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Biomedical subjects

J A Clements

Publications and source records attributed to J A Clements.

At least 73 records · Page 4Linked to original sources
PubMed1989

Regulation of liver angiotensinogen mRNA by glucocorticoids and thyroxine.

This study examines the immediate and longer-term changes in angiotensinogen mRNA and in plasma angiotensinogen which follow the withdrawal and replacement of glucocorticoids or thyroxine. RNA from rat liver was analysed by Northern blot and dot-blot hybridization with a 40-mer oligodeoxynucleotide probe specific for angiotensinogen mRNA. Adrenalectomy decreased plasma angiotensinogen and angiotensinogen mRNA to 55 and 50% of control values respectively over a period of 16 days. Similar decreases were obtained after propylthiouracil (1 mg/kg for 16 days) treatment, except when injected simultaneously with T3 (3 micrograms/kg/day). Over the same period plasma renin activity increased in adrenalectomized rats from 4.1 +/- 0.8 to 7.0 +/- 1.5 pmol angiotensin I (AI)/ml/h, and decreased in propylthiouracil-treated rats from 3.8 +/- 0.4 to 1.6 +/- 0.4 pmol AI/ml/h. Approximate half-times of 2-3 days were calculated for both plasma angiotensinogen and angiotensinogen mRNA post-adrenalectomy or after propylthiouracil treatment. Dexamethasone (400 micrograms/kg, i.m.) given to intact rats rapidly increased angiotensinogen mRNA to a maximum of 250% of control by 8 h and with a half-maximal response of 2.8 h. Plasma angiotensinogen responded similarly, apart from an initial delay of 2 h. Treatment with different doses of propylthiouracil and dexamethasone showed that responses were dose-related. We conclude that changes in plasma angiotensinogen and angiotensinogen mRNA are closely correlated, and that under various physiological circumstances angiotensinogen mRNA has a rapid rate of accumulation but a slow rate of decay.

Adrenalectomy↗
PubMed1989

Effect of ethane dimethane sulphonate on proopiomelanocortin (POMC) mRNA and POMC-derived peptides in the rat testis.

Recent studies have shown that both proopiomelanocortin (POMC)-derived peptides and a range of POMC gene transcripts are present in the testis. Previous immunocytochemical studies have reported immunoreactive (ir)-beta-endorphin (EP) and ir-ACTH to be localized in the Leydig cells, and ir-NacEP in spermatogonia and primary spermatocytes. In the present study, we have further examined the hypothesis that testicular Leydig cells are the principal site of synthesis of these peptides, by determining the effects of the administration of the cytotoxic drug ethane dimethane sulphonate (EDS) which selectively destroys the Leydig cells of the testis. As expected, serum testosterone levels fell and serum FSH/LH levels increased within 3 days of EDS administration, returning to normal levels 4-8 weeks later. In contrast, the testicular content of POMC-derived peptides and POMC mRNA levels in these animals was not significantly altered throughout the experimental period. In addition, POMC mRNA was not detected in a purified Leydig cell preparation derived from adult male rats, and POMC-derived peptides were also undetectable in the media of a similar preparation following cell culture. These data suggest that in the adult the predominant site of rat POMC gene expression is in testicular interstitial cells other than Leydig cells.

Adrenocorticotropic Hormone↗
PubMed1989

Determination of pipothiazine in human plasma by reversed-phase high-performance liquid chromatography.

A method is described for the determination of pipothiazine in human plasma, based on reversed-phase HPLC. The method has been applied in a pharmacokinetic study of pipothiazine in six psychiatric patients receiving repeated depot intramuscular injections for six months. A number of compounds likely to be taken concurrently by patients were tested for potential to interfere with the assay. There was no evidence of "dose-dumping" in the period following injection. Comparison of the pharmacokinetic profiles after the first and sixth injections showed no evidence of drug accumulation.

Adult↗
PubMed1989

Studies of the structure of lung surfactant protein SP-A.

SP-A, a glycoprotein of pulmonary surfactant, consists of an NH2-terminal domain containing a collagen-like sequence and a COOH-terminal domain with sequence homology to several Ca2(+)-dependent lectins. We have compared the size, thermal stability, and secondary structure of recombinant SP-A, the product of a fibroblast line transfected with a single human gene encoding SP-A, with natural SP-A isolated from canine and human lungs. Our results suggest both recombinant and natural SP-A are assembled as large oligomers. More variability in the degree of oligomerization was observed with recombinant human SP-A than with natural canine SP-A. As shown by collagenase digestion, the full assembly of protein subunits was dependent on an intact collagen-like domain. The cysteines in the noncollagen domain of SP-A form intrachain bonds between residues 135-226 and 204-218. The circular dichroism spectra of both recombinant and natural SP-A were consistent with the presence of a collagen-like triple helix. As determined by the change in ellipticity at 205 nm, the thermal transition temperatures of canine, natural human, and recombinant SP-A were 51.5, 52.3, and 42.0 degrees C, respectively. These results suggest differences in the assembly and stability of the natural and recombinant proteins.

Amino Acid Sequence↗
PubMed1989

Cellular uptake and processing of surfactant lipids and apoprotein SP-A by rat lung.

The intracellular pathways and the kinetics of metabolism of surfactant apoprotein and lipid, which may be recycled from the alveolar space, are largely unknown. We used a lipid-apoprotein complex made from liposomes of pure lipids in a ratio found in mammalian pulmonary surfactant plus surfactant apoprotein (SP-A, Mr = 26,000-36,000) to test some possible relationships in the recycling of these major surfactant components between intrapulmonary compartments. After intratracheal instillation of 80 microliters of an apoprotein-liposome mixture with separate radiolabels in the lipid and the apoprotein, rats were killed at times from 8 min to 4 h later. The lungs were lavaged with saline, and subcellular fractions were isolated on discontinuous sucrose density gradients. Both the [14C]lipid radiolabel and the 125I-apoprotein radiolabel demonstrated a time-dependent increase in radioactivity recovered in a lamellar body-enriched fraction. Uptake of the radiolabels into other subcellular fractions did not exhibit a clear-cut time dependence; more of the protein than the lipid radiolabel was found in the Golgi-rich and microsomal fractions. We conclude that both the lipid and apoprotein portions of lung surfactant are taken up by lung cells and are incorporated into secretory granules of the cells.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

Surfactant secretion and clearance in the newborn.

Pregnant rabbits (30 days) were injected intravenously with [3H]choline 8 h before delivery. The fetuses were delivered, and lung lavage and lamellar body phospholipids (PL) were analyzed. Some newborns also received radioactively labeled surfactant intratracheally on delivery and were permitted to breathe. With time, intratracheal label decreased in lavage and appeared in the lamellar body fraction, and intravenous label accumulated in both pools. Using a tracer analysis for non-steady state, we calculated surfactant secretion and clearance rates for the newborn period. Before birth, both rates rose slightly from 1.8 micrograms PL.g body wt-1.h-1 at 6 h before birth to 7.3 at birth. Immediately after birth, secretion rate rose to 37.7 micrograms PL.g body wt-1.h-1. Between 1.5 and 2 h after birth it fell to a minimum of 1.8 micrograms PL.g body wt-1.h-1 and then rose slowly to 6.0 at 12 h. After birth, clearance rate increased less than secretion rate (maximum 24.7 micrograms PL.g body wt-1.h-1 shortly after birth) then followed the same pattern but did not balance secretion rate in the 1st day.

Animals↗
PubMed1989

Glucocorticoid regulation of proopiomelanocortin gene expression in the pituitary gland of hypothalamopituitary intact and hypothalamopituitary disconnected sheep.

Proopiomelanocortin (POMC) gene expression in the anterior pituitary (AP) gland has previously been shown to be positively regulated by CRF and AVP and negatively regulated by glucocorticoids. In the neurointermediate lobe (NIL) of the pituitary, however, POMC gene expression is under tonic inhibitory dopaminergic control. In the present study we have used hypothalamopituitary intact (HPI), ovariectomized (OVX), and OVX/hypothalamopituitary disconnected (OVX/HPD) ewes to examine direct (i.e. nonhypothalamic) effects of glucocorticoids on POMC gene expression in both the AP and the NIL. There was no difference between POMC mRNA levels in intact and OVX sheep. In intact animals treated with dexamethasone, AP POMC mRNA levels were half those of controls. POMC mRNA levels were increased 3-fold in OVX/HPD sheep, compared with OVX, and lowered by dexamethasone to half OVX/HPD levels. In the NIL, hypothalamopituitary disconnection resulted in slightly higher mean POMC mRNA levels than in intact animals but the large intragroup variation did not allow a significant change. Dexamethasone administration had no effect on NIL levels of POMC mRNA in intact or OVX/HPD sheep.

Animals↗
PubMed1989

Regulation of follicle-stimulating hormone beta and common alpha-subunit messenger ribonucleic acid by gonadotropin-releasing hormone and estrogen in the sheep pituitary.

The effect of gonadotropin-releasing hormone (GnRH) and/or estradiol (E2) on pituitary messenger ribonucleic acid (mRNA) levels of luteinizing hormone beta (LH beta), follicle-stimulating hormone beta (FSH beta) and the common alpha-subunit were determined in anterior pituitary glands from ovariectomized (OVX) ewes. Hypothalamo-pituitary disconnected (HPD) ewes receiving appropriate hormonal treatment were used to assess the relative roles of GnRH and E2 in directly regulating FSH beta and alpha-subunit mRNA levels. Levels of LH beta mRNA were increased in OVX animals compared with intact controls, and E2 treatment of OVX animals significantly reduced mRNA levels of LH beta and FSH beta. HPD substantially reduced FSH beta and alpha-subunit mRNA levels. Treatment of OVX/HPD animals with pulses of GnRH (250 ng/2 h) for 1 week restored FSH beta and alpha-subunit mRNA to OVX levels. Combined GnRH and E2 treatment significantly lowered FSH beta mRNA levels, but resulted in a rise in alpha-subunit mRNA levels. Treatment of OVX/HPD ewes with E2 alone had no effect on FSH beta and alpha-subunit mRNA levels. These findings indicate that E2 acts directly on the pituitary to negatively regulate FSH beta mRNA levels, and to positively regulate alpha-subunit mRNA levels in the presence of GnRH.

Animals↗
PubMed1989

Effect of lung collapse on alveolar surfactant in rabbits subjected to unilateral pneumothorax.

To determine whether atelectasis might modify lung surfactant, we injected N2 into the right pleural space of adult rabbits. Daily, under sedation, pleural gas volume and pressure were measured and adjusted to 20 ml/kg and 0 to +2 cm H2O with N2. On the sixth day, pHa, PaCO2, PaO2, and FRC were measured. Pressure-volume diagrams or bronchoalveolar lavages (BAL) were performed separately on right and left lungs. Surfactant subfractions were obtained from BAL fluid, and total protein, LDH, and cell counts were determined. Phospholipid (PL) was assayed in lung homogenate, BAL fluid, and subfractions, and PL composition was determined on the largest BAL subfraction (P4). On the sixth day the pleural gas volume was 19.7 +/- 2.7 (SD) ml/kg, and PaO2 and FRC were significantly decreased. Air volume in excised right lungs at 30 cm H2O was 13.1 +/- 2.8 (SE) ml/kg with pneumothorax (PN) and 22.8 +/- 1.9 (SE) ml/kg in controls. Total PL was decreased 43% in BAL and 59% in P4 of collapsed lungs. Phosphatidylglycerol to phosphatidylinositol (PI) plus phosphatidylserine (PS) ratio of P4 was substantially decreased in both lungs of PN animals. Cell counts, LDH, and protein in BAL did not suggest inflammation or epithelial damage. We conclude that pneumothorax decreases the quantity of alveolar surfactant in the collapsed lung and alters its phospholipid composition toward the fetal pattern in both lungs, possibly due in part to the proliferative response of the lungs to pneumothorax.

Animals↗
PubMed1988

Transfer of phospholipids by a protein fraction obtained from canine pulmonary lavage.

Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.

Animals↗
PubMed1988

Androgen dependence of specific kallikrein gene family members expressed in rat prostate.

We have used oligonucleotide probes specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, and P1) to establish which arginyl esteropeptidase (kallikrein-like) genes are expressed in the prostate. We have also compared the expression and androgen dependence of these genes in prostate, submaxillary gland (SMG) and kidney. Only S3 (tonin-like) and P1 (kallikrein-like) are expressed in the prostate, with S3 very much more abundant. Prostatic S3 mRNA disappears after 8 days castration and is restored to intact levels by dihydrotestosterone (DHT) but not estradiol benzoate (EB) for 8 days. Prostate P1 mRNA levels were similarly but not identically affected. All six genes are expressed in the SMG, with PS (true kallikrein) the most abundant. Levels of PS mRNA in SMG are unaffected by castration, DHT, or EB treatment, although mRNA levels of other kallikrein-like (S1, K1, and P1), tonin (S2), and tonin-like (S3) genes fall 40-60% after castration, and are unaffected or partially restored by DHT and/or EB administration. Only PS and K1 are expressed in the kidney, at much lower levels than in the SMG and unaffected by castration or steroids. These studies thus confirm and extend the concept of tissue specificity of arginyl esteropeptidase gene expression, and further demonstrate that the same gene(s) is differentially regulated by androgens in the rat prostate, SMG, and kidney.

Animals↗
PubMed1988

Kallikrein gene expression in estrogen-induced pituitary tumors.

Anterior pituitary kallikrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.

Animals↗
PubMed1988

Luteinizing hormone-beta mRNA levels are regulated primarily by gonadotropin-releasing hormone and not by negative estrogen feedback on the pituitary.

Long-term effects of gonadotropin-releasing hormone (GnRH) and/or estrogen on pituitary mRNA levels for the beta-subunit of luteinizing hormone (LH-beta) were determined in anterior pituitary glands from ovariectomized (OVX) ewes. The relative roles of these two factors were assessed by studying hypothalamopituitary disconnected (HPD) ewes with appropriate hormonal treatments. Levels of LH-beta mRNA were increased by ovariectomy and substantially reduced by HPD. Treatment of OVX-HPD ewes with pulses of GnRH (250 ng each 2 h) for 1 week restored LH-beta mRNA levels to OVX levels, whereas treatment with estrogen alone did not alter the low levels found in OVX-HPD ewes. Combined GnRH and estrogen treatment for one week produced LH-beta mRNA levels that were similar to those found in OVX-HPD ewes given GnRH alone; plasma LH pulse amplitudes were also similar in these two groups. From these data we conclude that the long-term negative feedback effect of estrogen to reduce LH secretion is due to a primary inhibition of GnRH secretion and is not a pituitary effect of estrogen. Long-term regulation of LH-beta mRNA is thus primarily regulated by GnRH.

Animals↗
PubMed1988

Post-translational processing of pro-opiomelanocortin in the Brattleboro (di/di) rat pituitary.

In homozygous (di/di) Brattleboro rats, pro-opiomelanocortin (POMC) synthesis and processing, and the release of its products, is by definition not under the control of hypothalamic arginine vasopressin (AVP) in contrast with normal rats or heterozygote (di/+) littermates. To explore the role of AVP on these parameters, we have compared homozygotes and heterozygotes in terms of POMC mRNA levels, pituitary content of immunoreactive (ir)-ACTH, ir-beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH) and ir-N-acetyl-EP (NacEP), plasma levels of ir-ACTH and ir-beta-EP, and RP-HPLC profiles of the various forms of ir-alpha-MSH and ir-NacEP. Homozygous rats had immeasurably low levels of hypothalamic ir-AVP, in contrast with their heterozygote littermates; hypothalamic ir-corticotropin releasing factor did not differ between strains. No difference between-strains was seen in levels of POMC mRNA; elevated levels of all pituitary peptides, except ir-ACTH, were found in di/di rats; plasma levels of both ir-ACTH and ir-beta-EP were lower in di/di rats; pituitary ir-alpha-MSH RP-HPLC profiles were similar in both strains, but those for ir-NacEP showed a striking increase in Nac alpha-EP in di/di rats. We interpret these data as evidence for decreased degradation/retarded release of POMC products from the di/di anterior pituitary, for increased processing of POMC products to shorter forms in both anterior pituitary and neuro-intermediate lobe, and thus for a role of AVP in the processing of POMC, as well as POMC synthesis and ACTH/beta-EP release.

Adrenocorticotropic Hormone↗
PubMed1987

Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells.

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.

Animals↗
PubMed1987

Rapid and specific lowering of pituitary FSH beta mRNA levels by inhibin.

Hypothalamo-pituitary disconnected sheep were given gonadotropin releasing hormone pulses every 2 h for 1 week, and the effects of inhibin on mRNA for FSH beta, LH beta, alpha-subunit and prolactin examined. Levels of FSH beta mRNA were reduced to approximately 20% 6 h after administration, and to approximately 10% by 30 h; no change was seen in LH beta, alpha-subunit or prolactin mRNA. These data show that inhibin has a very rapid and specific effect on FSH beta mRNA levels directly at the level of the pituitary gland.

Animals↗
PubMed1987

Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption.

Pulmonary surfactant is a lipid-rich material that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. The turnover of surfactant phospholipids in the alveolar space is fast, and several lines of evidence suggest there is rapid formation and replenishment of the phospholipid surface film during normal respiration. Specific proteins may regulate these dynamic surface properties. The predominant surfactant protein is a well-characterized, lipid-associated glycoprotein, SP 28-36 (28-36 kDa). A second group of very hydrophobic proteins has recently been shown to affect the surface activity of surfactant phospholipids. We have isolated this group of hydrophobic proteins, herein called SP 5-18 (5-18 kDa), from canine surfactant and have shown by NH2-terminal sequence analysis that at least two proteins, SP 5-8 and SP 18, are present in this group. We have derived the full amino acid sequence of SP 18 from the nucleotide sequence of the cDNAs identified with oligonucleotide probes that were based on the NH2-terminal amino acids of SP 18. The protein isolated from extracellular surfactant appears to be a fragment of a much larger precursor protein (40 kDa). The amino acid sequence of SP 18 is markedly hydrophobic and contains two possible bilayer-spanning domains. We have shown that SP 18 and the glycoprotein SP 28-36 have a cooperative, calcium-dependent action in promoting the formation of phospholipid surface films.

Amino Acid Sequence↗