Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats.

The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled single-wall carbon nanotubes (SWCNT) in rats. The lungs of rats were instilled either with 1 or 5 mg/kg of the following control or particle types: (1) SWCNT, (2) quartz particles (positive control), (3) carbonyl iron particles (negative control), (4) phosphate-buffered saline (PBS) + 1% Tween 80, or (5) graphite particles (lung tissue studies only). Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers and cell proliferation methods, and by histopathological evaluation of lung tissue at 24 h, 1 week, 1 month, and 3 months postinstillation. Exposures to high-dose (5 mg/kg) SWCNT produced mortality in ~15% of the SWCNT-instilled rats within 24 h postinstillation. This mortality resulted from mechanical blockage of the upper airways by the instillate and was not due to inherent pulmonary toxicity of the instilled SWCNT particulate. Exposures to quartz particles produced significant increases versus controls in pulmonary inflammation, cytotoxicity, and lung cell parenchymal cell proliferation indices. Exposures to SWCNT produced transient inflammatory and cell injury effects. Results from the lung histopathology component of the study indicated that pulmonary exposures to quartz particles (5 mg/kg) produced dose-dependent inflammatory responses, concomitant with foamy alveolar macrophage accumulation and lung tissue thickening at the sites of normal particle deposition. Pulmonary exposures to carbonyl iron or graphite particles produced no significant adverse effects. Pulmonary exposures to SWCNT in rats produced a non-dose-dependent series of multifocal granulomas, which were evidence of a foreign tissue body reaction and were nonuniform in distribution and not progressive beyond 1 month postexposure (pe). The observation of SWCNT-induced multifocal granulomas is inconsistent with the following: (1) lack of lung toxicity by assessing lavage parameters, (2) lack of lung toxicity by measuring cell proliferation parameters, (3) an apparent lack of a dose response relationship, (4) nonuniform distribution of lesions, (5) the paradigm of dust-related lung toxicity effects, (6) possible regression of effects over time. In addition, the results of two recent exposure assessment studies indicate very low aerosol SWCNT exposures at the workplace. Thus, the physiological relevance of these findings should ultimately be determined by conducting an inhalation toxicity study.

Evaluation of Orntide microspheres in a rat animal model and correlation to in vitro release profiles.

Orntide acetate, a novel luteinizing hormone-releasing hormone (LHRH) antagonist, was prepared and evaluated in vivo in 30-day and 120-day sustained delivery formulations using a rat animal model. Orntide poly(d,l-lactide-co-glycolide) (PLGA) and poly(d,l- lactide) (PLA) microspheres were prepared by a dispersion method and administered subcutaneously in a liquid vehicle to rats at 2.2 mg Orntide/kg of body weight (30-day forms) or 8.8 mg Orntide/kg (120-day forms). Serum levels of Orntide and testosterone were monitored by radioimmunoassays, and a dose-response study at 4 doses (3, 2.25, 1.5, and 1.75 mg Orntide/kg) was conducted to determine the effective dose of Orntide. Microspheres with diameters between 3.9 and 14 micron were prepared. The onset and duration of testosterone suppression varied for different microsphere formulations and were influenced both by polymer properties and by microsphere characteristics. Microspheres prepared with 50:50 and 75:25 copolymers effectively sustained peptide release for 14 to 28 days, whereas an 85:15 copolymer and the PLA microspheres extended the pharmacological response for more than 120 days. Increase in drug load generally accelerated peptide release from the microspheres, resulting in higher initial serum levels of Orntide and shorter duration of the release. In general, apparent release was faster in vivo than under in vitro conditions. Orntide microspheres effectively suppressed testosterone in rats, providing rapid onset of release and extended periods of chemical castration. Testosterone suppression occurred immediately after microsphere administration without the initial elevation seen with LHRH superagonists.

Treatment effects of intranasal growth hormone releasing peptide-2 in children with short stature.

Growth hormone-releasing peptide (GHRP)-2 is a synthetic six amino acid peptide that is a potent GH secretagogue. Although it shares no structural homology with GH-releasing hormone, in clinical studies its actions on the pituitary release of GH are similar. It is effective when administered orally and intranasally. For children with GH deficiency, such noninvasive treatments are most desirable and in need of development. Fifteen children with short stature participated in this study. All of the children had a height < 2 S.D. below mean for age, poor height velocity, delayed bone age, and low serum concentrations of IGF-1. These children had been tested with standard GH secretagogues, e.g. arginine, insulin, and L-dopa. Fifty percent of the children were GH deficient, the remainder had idiopathic short stature. The children received testing with GHRH and GHRP-2 as an acute i.v. bolus of 1 microgram/kg; all children in this study demonstrated a GH response > 20 micrograms/l. Each child in this study also demonstrated a GH response > 10 micrograms/l in response to intranasal GHRP-2, in the dose range of 5-20 micrograms/kg. The children were administered intranasal GHRP-2, 5-15 micrograms/kg, twice a day for 3 months, then three times a day. Fifteen children participated in the study for 6 months; six of the children have participated for 18-24 months. Height velocity, serum IGF-1, IGF-binding protein 3 (IGFBP-3) and GH-binding protein (GHBP) concentrations, and GH responses to GHRP-2 by i.v. bolus and intranasal spray were examined during treatment. Height velocity increased from 3.7 +/- 0.2 cm/year to 6.1 +/- 0.3 cm/year at 6 months, 6.0 +/- 0.4 cm/year at 18-24 months. There were no significant changes in IGF-1 or IGF-PB3 concentrations, or in acute GH responses to i.v. or intranasal GHRP-2. GHBP concentrations rose significantly, from 439 +/- 63 pmol/l to 688 +/- 48 pmol/l. In this study, intranasal GHRP-2 administration was well tolerated, and produced a modest but significant increase in growth velocity.

Diagnostic studies with intravenous and intranasal growth hormone-releasing peptide-2 in children of short stature.

GH secretion is primarily regulated by the hypothalamic-releasing hormones GHRH and somatostatin. Additionally, several neurotransmitters act at the hypothalamus and pituitary to modulate GH release. The agents commonly used in clinical practice to diagnose GH deficiency, such as arginine, insulin and L-dopa, act through the neural GH network. Many children with a poor GH response to conventional agents have a significant serum GH response to iv GHRH. GH-releasing peptides (GHRPs) are synthetic peptides that like GHRH act directly on pituitary somatotrophs to stimulate GH release. GHRP-2, an investigational drug, is one of the most potent members of the GHRP family. It has been shown to be effective in adults via the oral and intranasal as well as the iv route of administration. In this study, GH responses to GHRP-2 were compared with GH responses to other provocative agents in children of short stature. GHRP-2 was administered iv or intranasally to children with short stature. In the same subjects, GHRP-2 was administered iv in combination with GHRH. Twenty-four children undergoing evaluation for GH deficiency received at least one conventional agent (arginine, L-dopa/exercise, insulin) in addition to iv GHRH and GHRP-2. The GH responses to GHRH or GHRP-2 were similar in each child, and both were equally reliable predictors of pituitary reserve. The conventional agents used in GH testing were less likely to predict the capacity of the pituitary to release GH than were either GHRH or GHRP-2. There was no correlation between maximal GH response to standard tests with GH responses to GHRH or GHRP-2. A subset of the group of 21 children who had a robust response to iv GHRP-2 were later administered GHRH+GHRP-2 simultaneously. The GH response to GHRH+GHRP-2 was synergistic in this group of 12 children, similar to previously reported observations in adults of normal stature. Fifteen of the 21 children who had a robust response to the iv GH-releasing factors also received intranasal GHRP-2. All 15 of these children had a significant GH response to intranasal GHRP-2 over a dose range of 5-20 micrograms/kg per dose. The mean peak GH response to 15 micrograms/kg was 31.3 micrograms/L. The intranasal preparation was well tolerated.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological activities of thionated thyrotropin-releasing hormone analogs.

Analogs of thyrotropin-releasing hormone (Glp-His-Pro-NH2, TRH) have been prepared which contain thioamide moieties in the pyroglutamic acid ring, the carboxyamide proline terminus, and in both positions (dithio). These compounds have been tested for TSH-releasing activities (in vitro and in vivo), and for binding to TRH receptors in rat pituitary and cortex. The monothionated analogs showed no significant differences in TSH-releasing potency from TRH either in vitro or in vivo. However, with two thioamide replacements the potency decreases about 50%. Significantly, in terms of receptor selectivity, thionation has resulted in differentiation between brain receptors (pituitary and cortex). The Pro psi[CSNH2] and dithio analogs were more selective (higher affinity to pituitary receptors) than the parent hormone, while the analog containing a thioamide replacement in the pyroglutamyl ring had lower affinity and was not selective. These results suggest that the subtle exchange of sulphur for oxygen can have an important impact on both receptor selectivity and affinity within a biologically active peptide.

General practitioner outpatient referrals: do good doctors refer more patients to hospital?

OBJECTIVE: To investigate the relation between general practitioners' referral rates to individual specialties and the individual areas of expertise of the referring doctors. DESIGN: Data collected on referral patterns in one group practice over nine months. SETTING: General practice in suburban Birmingham consisting of five partners and a trainee. RESULTS: In 395 referrals there were large differences in referral patterns among partners for otorhinolaryngology, ophthalmology, general surgery, and dermatology. The doctors with particular expertise in otorhinolaryngology and ophthalmology had high referral rates to those specialties, and these differences persisted after allowing for case mix. CONCLUSION: A high referral rate does not necessarily imply a high level of inappropriate referral.

On the actions of the growth hormone-releasing hexapeptide, GHRP.

GH-releasing peptide (His-DTrp-Ala-Trp-DPhe-Lys-NH2 or GHRP) releases GH by a unique and complementary dual site of action on the hypothalamus and pituitary. These effects are mediated via non-GH-releasing hormone (non-GHRH) and nonopiate receptors in rats. Select types of opiates are known to release GH by a hypothalamic site of action, and thus, the dermorphin heptapeptide and benzomorphan opiate agonist 2549 used in this study presumably act on the hypothalamus to release GH. Neither dermorphin nor 2549 released GH or augmented the GH responses of GHRP or GHRH in vitro by a direct pituitary action, while GHRH antiserum inhibited the GH response of both dermorphin and 2549 in vivo. Evidence indicates that these opiates and GHRP administered together synergistically release GH, demonstrating the independent action(s) of GHRP and the opiates. Present data indicate that one of the major differences in the actions of dermorphin, 2549, and GHRP is the inhibition of somatostatin (SRIF) release by the opiates but not by GHRP. Although the actions of dermorphin, 2549, and GHRP on GH release are GHRH dependent, release of endogenous GHRH does not explain how GH is released synergistically by the combination of these peptides. It is proposed that dermorphin/2549 synergistically release GH with GHRP or GHRH because these opiates inhibit SRIF release. Since the GHRP plus GHRH synergistic GH release was not explained by inhibition of SRIF or stimulation of GHRH, an alternative mechanism is proposed to explain how GHRP synergistically release GH in combination with GHRH. The complementary, rather dramatic synergistic interaction of GHRP, GHRH, and dermorphin or GHRP, GHRH, and 2549 in releasing GH again strongly supports the independent actions of these compounds.

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes.

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.

Two novel annexins from Drosophila melanogaster. Cloning, characterization, and differential expression in development.

The annexins are a family of homologous Ca2(+)- and phospholipid-binding proteins that until now have only been found in vertebrates. cDNA clones encoding two novel annexins from Drosophila melanogaster were isolated and characterized. RNA blots indicate that the messages for the two Drosophila proteins are differentially expressed in development, with one message being expressed throughout development, while the other is only found in early embryos and adult flies. In situ hybridizations localize the two Drosophila genes to 93B and 19A-4,7. A similarly high degree of homology relates Drosophila annexins to different vertebrate annexins, indicating that the Drosophila annexins are not the invertebrate homologues of particular mammalian annexins but that they constitute novel members of the annexin gene family. In continuation with a recently established terminology, the Drosophila annexins will be named annexins IX and X. The biochemical properties of Drosophila annexin X were investigated using recombinant protein. Similar to vertebrate annexins, annexin X bound to liver membranes and liposomes containing phosphatidylserine in a calcium-dependent manner but not to liposomes containing phosphatidylcholine. In addition, annexin X partitioned into the detergent phase of Triton X-114 as a function of calcium. The conservation of the annexin family of Ca2(+)-binding proteins in invertebrates suggests that they have a basic function in cells which is not peculiar to vertebrate biology, and the availability of the Drosophila sequences will open avenues for mutational studies of these functions.

Growth hormone (GH)-releasing peptide stimulates GH release in normal men and acts synergistically with GH-releasing hormone.

The acute GH release stimulated by the synthetic hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH2 [GH releasing peptide (GHRP)], was determined in 18 normal men and compared with the effects of GH-releasing hormone, GHRH-(1-44)-NH2. Specificity of effect was assessed by measurement of serum PRL, LH, TSH, and cortisol. GHRP was administered at doses of 0.1, 0.3, and 1.0 microgram/kg by iv bolus. GHRH at a dose of 1.0 microgram/kg was administered alone and together with various does of GHRP. No adverse clinical effects of laboratory abnormalities were observed in response to GHRP. A side-effect of mild facial flushing of 1- to 3-min duration occurred in 16 of the 18 subjects who received GHRH-(1-44)-NH2. Mean (+/- SEM) peak serum GH levels after injection of placebo and 0.1, 0.3, and 1.0 microgram/kg GHRP were 1.2 +/- 0.3, 7.6 +/- 2.5, 16.5 +/- 4.1, and 68.7 +/- 15.5 micrograms/L, respectively. The submaximal dosages of 0.1 and 0.3 microgram/kg GHRP plus 1 microgram/kg GHRH stimulated GH release synergistically. Serum PRL and cortisol levels rose about 2-fold above basal levels only at the 1 microgram/kg dose of GHRP, and there were no changes in serum LH and TSH over the first hour after administration of the peptide(s). GHRP is a potent secretagogue of GH in normal men. Since GHRP and GHRH together stimulate GH release synergistically, these results suggest that GHRP and GHRH act independently. This supports our hypothesis that the GH-releasing activity of GHRP reflects a new physiological system in need of further characterization in animals and man.

Human 67-kDa calelectrin contains a duplication of four repeats found in 35-kDa lipocortins.

The 67-kDa calelectrin is the largest member of a family of Ca2+-binding proteins that associate with membranes and phospholipids in a Ca2+-dependent manner. Oligonucleotide probes based on peptide sequences obtained from purified bovine 67-kDa calelectrin were used to screen a human retina cDNA library, and the complete primary structure of human 67-kDa calelectrin was deduced by DNA sequence analysis. The protein consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to the human lipocortin I and II sequences, each of which contains four such repeats. The amino termini of the three proteins show no sequence similarity; however, in the repeated regions the proteins are 42-45% identical in sequence. Analysis of the 16 repeats from the three proteins provides insights into the structural basis for Ca2+-dependent phospholipid binding. These data place the calelectrins and the lipocortins into the same gene family and suggest that these proteins have similar functions and have evolved from a common ancestor.

Cigarette smoking-induced coronary vasoconstriction in atherosclerotic coronary artery disease and prevention by calcium antagonists and nitroglycerin.

In patients with coronary artery disease, cigarette smoking increases myocardial oxygen demand but may cause an inappropriate alpha-adrenergically mediated fall in myocardial oxygen supply. This study was performed to determine if smoking-induced coronary vasoconstriction is prevented by nitroglycerin, verapamil or nifedipine treatment. In 25 smokers with coronary artery disease (20 men, 5 women, aged 32 to 65 years), heart rate-systolic arterial pressure double product and coronary sinus blood flow (thermodilution) were measured before and during smoking both before and 30 to 60 minutes after administration of saline solution (n = 5, control subjects); nifedipine, 10 mg sublingually (n = 6); verapamil, 10 mg intravenously (n = 7); or nitroglycerin, 0.4 mg sublingually (n = 7). During the first smoking period, double product increased, but coronary sinus flow did not change or decreased. During the second smoking period, in the control subjects double product and coronary sinus flow responded in a manner similar to that observed previously. In those given nifedipine, double product did not change, but coronary sinus flow increased (-4 +/- 5% during the first smoking period [before nifedipine] and 17 +/- 12% during the second period [after nifedipine], p less than 0.01). In those given verapamil, double product and coronary sinus flow increased during smoking (-12 +/- 8% during the first smoking period [before verapamil], 10 +/- 9% during the second period [after verapamil], p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Variables determining the growth hormone response of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in the rat.

Previous studies of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GH-RP-6) have shown this synthetic hexapeptide to be a potent and specific stimulator of GH secretion both in vivo and in vitro. In this study the variables determining the in vivo responses were examined in the rat. The magnitude of the GH response to sc GH-RP-6 was dependent on the age and sex of the rat. Animals less than 15 days of age had much larger responses than did rats 21 days and older. At 10 days of age the male rat had a larger GH response than the female. At 21 days of age, bis(4-methyl 1-homo-piperazinyl-thiocarbonyl) disulfide (Fla-63)-pretreated females had larger responses than did Fla-63-pretreated males. In the Fla-63-pretreated adult rat, sc GH-RP-6 stimulated GH release in the female but not in the male. In the 10-day-old male, the ED50 for sc GH-RP-6 was 0.4 micrograms, and the maximal serum GH response was 800 ng/ml. In the 21-day-old female Fla-63-pretreated rat, the ED50 for sc GH-RP-6 was 3.0 micrograms, and the maximal GH response was 200 ng/ml. In the 21-day-old female pentobarbital-anesthetized rat, iv GH-RP-6 had an ED50 of 0.5 micrograms and a maximal serum GH response of 2500 ng/ml. A marked dose- and time-dependent decrease of subsequent GH-RP-6 responses occurred after a single sc GH-RP-6 injection. Decreases in pituitary GH or increases in somatostatin secretion would not explain this decreased response because the GH response of MRZ 2549, an opiate agonist, was unchanged by GH-RP-6 pretreatment. In contrast to the acute effect of GH-RP-6, chronic daily injections of GH-RP-6 resulted in an enhancement of the GH-RP-6 response.

Multiple mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A reductase determined by multiple transcription initiation sites and intron splicing sites in the 5'-untranslated region.

The current studies show that mRNAs with 16 different 5'-untranslated regions (varying in length from 68 to 670 nucleotides) are produced from the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in hamster UT-1 cells. This complex pattern of mRNAs results from a combination of multiple transcription initiation sites and multiple 5' splice donor sites for the intron in the 5'-untranslated region of the gene. Analysis of the multiple mRNAs was made possible by a modification of the S1 nuclease technique in which we used a series of progressively truncated uniformly labeled, single-stranded [32P]DNA probes in addition to the usual end-labeled 32P-probes. All of the reductase mRNAs are diminished when UT-1 cells are incubated with sterols, indicating that all of them are subject to coordinate control.

HMG CoA reductase: a negatively regulated gene with unusual promoter and 5' untranslated regions.

The rate-limiting enzyme of cholesterol biosynthesis, HMG CoA reductase, is controlled by negative feedback regulation of transcription. We have isolated the reductase gene from a bacteriophage lambda genomic library prepared from hamster UT-1 cells. The 25 kilobase gene is split into 20 exons. The 5' untranslated and promoter regions differ from those of previously characterized genes. The 5' untranslated region encompasses as many as 670 nucleotides; contains up to eight AUG codons upstream of the codon used to initiate translation; and has multiple transcription initiation sites as determined by S1 nuclease mapping and primer extension analysis. The promoter region lacks a characteristic TATA box and CCAAT box; is rich in G + C residues (65%); and contains repeat sequences homologous to the 21 base pair repeats of the SV40 promoter. These unusual features may be relevant to the mechanism of expression of "housekeeping" genes, particularly those that are subject to negative feedback regulation.

On the in vitro and in vivo activity of a new synthetic hexapeptide that acts on the pituitary to specifically release growth hormone.

His-DTrp-Ala-Trp-DPhe-LysNH2, [His1,Lys6] GHRP, is a new synthetic hexapeptide which specifically elicits a dosage-related release of GH in vitro and in vivo without a concomitant release of LH, FSH, TSH, or PRL and, in limited in vivo studies, insulin or glucagon. Our results indicate that this small peptide has the attributes of a hypophysiotropic hormone. In vitro the minimum and maximum active dosages ranged from 1-10 ng/ml in the pituitary incubate assay. It was active in rats, monkeys, lambs, calves, and under special experimental conditions chicks, indicating its lack of species dependency. It was active when administered iv, sc, or ip to rats. After iv injection, GH levels rose within 2 min, peaked at +10-20 min, and by 2 h usually had returned to normal. It was not possible to directly compare the potencies of [His1,Lys6]GHRP, and the GH-releasing factors GHRF-44 and GHRF-40 after a single sc injection in rats because the time course of the GH response of these peptides was different. The GH response of [His1,Lys6]GHRP was longer in duration than either of these larger peptides. Both SRIF-14 and SRIF-28 inhibited the GH response of the hexapeptide; however, SRIF-28 was about four times more active than SRIF-14 in vitro and 7.5 times more active in vivo. When this small peptide was administered sc once or twice daily to immature rats for 9 or 25 days, the BW gain increased above the control. At the end of the weight gain studies the pituitary remained fully responsive to the peptide. Thus, [His1,Lys6] GHRP may be a valuable peptide for investigating the function of the pituitary somatotrophs and, in addition, it has the potential for increasing BW gain of a variety of normal animals by inducing GH release via a direct pituitary site of action.

Conformational energy studies and in vitro and in vivo activity data on growth hormone-releasing peptides.

A series of growth hormone-releasing peptides have been designed and tested for both in vitro and in vivo activity. In vitro activity at 1-10 ng/ml was obtained for the pentapeptide, His-DTrp-Ala-Trp-DPhe-NH2 (I) and the hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH2 (II). These peptides, as well as others to be described, are active in releasing GH in vivo at low microgram dosages. In this manuscript, the conformational properties and in vitro and in vivo activity of a series of small peptides are reported. Results of the biological studies are reported in an accompanying paper.