoxic conditions, whereas GLUT4 expression was not affected. These results indicate that hypoxia induces an adaptive response of increasing cellular glucose uptake through elevated expression of GLUT1 in an attempt to maintain supply of glucose for utilization by nonoxidative pathways.


Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle.

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.


Phylogenetic conservation of isoform-specific regions within alpha-subunit of Na(+)-K(+)-ATPase.

Site-directed polyclonal antibodies were generated against four oligopeptides derived from the rat alpha-isoforms of Na(+)-K(+)-adenosinetriphosphatase (i.e., Na(+)-K+ pump) and were used to assess the phylogenetic conservation of the target regions. The first antibody was directed against a region near the phosphorylation site that is conserved among alpha-subunits from numerous species. The remaining antibodies were specific for the isoforms of alpha and recognized a region near the fluorescein isothiocyanate-reactive lysine of the adenine binding site. Immunoblots of protein from a number of species probed with these antibodies revealed dramatic differences in the conservation of the target sequences. The region derived from alpha 1 was found in four of the six mammals screened. In contrast, the analogous region derived from alpha 2 was found in all the screened mammals. Most conserved was the region derived from alpha 3, which was found in all the screened mammals as well as in chickens, catfish, dogfish, and sea slugs. Conservation of the region from alpha 3 suggests functional significance and implies that strong selective pressures have discouraged mutational change. Results from the immunoblots further suggest that isoforms are present in all vertebrates.


Regulated endocytosis in a chloride secretory epithelial cell line.

The colonic epithelial cell line T84 has been shown to be a good model to investigate the regulation of Cl- secretion by the adenosine 3',5'-cyclic monophosphate (cAMP)-mediated second messenger cascade. Regulated exocytic insertion and endocytic retrieval of transport proteins, or proteins that regulate transport proteins, is one mechanism proposed to regulate plasma membrane solute permeabilities. The aims of our studies were to characterize endocytic processes in T84 cells and to investigate their regulation by known activators of Cl- secretion that are mediated by the cAMP second messenger cascade. Forskolin, an activator of adenylate cyclase, caused a marked inhibition of endocytic uptake of the fluid-phase marker horseradish peroxidase (HRP) and the adsorptive marker wheat germ agglutinin conjugated to HRP. Similar inhibition was obtained with vasoactive intestinal peptide, a secretagogue whose receptor is coupled to adenylate cyclase, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable cAMP analogue. 1,9-Dideoxy-forskolin, a forskolin analogue that fails to activate adenylate cyclase, was without effect on endocytosis. Our data show that the net rate of endocytosis, as measured by fluid-phase uptake, is decreased by a cAMP-mediated mechanism. Because the number of Cl- channels or associated regulatory proteins in the plasma membrane reflects a balance between their exocytic insertion and endocytic retrieval, we propose that the cAMP-mediated decrease in endocytosis could contribute to the concomitant increase in plasma membrane Cl- permeability.


Recovery from exercise-induced desaturation in the quadriceps muscles of elite competitive rowers.

A simple muscle tissue spectrophotometer is adapted to measure the recovery time (TR) for hemoglobin/myoglobin (Hb/Mb) desaturation in the capillary bed of exercising muscle, termed a deoxygenation meter. The use of the instrument for measuring the extent of deoxygenation is presented, but the use of TR avoids difficulties of quantifying Hb/Mb saturation changes. The TR reflects the balance of oxygen delivery and oxygen demand in the localized muscles of the quadriceps following work near maximum voluntary contraction (MVC) in elite male and female rowers (a total of 22) on two occasions, 1 yr apart. TR ranged from 10 to 80 s and was interpreted as a measure of the time for repayment of oxygen and energy deficits accumulated during intense exercise by tissue respiration under ADP control. The Hb/Mb resaturation times provide a noninvasive localized indication of the degree of O2 delivery stress as evoked by rowing ergometry and may provide directions for localized muscle power output improvement for particular individuals in rowing competitions.


Effects of D-beta-hydroxybutyrate and long- and medium-chain triglycerides on leucine metabolism in humans.

Ketone bodies and/or fatty acids might play a protein-sparing role during prolonged fasting or parenteral nutrition. To assess this problem, we studied whole body leucine metabolism, using L-[1-13C]leucine in normal postabsorptive volunteers who received either long-chain triglycerides (LCT, 0.15 g.kg-1.h-1, 6 subjects), a 50-50 mixture of medium-chain triglycerides (MCT) and LCT (0.15 g.kg-1.h-1, 6 subjects), D-beta-hydroxybutyrate (540 mumol.kg-1.h-1, 6 subjects), or saline (4 subjects). Leucine concentration decreased only with MCT-LCT. Leucine flux decreased by 10-20% from basal in all groups. Leucine oxidation, which was corrected for the contribution to 13CO2 of the 13C natural abundance of the infused substrates, decreased during LCT infusion (0.31 +/- 0.02 to 0.24 +/- 0.01 mumol.kg-1.min-1, P less than 0.01), but was unaffected by MCT-LCT (despite plasma free fatty acid levels similar to those obtained with LCT), D-beta-hydroxybutyrate, or saline infusion. Therefore, 1) the effect of fatty acids on amino acid oxidation is not mediated by ketone bodies, 2) it depends on the fatty acid chain length, 3) long-chain fatty acids but not medium-chain fatty acids could play a protein-sparing role during parenteral nutrition.


Upregulation of D-myo-inositol transport in diabetic rat glomerular cells.

In the streptozotocin (STZ)-treated diabetic rat, reduced glomerular arteriolar resistance leads to raised intraglomerular pressure. Because vasoconstrictor hormones, such as angiotensin II, stimulate arteriolar smooth muscle and mesangial cell contraction via the D-myo-inositol (MI)-dependent transmembrane signaling pathway, in diabetes extracellular D-glucose may inhibit MI transport causing MI depletion, reduced signaling, and hypocontractility. Therefore we studied the regulation of Na-dependent MI transport in the intact cells (mesangial and endothelial) of isolated glomeruli from STZ rats after 2 wk of diabetes, with and without insulin, compared with controls. Specific [3H]MI uptake per milligram glomerular protein (10-150 min, 37 degrees C) was observed in the presence of 0, 5.5, and 30 mM D-glucose using L-[14C]glucose as a marker of nonspecific uptake. D-Glucose competitively inhibited Na-dependent MI transport (maximum velocity) into diabetic and normal glomerular cells. At 5.5 mM D-glucose, MI uptake by diabetic non-insulin-treated glomeruli was increased to 191 +/- 14% (SE) above normal glomeruli. Insulin treatment resulted in less upregulation (116 +/- 11%) of normal MI transport. High glucose concentration did not alter the rate of [3H]MI efflux from preloaded glomerular cells. To determine whether diabetes alters available substrate for transmembrane signaling, after incubation for 120 min, the incorporation of [3H]MI into cellular membrane phosphoinositides and soluble D-myo-inositol phosphates of isolated diabetic and control glomerular cells was compared. Diabetic glomerular cells displayed a significant increase (P less than 0.005) in [3H]MI incorporation into these fractions compared with controls.


Role of brain kallikrein-kinin system in regulation of adrenocorticotropin release.

We evaluated whether the brain kallikrein-kinin system plays a role in the regulation of adrenocorticotropin (ACTH) release in rats. Intracerebroventricular (icv) injection of bradykinin (0.24 nmol) increased plasma immunoreactive ACTH (irACTH) levels (from 93 +/- 4 to 200 +/- 12 pg/ml, P less than 0.01). This effect was prevented by icv kinin antagonist at 15.4 nmol/h (from 98 +/- 5 to 108 +/- 6 pg/ml; not significant). The antagonist did not alter the increase in plasma irACTH levels induced by icv corticotropin-releasing factor (CRF), arginine vasopressin, or prostaglandin E2. Melittin (7 nmol/h icv) increased plasma irACTH from 95 +/- 4 to 268 +/- 7 pg/ml (P less than 0.01). This effect was prevented by icv kinin antagonist (15.4 nmol/h), kallikrein antibodies (13 pmol/h), or indomethacin (0.28 mmol/h). ACTH response to melittin was not altered by antagonists of CRF or vasopressin. Intra-arterial injection of insulin (0.3 IU/kg body wt) reduced plasma glucose levels to a similar extent in rats given icv kinin antagonist or vehicle; the ACTH response to insulin-induced hypoglycemia was slightly less in rats given kinin antagonist than in those given vehicle (55 +/- 5 vs. 86 +/- 4 pg/ml, P less than 0.05). The brain kallikrein-kinin system may play a role in the regulation of ACTH secretion in stimulated conditions.


ACTH and cortisol responses to sequential CRF injections in fetal sheep.

To determine whether an initial ovine corticotropin-releasing factor (oCRF) injection modifies adrenocorticotropic hormone (ACTH) and cortisol responses to a second injection and to establish whether the effect changes throughout gestation, we studied chronically cannulated fetal lambs of 103-113 and 133-137 days gestation. Experimental groups underwent an injection (500 ng/kg iv) of oCRF, arterial blood sampling for 6 h, then a similar oCRF injection followed by sampling. In control studies, vehicle was the initial injection. After the first oCRF injection, plasma cortisol levels went from 1.7 +/- 0.4 to 9.5 +/- 5.2 (SE) ng/ml ("immature") and from 22.3 +/- 4.9 to 52.5 +/- 5.8 ng/ml ("mature"), remaining elevated for 6 h. In immature fetuses, the first oCRF injection did not alter the ACTH response to a second injection. Cortisol increases were reduced. In mature animals, ACTH and cortisol response to oCRF were eliminated by prior oCRF. Thus a large increase in cortisol after oCRF in mature fetuses is associated with inhibition of the ACTH response to a second oCRF injection, whereas in immature animals a small increase in cortisol after the first oCRF injection is not.


ACTH and cortisol responses to hypotension in fetal sheep after a prior CRF injection.

To determine whether an ovine corticotropin-releasing factor (oCRF) injection modifies adrenocorticotropic hormone (ACTH) and cortisol responses to hypotension and whether the effect of any interactions between these stimuli changes across gestation, we studied chronically cannulated fetal lambs of 103-113 ("immature") and 133-139 days gestation ("mature"). Experimental groups received 500 ng/kg oCRF injections and 6 h later had arterial pressure reduced 20% for 10 min with nitroprusside. Blood samples were obtained before and after each manipulation. Controls received vehicle instead of oCRF. The oCRF increased plasma cortisol levels from 2.1 +/- 0.4 to 14.2 +/- 4.7 (SE) ng/ml in immature and 44.9 +/- 2.2 to 102.8 +/- 15 ng/ml in mature animals. In mature fetuses the oCRF did not alter plasma ACTH and cortisol increases due to hypotension. In immature animals ACTH increases were normal but cortisol increases were eliminated. This suggests that the CRF caused maximal stimulation of the adrenal gland. In older fetuses, it appears that the action of ACTH-releasing factors, secreted in response to arterial hypotension, can overcome the negative feedback effects of elevations in endogenous cortisol.


Cl- fluxes related to fluid secretion by the rat parotid: involvement of Cl(-)-HCO3- exchange.

Muscarinic-induced 36Cl- and 86Rb+ (K+ substitute) fluxes were studied in rat parotid acini. Stimulation resulted in a rapid [half time (t1/2) less than 30 s] decrease in both Cl- and Rb+ content (approximately 50 and 30%, respectively) followed by a slower partial recovery (t1/2 approximately 3-4 min) to approximately 80% of resting levels for both ions. Cl- loss was inhibited by the venom of Leiurus quinquestriatus, which contains the maxi-K+ channel blocker charybdotoxin. Cl- recovery was blunted in the presence of bumetanide, an inhibitor of Na(+)-K(+)-Cl- cotransport, or on HCO3- removal and was completely blocked in the presence of bumetanide and 4,4' diisothiocyanostilbene-2,2' disulfonic acid (DIDS), an inhibitor of Cl(-)-HCO3- exchange. In HCO3(-)-containing medium a rapid (t1/2 less than 1 min), DIDS-inhibitable cytoplasmic alkalinization (approximately 0.4 pH unit) was observed in acini switched to a Cl(-)-free solution. This alkalinization was not seen in HCO3(-)-free medium but persisted in the absence of Na+, consistent with the presence of a potent Na(+)-independent Cl(-)-HCO3- exchanger. Kinetic studies indicated that the half-maximal effect of this exchanger for extracellular Cl- was approximately 18 mM. These results are consistent with the hypothesis that secretagogue-induced KCl loss by salivary acinar cells occurs via electrically coupled K+ and Cl- channels. In addition, they provide strong evidence that Cl- entry into, and thus fluid secretion by, these cells is mediated by both Cl(-)-HCO3- exchange and Na(+)-K(+)-Cl- cotransport.


Effects of hypoxia on function and morphology of in vitro frog gastric mucosa.

The effects of gaseous hypoxia and reoxygenation on oxynticopeptic (OPC) and surface mucous cells (SMC) were examined in in vitro bullfrog gastric fundic mucosae mounted in Ussing chambers. Forskolin-stimulated H+ secretion, transmucosal potential difference (PD), and electrical resistance (R) were monitored in tissues incubated in HCO3(-)-free or HCO3(-)-containing buffer. At serosal pH (pHs) 7.2, 1 h of hypoxia with 100% N2 resulted in a decrease in PD, increase in R, and complete inhibition of H+ secretion. After 30 min of hypoxia, the morphology of OPC changed from the secretory to the nonsecretory state without recognizable cytopathology. Destructive changes in OPC increased progressively at pHs 7.2 as the hypoxic period was prolonged from 4 to 24 h. After 4 h of reoxygenation following 12-24 h of hypoxia, OPC remained necrotic and H+ secretion showed no recovery, whereas in some areas where SMC were exfoliated adjacent SMC showed epithelial restitution. The recovery of H+ secretion and PD during 2 h of reoxygenation after 4 h of hypoxia at pHs 6.0 and 6.8 was less than that at 7.2 and 8.0 and was greater in the presence of serosal HCO3- than its absence at pHs 7.2. These results suggest that, in in vitro frog gastric mucosa, 1) OPC are more vulnerable to hypoxia than SMC, 2) basolateral acidosis exaggerates hypoxic injury of OPC, and 3) serosal HCO3- protects OPC from hypoxic injury.


Characterization of opioid receptors on smooth muscle cells from guinea pig stomach.

On the basis of opioid-stimulated contraction of dispersed gastric smooth muscle cells it has been suggested that these cells possess opioid receptors of three subtypes: kappa (kappa), mu (mu), and delta (delta). We have used selective peptidase-resistant radioligands, agonists and antagonists, to examine receptor subtypes on dispersed gastric smooth muscle cells from guinea pigs prepared by collagenase digestion. The kappa-agonist U-50488H, the mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO), and the delta-agonist [D-Pen2,Pen5]enkephalin (DPDPE) each caused muscle contraction. The concentrations required to caused half-maximal contraction were U50488H (6 pM) greater than DAGO (13 pM) greater than DPDPE (6 nM). The abilities of these agonists to inhibit binding of [3H]U-69593 (kappa-preferring) by 50% were U50488H (43 nM) greater than DAGO (43 microM) greater than DPDPE (200 microM). Their abilities to inhibit binding of [3H]naloxone (mu-preferring) by 50% were DAGO (0.2 microM) greater than U50488H (10 microM) greater than DPDPE (greater than 100 microM). No binding could be detected with the delta-selective ligand [3H]DPDPE. The kappa-preferring antagonist Mr2266 (10 nM) preferentially inhibited contraction stimulated by the kappa-agonist U50488H, and naltrexone (10 nM) (mu-selective antagonist) preferentially inhibited contraction stimulated by the mu-agonist DAGO. ICI 174864 (200 microM; delta-selective antagonist) had no effect on contraction stimulated by mu-, kappa-, or delta-agonists. Contraction stimulated by the delta-agonist DPDPE was inhibited by both kappa- and mu-receptor antagonists. Studies on the effect of the antagonists on binding of [3H]naloxone and [3H]U69593 also provided evidence for kappa- and mu-sites but nor for delta-sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurotensin and neuromedin N stimulate mucin output from human goblet cells (Cl.16E) via neurotensin receptors.

The stably differentiated human intestinal goblet cell line Cl.16E was used to study the effects of two structurally related regulatory peptides, neurotensin (NT) and neuromedin N (NN), on mucus secretion. NT and NN stimulated rapid release of mucins from filter-grown Cl.16E cells, this effect being dose related with a mean effective dose of 36 nM for NT and 422 nM for NN. The order of potency of NT, three NT fragments corresponding to the NH2-terminal part [NT-(1-11)] or to the COOH-terminal part [NT-(8-13) and NT-(9-13)], and NN in promoting mucin release and in inhibiting 125I-labeled NT binding to Cl.16E cell membranes was identical with NT greater than or equal to NT-(8-13) greater than NN greater than NT-(9-13) much greater than NT-(1-11) supporting the hypothesis that NT and NN stimulate mucin output through interaction with a common NT-preferring receptor. Scatchard analysis of equilibrium binding data showed one population of NT binding sites in Cl.16E cell membranes with the following characteristics: binding capacity (Bmax) was 141 fmol/mg of protein and dissociation constant (Kd) was 1.00 nM.(ABSTRACT TRUNCATED AT 250 WORDS)


Action of alpha 2A-adrenergic receptors in circular smooth muscle of canine proximal colon.

Addition of the alpha 2-adrenergic receptor agonist clonidine (1 microM) to tetrodotoxin-treated strips of canine colonic circular smooth muscle resulted in a significant increase in contractile force that was blocked by addition of the alpha 2-antagonist yohimbine (0.1 microM). The alpha 2-receptor antagonist radioligand [3H]rauwolsine bound rapidly and reversibly to a single class of saturable sites (Bmax, 38.4 +/- 6.2 fmol/mg protein) in colonic circular smooth muscle membranes with an affinity (KD = 5.1 +/- 0.9 nM) characteristic of alpha 2-adrenergic receptors in smooth muscle. Studies in cells freshly isolated from circular muscle of canine proximal colon verified the smooth muscle origin of these receptors. Rank order of potency of alpha 2-adrenergic receptor antagonists in competition for [3H]rauwolsine binding was yohimbine greater than oxymetazoline much greater than prazosin. Affinity of alpha 2-receptors for yohimbine was indistinguishable from that of its optical isomer, rauwolsine, in both competition studies (KI = 3.4 nM) and in saturation-binding experiments employing [3H]yohimbine directly (KD = 4.2 nM). The alpha-receptor agonist epinephrine, in competition studies employing [3H]rauwolsine, revealed high-affinity binding sites that were converted to low-affinity binding sites for agonist by addition of 100 microM GTP gamma S. Addition of the alpha 2 more-selective agonist clonidine (100 microM) resulted in inhibition of adenylate cyclase activity that was abolished by pretreatment of tissue strips with pertussis toxin suggesting coupling of the alpha 2-receptor in colon to adenylate cyclase via the GTP-binding protein Gi. Our data demonstrate a physiological role for adenylate cyclase-coupled receptors of the alpha 2A-subtype in canine colon circular smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)